Conformationally constrained analogues of diacylglycerol. 18. The incorporation of a hydroxamate moiety into diacylglycerol-lactones reduces lipophilicity and helps discriminate between sn-1 and sn-2 binding modes to protein kinase C (PK-C). Implications

Article abstract of DOI:10.1021/jm0103965

An approach to reduce the log P in a series of diacylglycerol (DAG)-lactones known for their high binding affinity for protein kinase C (PK-C) is presented. Branched alkyl groups with reduced lipophilicity were selected and combined with the replacement o

Full text of DOI:10.1021/jm0103965

© Copyright 2001 by the American Chemical Society
Volume 44, Number 25
December 6, 2001
Letters
the presence of DAG fully activates both calcium-
dependent classical isozymes R, â, and γ and novel or
calcium-independent δ, ꢀ, η, and θ isozymes.3,4 The
transiently generated DAG, however, binds only weakly
to the C1 domains of the enzyme(s), which is in sharp
contrast to the well-known, high binding affinity dis-
played by the phorbol esters. Phorbol esters function as
potent and metabolically stable DAG surrogates with
the capacity to bind to PK-C with affinities several
orders of magnitude greater than those of the commonly
studied DAGs.5
Con for m a tion a lly Con str a in ed An a logu es
of Dia cylglycer ol. 18. Th e In cor p or a tion
of a Hyd r oxa m a te Moiety in to
Dia cylglycer ol-La cton es Red u ces
Lip op h ilicity a n d Help s Discr im in a te
betw een sn -1 a n d sn -2 Bin d in g Mod es to
P r otein Kin a se C (P K-C). Im p lica tion s for
Isozym e Sp ecificity
J eewoo Lee,*,† Kee-Chung Han,† J i-Hye Kang,†
Larry L. Pearce,‡ Nancy E. Lewin,‡ Shunqi Yan,§
Samira Benzaria,§ Marc C. Nicklaus,§
Over the past few years, we have attempted to bridge
the affinity gap between phorbol esters and DAGs by
two independent but mutually complementary ap-
proaches.6,7 The first approach seeks to reduce the
entropic penalty associated with DAG binding by con-
straining the glycerol backbone into five-member DAG-
lactones. The second approach involves the use of highly
branched alkyl chains to improve the interaction of the
DAG-lactone ligand with a cluster of conserved hydro-
phobic amino acids in the space between the two â
sheets of the C1 domain.8 Modeling experiments using
the crystal coordinates of the C1b domain of PK-Cδ in
complex with phorbol-13-acetate8 revealed two possible
binding orientations when the DAG-lactones were docked
into the empty C1b domain.7 These two apparently
equivalent binding modes, sn-1 and sn-2, form a net-
work of hydrogen bonds with amino acids Thr242,
Leu251, and Gly253 identical to those formed by phor-
bol-13-acetate in the crystal structure of the complex,
as schematically represented in Figure 1. On the basis
of the two nonequivalent carbonyl moieties of the DAG-
lactones, the sn-1 binding mode is defined as that in
which the sn-1 carbonyl and the hydroxyl group form
H-bonds with the protein. In the alternative sn-2
binding mode, the sn-2 carbonyl and the hydroxyl group
are engaged in H-bonding with the protein. A prediction
is that these two binding orientations can direct the
hydrophobic groups (R1 and R2) of the ligand into rather
different directions and that differences in binding
affinities should reflect preferences between these two
binding modes. It should be noted that molecular
Peter M. Blumberg,‡ and Victor E. Marquez*,§
Laboratory of Medicinal Chemistry, College of Pharmacy,
Seoul National University, Shinlin Dong, Kwanak-ku,
Seoul 151-742, South Korea, Laboratory of Cellular
Carcinogenesis and Tumor Promotion, Center for Cancer
Research, National Cancer Institute, Bethesda, Maryland
20892, and Laboratory of Medicinal Chemistry, Center for
Cancer Research, National Cancer Institute at Frederick,
376 Boyles St., Rm. 104, Frederick, Maryland 21701
Received August 20, 2001
Abstr a ct: An approach to reduce the log P in a series of
diacylglycerol (DAG)-lactones known for their high binding
affinity for protein kinase C (PK-C) is presented. Branched
alkyl groups with reduced lipophilicity were selected and
combined with the replacement of the ester or lactone oxygens
by NH or NOH groups. Compound 6a with an isosteric
N-hydroxyl amide arm represents the most potent and least
lipophilic DAG analogue known to date.
In tr od u ction a n d Ba ck gr ou n d . Membrane-gener-
ated diacylglycerol (DAG) resulting from the stimulus-
initiated activation of phospholipase C induces the
translocation of cytosolic protein kinase C (PK-C) to the
inner leaflet of the cellular membrane.1,2 The resulting
association of PK-C with membrane phospholipids in
* To whom correspondence should be addressed. J . L.: phone, 301-
880-7846; fax, 301-888-0649; e-mail, jeewoo@snu.ac.kr. V.E.M.: phone,
301-846-5954; fax, 301-846-6033; e-mail, marquezv@dc37a.nci.nih.gov.
† Seoul National University.
‡ National Cancer Institute.
§ National Cancer Institute at Frederick.
10.1021/jm0103965 CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/09/2001

4310 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Letters
effectively discriminated between both sn-1 and sn-2
binding alternatives since transposition of the NOH
group to replace the lactone oxygen resulted instead in
a very weak ligand. This situation contrasts with
compounds where both chemical groups are esters (side
chain ester and lactone) which bind in a combination
of sn-1 and sn-2 modes.7 In the present work, we propose
that the N-hydroxyl amide group is able to direct the
binding of DAG-lactones into the C1 domain of PK-C
exclusively in the sn-2 mode.
Dr u g Design Con sid er a tion s a n d Biologica l Re-
su lts. The gradual steps taken in the structure-activity
analysis leading to the potent N-hydroxyl amide DAG-
lactone 6a are described in Table 1. For the sake of
simplicity, only the geometric Z-isomers will be dis-
cussed in the analysis, although both Z- and E-isomers
were synthesized and tested. The DAG-lactone 1a ,
selected as the parent compound for this approach, was
identified in a previous study as a high binding affinity
ligand for PK-CR (Ki ) 2.89 nM) in a competition assay
that measures the ability of the ligand to displace bound
[20-3H-PDBU] from the enzyme.7 This molecule contains
two similar branched alkyl chains, one acyl (R1) and one
R-alkylidene (R2). The corresponding E-isomer 1b was
also a potent ligand for PK-CR, but only the Z-isomer
1a displayed potent and selective antitumor activity in
the NCI 60-cell line in vitro screeen.7 Since we also
discovered that DAG-lactones bearing linear acyl chains
were devoid of cellular activityspresumably caused by
cleavage of the acyl group by cellular esterasessit was
important to maintain R1 as a highly branched alkyl
group. Thus, the first step toward reducing log P
involved trimming four carbons off the alkyl group of
1a to give the smallest possible and highest branched
R1 group (i.e., pivaloyl). Relative to compound 1a , such
change lowered the lipophilicity of 2a by almost 2 log P
units without compromising binding affinity, which was
only reduced ca. 3-fold. Since the pivaloyl group was
considered an ideal small group stable toward hydroly-
sis, it was maintained constant. To recover the 3-fold
loss in potency caused by the smaller pivaloyl group,
the branched R-alkylidene (R2) group was enlarged by
two additional carbons to give compound 3a . This
compound equaled 1a in potency and was still 0.86 log
P units lower (more hydrophilic). Support for the
pivaloyl group as the smallest and most effective acyl
group was provided by compound 4a , which contained
the original larger branched acyl group (R1) of 1a and
the same R-alkylidene (R2) chain of 3a . This change was
not advantageous since the resulting compound (4a ) was
not only less potent but more lipophilic. Having reached
what appeared to be the ideal set of branched acyl and
R alkylidene chains in compound 3a , we turned our
attention to the ester moiety. Changing the ester moiety
into an amide (X ) O f NH) was desirable, since for
the same set of branched acyl and R-alkylidene chains
the log P was reduced by ca. 1 log P unit. Unfortunately,
the affinity of compound 5a for PK-C plummeted.
However, when the amide hydrogen in compound 5a
was replaced by a hydroxyl group (X ) NH f NOH),
potency was restored, and, relative to the initial DAG-
lactone 1a , the new compound (6a ) was 2.3 log P units
less lipophilic! This change has produced the most
potent and least lipophilic DAG analogue known to date.
F igu r e 1. H-bonding interactions of DAG-lactones at the C1
domain of PK-C in alternative binding modes sn-1 (left) and
sn-2 (right).
modeling is limited to examining only binary complexes
between the C1 domain and the DAG-lactone ligands,
since the phospholipid component of the ternary com-
plex is missing in the experimentally available X-ray
structure.8
In our previous studies with DAG-lactones, we com-
bined linear and branched R1 and R2 alkyl chains, but
were only able to guess at the preferred binding mode.
In fact, in most instances it appeared that both binding
alternatives coexisted.7 To increase our chances of
designing C1 domain, isozyme-specific DAG-lactones, we
felt that it was imperative to know which specific
binding mode, sn-1 or sn-2, was operating.
While the role of the alkyl chain in DAGs has been
principally correlated with providing adequate lipophi-
licity to facilitate partitioning into a lipid-rich environ-
ment, docking of the DAG-lactones into the C1 domain
of PK-C revealed potentially important hydrophobic
contacts with the protein that could provide the means
of achieving discrimination between sn-1 and sn-2
binding modes.7 Thus, an increase in binding affinity
due to the branched alkyl chains would probably result
from a combination of two factors: adequate membrane
partitioning and specific hydrophobic contacts with the
protein. We anticipated that by reducing the lipid
dependency required for membrane distribution, the
compounds would be more effectively discriminated by
the protein in terms of the two possible binding alterna-
tives (sn-1 versus sn-2), since this process of recognition
would allow protein-ligand interactions to become the
dominant forces.
While working with a set of DAG analogues, we
recently showed a practical way to minimize lipid-
dependency as a way to increase interaction of the
ligand with the protein through the gradual reduction
of log P.9 In the present work, the application of this
approach to DAG-lactones started with the progressive
reduction of log P from the potent, symmetrically
branched DAG-lactone (1a , Table 1).7 We will demon-
strate that this approach, coupled with other structural
changes, reduces lipophilicity by more than 2 orders of
magnitude without sacrificing binding affinity for PK-
C. A critical change that allowed this reduction in log
P was the isosteric replacement of the side chain ester
group with an N-hydroxyl amide group [RC(O)O f RC-
(O)NOH], which lowered the log P to an unprecedented
calculated value of 3.58, almost matching the value of
the prototypic phorbol ester, phorbol-12,13-dibutyrate
(PDBU, calculated log P ) 3.43). In addition, the N-OH
group provided a fourth pharmacophoric group that

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4311
Ta ble 1. Structure-Activity Analysis of log P and Ki as a Function of Size of the Acyl and R Alkylidene Chainsa
no.
R1
R2
X
Y
E/ Z
log P
Ki (nM)
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
CH2CH(i-Pr)2
CH2CH(i-Pr)2
(CH3)3C
(CH3)3C
(CH3)3C
CH2CH(i-Pr)2
CH2CH(i-Pr)2
CH2CH(i-Pr)2
CH2CH(i-Pr)2
O
O
O
O
O
O
O
O
NH
NH
NOH
NOH
O
O
O
O
O
O
O
O
O
O
O
O
O
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
E
5.89
5.89
4.04
4.04
5.03
5.03
6.88
6.88
3.95
3.95
3.58
3.58
5.01
5.01
4.64
2.89 ( 0.2
2.70 ( 0.4
8.32 ( 0.7
11.25 ( 0.7
2.90 ( 0.4
4.51 ( 0.5
6.87 ( 0.6
4.46 ( 0.4
429.7 ( 5.5
1260 ( 90
5.42 ( 0.3
4.81 ( 0.4
4330 ( 280
4840 ( 330
10430 ( 450
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
CH2CH[CH2(i-Pr)]2
(CH3)3C
CH2CH(i-Pr)2
CH2CH(i-Pr)2
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
NH
NH
NOH
O
O
a
log P was calculated according to the fragment-based program KOWWIN 1.63.10
NOH group and the amide carbonyl of Gln257 that is
absent in the phorbol ester PK-Cδ complex.8 This single
binding mode of 6a represents a significant step toward
achieving isozyme selectivity, since knowing exactly
what orientation the ligand prefers at the binding site
will help select appropriate acyl or R-alkylidene groups
designed to interact specifically with key hydrophobic
amino acids of the various isoforms. As anticipated, the
N-OH lactam 8a failed completely to dock at the
expected active site, confirming the validity of the
model.
Recent results by Kazanietz et al.12 show that al-
though no differences in the activity of 6a toward
isozymes R and δ were observed in in vitro inhibition
constants (Ki) from competitive binding curves against
PDBU, as well as in its capacity to stimulate PK-C-
catalyzed phosphorylation of the R-pseudosubstrate
peptide,13 the compound was exquisitely selective in
directing the translocation of these isozymes specifically
to either the cellular membrane (PK-CR) or the nuclear
membrane (PK-Cδ). The consequences of this specific
translocation are discussed in a companion paper de-
scribing the strong apoptosis inducing activity for 6a
in LNCaP prostate cancer cells.12 Since molecular
modeling is limited to examining only binary complexes
between the C1 domain and the ligand, the role of the
membrane in influencing isozyme specificity remains
unresolved. An additional structural element that could
be responsible for the specific membrane interaction of
6a is the subtle but important orientation of the
R-alkylidene chain induced by the Z- or E-stereochem-
isty of the double bond. This structural element confirms
the crucial role played by the branched hydrophobic
chains as only the Z-isomer was found active in inducing
apoptosis. Since no significant differences between 6a
and 6b were detected in the PK-C in vitro binding assay
(Table 1), this method has to be used only as a first
screening step. It appears that isozyme selectivity for
this class of compounds is best assessed by using PK-C
isoforms tagged with green fluorescent protein to visu-
alize cellular compartmentalization.1,2,9,12
F igu r e 2. Docking results showing H-bonding interactions
of 6a with the C1b domain of PK-Cδ exclusively in the sn-2
binding mode.
In addition, the extra NOH provides for an effective
discrimination between sn-1 and sn-2 binding modali-
ties, must likely by engaging in specific H-bonding with
the protein (vide infra). When a similar change in the
lactone ring was implemented by replacing the lactone
oxygen for NH (7a ,b) or NOH (8a ), the resulting ligands
behaved extremely poorly and displayed high Ki values
(Table 1). The implications of these changes in terms of
the two alternative binding modes are discussed below.
Com p u ta tion a l Mod elin g a n d Discu ssion . The
use of AutoDock version 2.411 for the docking of 6a into
the empty C1b cavity of PK-Cδ8 revealed the existence
of a single binding mode (Figure 2). In this unique sn-2
binding mode, the binding of the lactone carbonyl (sn-
2) to the NH group of Gly253 appears reinforced by a
strong H-bond coming from the sn-1 NOH group. There
is also an additional H-bond between the oxygen of the

4312 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Letters
(7) Nacro, K.; Bienfait, B.; Lee, J .; Han, K.-C.; Kang, J .-H.; Benzaria,
S.; Lewin, N. E.; Bhattacharyya, D. K.; Blumberg, P. M.;
Marquez, V. E. Conformationally constrained analogues of
diacylglycerol (DAG). 16. How much structural complexity is
necessary for recognition and high binding affinity to protein
kinase C? J . Med. Chem. 2000, 43, 921-944.
(8) Zhang, G. G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J . H.
Crystal structure of the cys2 activator-binding domain of protein
kinase C delta in complex with phorbol ester. Cell 1995, 81, 917-
914.
Ack n ow led gm en t. This work was partially sup-
ported by Grant KOSEF 2000-2-21500-001-3 from the
Korea Science and Engineering Foundation.
Su p p or tin g In for m a tion Ava ila ble: Detailed discussion
of the syntheses and analytical data for all target compounds
presented in Table 1 are provided. This material is available
free of charge via the Internet at http://pubs.acs.org.
(9) Nacro, K.; Sigano, D. M.; Yan, S.; Nicklaus, M. C.; Pearce, L.
L.; Lewin, N. E.; Gardfield, S. H.; Blumberg, P. M.; Marquez,
V. E. An optimized protein kinase C activating diacylglycerol
combining high binding affinity (Ki) with reduced lipophilicity
(log P). J . Med. Chem. 2001, 44, 1892-1904.
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J M0103965