Synthesis and modeling study of (2S,5R,6R)- and (2S,5R,6S)-6-Hydroxy-8-(1-decynyl)-benzolactam-V8 as protein kinase C modulators

Article abstract of DOI:10.1021/ol026013u

(matrix presented) Both (2S,5R,6R)- and (2S,5R,6S)-6-hydroxy-8-(1-decynyl)benzolactam-V8 were designed and synthesized as PKC modulators. Biological assays reveal the (6R)-ligand to be 20-fold more potent than its (6S)-counterpart in binding to PKCα.

Full text of DOI:10.1021/ol026013u

ORGANIC
LETTERS
2002
Vol. 4, No. 13
2169-2172
Synthesis and Modeling Study of
(2S,5R,6R)- and (2S,5R,6S)-6-Hydroxy-
8-(1-decynyl)-benzolactam-V8 as Protein
Kinase C Modulators
Zhi-Liang Wei,† Sukumar Sakamuri,† Pavel A. Petukhov,† Clifford George,‡
,†
Nancy E. Lewin,§ Peter M. Blumberg,§ and Alan P. Kozikowski*
Drug DiscoVery Program, Department of Neurology, Georgetown UniVersity Medical
Center, 3900 ReserVoir Road, NW, Washington, DC 20007, NaVal Research
Laboratory, 4555 OVerlook AVe., SW, Washington, DC 20375, and Laboratory of
Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute,
Bethesda, Maryland 20892
kozikowa@georgetown.edu
Received April 15, 2002
ABSTRACT
Both (2S,5R,6R)- and (2S,5R,6S)-6-hydroxy-8-(1-decynyl)benzolactam-V8 were designed and synthesized as PKC modulators. Biological assays
reveal the (6R)-ligand to be 20-fold more potent than its (6S)-counterpart in binding to PKCr.
Protein kinase C (PKC) is a phospholipid-dependent serine/
threonine kinase family consisting of at least 11 closely
related isozymes that mediate a wide range of cellular signal
transduction processes.1 However, complete details of func-
tional significance of each PKC isozyme in these cellular
events and in tumor promotion are not fully understood,
which is attributable in part to the lack of isozyme-selective
modulators. The discovery of isozyme-selective activators
and inhibitors of PKC is crucial to both dissecting and
manipulating their physiological pathways. The teleocidins,
as well as analogues of indolactam V (ILV) (Figure 1), are
a class of natural products that show high affinity for PKC
but possess little isozyme selectivity.2 The teleocidins thus
serve as important lead structures in the search for isozyme-
selective modulators of PKC.3 Through studies of the active
conformation of ILV, benzolactam-V8 (1), a molecule whose
constrained eight-membered lactam is forced to adopt the
active twist conformation, was designed by Endo and co-
workers.4 Such benzolactams provide a synthetically more
accessible core structure in the search for isozyme-selective
† Georgetown University Medical Center.
‡ Naval Research Laboratory.
§ National Cancer Institute.
(1) (a) Nishizuka, Y. Science 1992, 258, 607. (b) Lester, D. S.; Epand,
R. M. Protein Kinase C. Current Concepts and Future PerspectiVes; Ellis
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(d) Mochly-Rosen, D.; Kauvar, L. M. AdV. Pharmacol. 1998, 44, 91. (e)
Glazer, R. I. Curr. Pharm. Des. 1998, 4, 277. (g) Armato, U.; Whitfield, J.
F.; Charini, A.; Pra, I. D. Curr. Top. Biochem. Res. 1999, 1, 1. (f) Newton,
A. C. Chem. ReV. 2001, 101, 2353.
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10.1021/ol026013u CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/04/2002

The ortho functionalization of N-(tert-butoxycarbonyl)-
aniline was used as the key strategy in our synthesis. Thus,
ortho metalation of 5 with excess tert-butyllithium at low
temperature provided the corresponding dilithio intermedi-
ate,9 which was treated with the aldehyde 68c to give rise to
a mixture of the alcohols 7/8. The diastereoisomers of 7 and
8 could not be separated efficiently by flash column
chromatography. Acid hydrolysis of 7/8 by treatment with
1 M HCl in THF at 70 °C yielded 9 and 10 in a ratio of ca.
1.3:1, which were readily separated chromatographically. The
absolute configuration of the newly generated hydroxyl group
of the two isomers was assigned by the X-ray crystal-
lographic analysis of 9. Coupling of the amines 9 and 10
with D-valine-derived triflate 1110 followed by simultaneous
removal of the benzyl ester and N-Cbz protecting groups by
catalytic hydrogenation provided the amino acids 12 and 13,
respectively (Scheme 1).
Figure 1.
PKC modulators. In connection with such efforts, we
reported that 8-(1-decynyl)benzolactam-V8 (2) exhibited
improved potency and selectivity for the classical isozymes.5
These results prompted us to synthesize additional benzolac-
tam-V8 analogues bearing substituents that may confer
enhanced isozyme selectivity.6
Scheme 1a
The X-ray crystal structure of the complex of the C1b
activator-binding domain of PKCδ with phorbol 13-acetate
has revealed how this phorbol ester binds to PKC.7 Although
the structure is incomplete from the standpoint of revealing
interactions with lipids, it nonetheless opens the possibility
for the rational design of selective PKC modulators through
the evaluation of their binding to the homologous PKC
isozymes using computer assisted docking methods.5,8 Herein
we describe the synthesis and modeling of the 6-hydroxyl-
substituted benzolactam-V8 analogues 3 and 4 as PKC
modulators. These compounds were designed with the idea
that the extra hydrogen bond donor group might enhance
PKC affinity and selectivity.
(3) (a) Meseguer, B.; Alonso-D´ıaz, D.; Griebenow, N.; Herget, T.;
Waldmann, H. Chem. Eur. J. 2000, 6, 3943. (b) Irie, K.; Isaka, T.; Iwata,
Y.; Yanai, Y.; Nakamura, Y.; Koizumi, F.; Ohigashi, H.; Wender, P. A.;
Satomi, Y.; Nishino, H. J. Am. Chem. Soc. 1996, 118, 10733. (c)
Kozikowski, A. P.; Ma, D.; Du, L.; Lewin, N. E.; Blumberg, P. M. J. Am.
Chem. Soc. 1995, 117, 6666. (d) Quick, J.; Saha, B.; Driedger, P. E.
Tetrahedron Lett. 1994, 35, 8549. (e) Kozikowski, A. P.; Ma, D.; Pang,
Y.-P.; Shum, P.; Likic, V.; Mishra, P. K.; Macura, S.; Basu, A.; Lazo, J.
S.; Ball, R. G. J. Am. Chem. Soc. 1993, 115, 3957. (f) Webb, R. R., II;
Venuti, M. C.; Eigenbrot, C. J. Org. Chem. 1991, 56, 4706. (g) Kozikowski,
A. P.; Shum, P. W.; Basu, A.; Lazo, J. S. J. Med. Chem. 1991, 34, 2420.
(h) Kozikowski, A. P.; Sato, K.; Basu, A.; Lazo, J. S. J. Am. Chem. Soc.
1989, 111, 6228.
(4) (a) Endo, Y.; Ohno, M.; Hirano, M.; Itai, A.; Shudo, K. J. Am. Chem.
Soc. 1996, 118, 1841. (b) Endo, Y.; Takehana, S.; Ohno, M.; Driedger, P.
E.; Stabel, S.; Mizutani, M. Y.; Tomioka, N.; Itai, A.; Shudo, K. J. Med.
Chem. 1998, 41, 1476.
(5) Kozikowski, A. P.; Wang, S.; Ma, D.; Yao, J.; Ahmad, S.; Glazer,
R. I.; Bogi, K.; Acs, P.; Modarres, S.; Lewin, N. E.; Blumberg, P. M. J.
Med. Chem. 1997, 40, 1316.
(6) (a) Ma, D.; Tang, W.; Kozikowski, A. P.; Lewin, N. E.; Blumberg,
P. M. J. Org. Chem. 1999, 64, 6366. (b) Ma, D.; Wang, G.; Wang, S.;
Kozikowski, A. P.; Lewin, N. E.; Blumberg, P. M. Bioorg. Med. Chem.
Lett. 1999, 9, 1371. (c) Ma, D.; Zhang, T.; Wang, G.; Kozikowski, A. P.;
Lewin, N. E.; Blumberg, P. M. Bioorg. Med. Chem. Lett. 2001, 11, 99.
(7) Zhang, G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J. H. Cell
1995, 81, 917.
(8) (a) Qiao, L.; Wang, S.; George, C.; Lewin, N. E.; Blumberg, P. M.;
Kozikowski, A. P. J. Am. Chem. Soc. 1998, 120, 6629. (b) Wang, S.; Liu,
M.; Lewin, N. E.; Lorenzo, P. S.; Bhattacharrya, D.; Qiao, L.; Kozikowski,
A. P.; Blumberg, P. M. J. Med. Chem. 1999, 42, 3436. (c) Zhao, L.; Qiao,
L.; Rong, S.-B.; Kozikowski, A. P. Tetrahedron Lett. 2000, 41, 8711. (d)
Qiao, L.; Zhao, L.-Y.; Rong, S.-B.; Wu, X.-W.; Wang, S.; Fujii, T.;
Kazanietz, M. G.; Rauser, L.; Savage, J.; Roth, B. L.; Flippen-Anderson,
J.; Kozikowski, A. P. Bioorg. Med. Chem. Lett. 2001, 11, 955.
a (i) t-BuLi (2.2 equiv), THF, -78 to -20 °C, 2 h, then 6, -20
°C, 5 h, 31%. (ii) 1 M HCl, THF, 70 °C, 5 h, 87%. (iii) 11, 2,6-
lutidine, 1,2-dichloroethane, 70 °C, 72 h, 65-67%. (iv) 10% Pd-
C, H2 (1 atm), ethanol, rt, 24 h, 100%. (v) See Table 1.
With the amino acids 12 and 13 in hand, their lactamiza-
tion to 14 and 15 was investigated. Two procedures, BOP/
HOBt/NMM/DMF and DPPA/Et3N/DMF, were examined
for this cyclization reaction. The results are summarized in
Table 1. The erythro-isomer 12 could be readily lactamized
(9) Muchowski, J. M.; Venuti, M. C. J. Org. Chem. 1980, 45, 4798.
(10) Kogan, T. P.; Somers, T. C.; Venuti, M. C. Tetrahedron 1990, 46,
6623.
2170
Org. Lett., Vol. 4, No. 13, 2002

(16). Treatment of 16 with Ac2O and pyridine followed by
bromination at room temperature using benzyltrimethylam-
monium tribromide (BTMA Br3)11 gave 18 in high yield.
The latter was coupled with 1-decyne in the presence of
PdCl2(PPh3)2 and CuI followed by removal of the acetate
groups with 2 M NaOH in methanol to afford (2S,5R,6S)-
6-hydroxy-8-(1-decynyl)benzolactam-V8 (3).
In the same manner as described above, the other isomer,
(2S,5R,6R)-6-hydroxy-8-(1-decynyl)benzolactam-V8 (4), was
synthesized from 15 (Scheme 2).
Compounds 3 and 4 have been evaluated for their ability
to displace phorbol 12,13-dibutyrate (PDBu) binding from
recombinant PKCR. The Ki values for these analogues were
found to be 1571 ( 147 nM and 76.1 ( 3.8 nM, respectively,
while the Ki value for 2 was 14.7 ( 1.3 nM. While both of
the hydoxylated benzolactams were unexpectedly found to
be less potent than the parent compound, the (6R)-isomer is
20-fold more potent than the (6S)-isomer.
To understand these results, the ligands 2, 3, and 4 were
docked to the PKCR C1b domain using the FlexX12 module
in SYBYL. Loop A comprising amino acids Thr 108, Tyr
109, Ser 110, Ser 111, Pro 112, Thr 113, and Phe 114 and
loop B comprising amino acids Leu 121, Leu 122, Tyr 123,
Gly 124, Leu 125, Ile 126, His 127, and Gln 128 were used
to create the binding site for the docking experiments. The
poses of ligands 2, 3, and 4 ranked as best are shown in
Figure 2.
Table 1. Lactamization of Amino Acids 12 and 13
entry
substrate
conditionsa
product
yieldb
1
2
3
4
12
12
13
13
A
B
A
B
14
14
15
15
91%
75%
18%
39%
a Condition A: BOP, HOBt, NMM, DMF, rt, 72 h; condition B: DPPA,
Et3N, DMF, rt, 48 h. BOP, benzotriazol-1-yloxy-tris(dimethylamino)phos-
phonium hexafluorophosphate; HOBt, hydroxybenzotriazole; NMM, N-
methylmorpholine; DPPA, diphenylphosphoryl azide. b Isolated yield.
by use of either the BOP or DPPA reagent to afford 14 in
good yield, although higher yields were achieved by using
the BOP reagent (entries 1 and 2). However, only low to
moderate yields were achieved in the lactamization of the
threo-isomer 13 to 15 (entries 3 and 4). The lower yields in
this cyclization reaction may reflect increased steric hin-
drance in the transition state.
Completion of the synthesis of (6S)-hydroxy-benzolactam-
V8 (3) is outlined in Scheme 2. Reductive methylation of
14 afforded the (6S)-hydroxylated parent benzolactam-V8
Scheme 2a
The molecular modeling and docking studies reveal that
all ligands 2, 3, and 4 are able to form a network of hydrogen
bonds with the PKCR C1b domain (Table 2 and Figure 2).
Table 2. Hydrogen Bond Distance (Å) between Acceptor and
Hydrogen Atoms in PKCR C1b Domain and Ligands 2, 3, and
4
2
3
4
r(O6-OH‚‚‚NH2Gln128
)
n/aa
n/aa
n/aa
1.75
1.92
1.88
r(H6-OH‚‚‚COGly124
r(H6-OH‚‚‚COTyr109
)
)
1.85
1.95
2.11
2.38
1.75
r(H11-OH‚‚‚COTyr109
r(H11-OH‚‚‚COSer111
)
)
2.29
1.60
1.83
1.77
r(H4-NH‚‚‚COLeu122
r(O3-CO‚‚‚NHGly124
)
)
2.15
1.64
a n/a, not applicable, group absent in the ligand.
The amido group in all three ligands forms two hydrogen
bonds with Leu 122 and Gly 124. The primary hydroxyl
group at C-11 in ligands 3 and 4, acting as a hydrogen bond
donor, forms two hydrogen bonds with Tyr 109 and Ser 111,
although in ligand 2 it participates in only one interaction
with Tyr 109. The (6S)-hydroxyl group in ligand 3 forms
only one hydrogen bond with Tyr 109, whereas the (6R)-
hydroxyl group in ligand 4 forms two hydrogen bonds with
a (i) HCHO, HOAc, NaCNBH3, CH3CN, 0 °C, 1 h, 89-90%.
(ii) Ac2O, pyridine, Et3N, rt, 12 h, 81-83%. (iii) BTMA Br3,
CH2Cl2, MeOH, CaCO3, rt, 2 h, 89-90%. (iv) PdCl2(PPh3)2, CuI,
1-decyne, Bu4NI, Et3N, DMF, 70 °C, 72 h, 69-75%. (v) 2 M
NaOH, MeOH, rt, 30 min, 88-93%.
(11) Kajigaeshi, S.; Kakinami, T.; Inoue, K.; Kondo, M.; Nakamura, H.;
Fujikawa, M.; Okamoto, T. Bull. Chem. Soc. Jpn. 1988, 61, 597.
(12) Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol. Biol. 1996,
261, 470.
Org. Lett., Vol. 4, No. 13, 2002
2171

Figure 2. Hydrogen bonding interactions between PKCR C1b domain and the best docked poses of 2 (A), 3 (B), and 4 (C).
Gly 124 and Gln 128. While ligand 3 forms five hydrogen
bonds with PKCR and ligand 4 forms six, this difference
alone is unlikely to explain the poorer binding affinity of 3.
The lower activity of 3 may also arise from the ability of its
hydroxyl groups to engage in intramolecular hydrogen bond
formation in the binding conformation (r[H11-OH‚‚‚O6-OH
]
) 1.85 Å, Figure 2B). Consequently, this would make the
intermolecular interactions of these groups less favorable.
On the other hand, the lower activity of 4 compared to the
activity of 2 may originate from additional bulk and
unfavorable electrostatic interactions as well as desolvation
factors due to the presence of the 6-hydroxyl group. The
surface of the binding site in the PKCR C1b domain is
negatively charged (Figure 3A), thus resulting in an unfavor-
able interaction with the oxygen atom of the 6-hydroxyl
group in 4 (note the additional blue, negatively charged spot
in Figure 3C compared to Figure 3B).
In summary, we have described an efficient route to the
6-hydroxylated benzolactam-V8 isomers 3 and 4. Biological
assays reveal that the (6R)-isomer shows higher affinity for
PKCR than the (6S)-isomer, a result that is rationalized by
our modeling studies.
Acknowledgment. We thank the National Institutes of
Health (CA 79601) for their support of these studies.
Supporting Information Available: Modeling methods,
crystal data for compound 9, and detailed experimental
procedures with spectroscopic data. This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 3. Electron density surface of the PKCR C1b domain
mapped by electrostatic potential, docked ligand 4 depicted by
capped sticks (A); electron density surfaces of 2 (B) and 4 (C)
mapped by electrostatic potential (blue is negative and red is
positive).
OL026013U
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Org. Lett., Vol. 4, No. 13, 2002