Modular Total Synthesis of Protein Kinase C Activator (-)-Indolactam v

Article abstract of DOI:10.1021/acs.orglett.6b00614

A concise, eight-step total synthesis of (-)-indolactam V, a nanomolar agonist of protein kinase C, is reported. The synthesis relies upon an efficient copper-catalyzed amino acid arylation to establish the indole C4-nitrogen bond. This cross-coupling met

Full text of DOI:10.1021/acs.orglett.6b00614

Letter
pubs.acs.org/OrgLett
Modular Total Synthesis of Protein Kinase C Activator
(−)-Indolactam V
Jeremy Haynes-Smith, Italia Diaz, and Kelvin L. Billingsley*
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132, United States
S
* Supporting Information
ABSTRACT: A concise, eight-step total synthesis of (−)-in-
dolactam V, a nanomolar agonist of protein kinase C, is
reported. The synthesis relies upon an efficient copper-
catalyzed amino acid arylation to establish the indole C4−
nitrogen bond. This cross-coupling method is applicable to a
range of hydrophobic amino acids, providing a platform for
further diversification of indolactam alkaloid scaffolds and
studies on their potent biological activity.
rotein kinase C (PKC) is a family of serine and threonine
kinases that play critical roles in cellular signal trans-
these dichotomous effects, tumor growth promotion versus
suppression, for PKC ligands remains poorly understood,10
which has to date limited the therapeutic potential of the
indolactam alkaloids.
P
duction,1 and dysfunction of specific isozymes has been
implicated in a range of disease states.2 Small molecule
therapeutics that regulate PKC activity have correspondingly
been employed for the treatment of numerous disorders,
including various cancers, Alzheimer’s disease, and HIV/AIDS.3
In addition, these molecular agents have been applied in
biochemical studies to understand disease onset and pro-
gression.4 For example, the indolactam alkaloids (Figure 1)
The biological significance of the indolactam alkaloids has
resulted in numerous synthetic approaches for accessing these
natural products. The majority of these strategies target the 9-
membered lactam parent structure, (−)-indolactam V (1),
which is an agonist of both conventional and novel PKC
isoforms at nanomolar concentrations.11 These previous
syntheses have primarily relied upon an intramolecular peptide
coupling to establish the macrocycle.11 Other innovative
approaches to 1 have included (1) photochemically induced
9-membered ring formation11f or (2) trapping of indolyne
intermediates to establish the C4−nitrogen bond followed by
Lewis acid mediated macrocyclization.11k However, while
further hydrophobic substitution on the scaffold, particularly
at C6, C7, or C12, has been shown to have profound effects on
the potency and tumor promoting capacity of these
molecules,12 the lack of general synthetic strategies to
selectively diversify at these specific positions has restricted
systematic structure−activity relationship studies.13 Further-
more, biological evaluation of tumor promotion properties for
PKC modulators has relied upon a variety of assays,14 and
inconsistencies among previous reports has further complicated
the preclinical analyses of indolactam alkaloids.15 To fully
appreciate the molecular features that confer potency and/or
selectivity to these scaffolds and expand their therapeutic
potential, a systematic structure−activity platform is required.
A primary objective of our research program is to develop
modular strategies toward indolactam natural products and
analogues. Through their biochemical assessment, we aim to
determine the structural features that govern the biological
profile of indolactam-based agents and, in turn, increase the
Figure 1. Representative indolactam alkaloids. Pharmacophore shown
in blue.
have been used in mechanistic investigations of stem cell
differentiation,5 tumor growth,6 and neurodegeneration.7 These
natural products regulate kinase activity through binding to the
PKC regulatory C1 domain, mimicking the endogenous ligand
diacylglycerol.8 However, unlike other PKC modulators (e.g.,
bryostatin 1) that suppress cancer cell proliferation, indolactam
alkaloids promote tumor growth.9 The mechanistic basis for
Received: March 3, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b00614
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
therapeutic relevance of this family of PKC activators. The
initial aim in this program was to establish a concise synthetic
approach to access the core indolactam scaffold, 1. We
envisioned that indolactam macrocycle in 1 could be accessed
through intramolecular indole alkylation at C3 with a C8
electrophile derived from dipeptide precursor 2 (Scheme 1).
in this initial report afforded the cross-coupling product in
modest yield (62%), employed long reaction times (52 h), and
was not applicable to other hydrophobic alkyl amino , feature
critical for indolactam analogue synthesis. To develop a more
general protocol, we first examined the arylation of valine with
4-bromo-N-tosylindole (2). While we successfully isolated the
desired N-arylvaline product (3) in a 56% yield after 52 h of
reaction time, we also observed the presence of a byproduct (4-
amino-N-tosylindole, 6) in a ∼2:1 ratio (56% and 30% isolated
yields of 5 and 6, respectively) (Figure 2A). Through further
Scheme 1. Retrosynthetic Analysis of 1
Dipeptide 2 would in turn be readily assembled from the N-
indolated amino acid 3. We reasoned that the amino acid
moiety in 3 might be introduced through a metal-catalyzed
arylation with a 4-haloindole electrophile. Critically, use of a
cross-coupling method that is amenable to introduction of
other hydrophobic amino acids permits further diversification
of the indolactam scaffold. In this report, we describe a modular
cross-coupling protocol to assemble a collection of substituted
N-aryl amino acids and apply our synthetic strategy in the
concise total synthesis of natural product indolactam V (1).
Establishing an effective, high-yielding method for amino acid
arylation was our first key objective, as indolactam alkaloids
typically incorporate a hydrophobic amino acid side chain at the
C12-position of the 9-membered lactam ring. For example,
while 1 contains a valine moiety and corresponding isopropyl
substituent at the C12-position, other naturally occurring
variants (e.g., (−)-indolactam I) integrate isoleucine, exhibiting
a sec-butyl C12 alkyl chain (Figure 1). In addition, since the 9-
membered ring exists as two stable conformers (cis- and trans-
amide) at room temperature, conformationally restricted
analogues possessing hydrophobic substituents at appropriate
positions have been designed and displayed increased selectivity
for novel PKC isozymes.16 While C12 hydrophobicity has been
observed to play a key role in the potency of these compounds,
the low yielding microbial-based synthetic methods currently
used to prepare indolactam scaffolds have precluded systematic
analysis of hydrophobic substituents.17 We sought to utilize a
copper-catalyzed cross-coupling method that allows for the
arylation of a diverse collection of hydrophobic amino acids
with commercially available 4-bromoindole.
The copper-catalyzed carbon−nitrogen bond-forming reac-
tion has been extensively researched over the past several
decades, and numerous catalysts and protocols have been
developed for the coupling of aryl halides with amine
nucleophiles.18 Our early studies demonstrated that both
unprotected and N-alkyl- or N-benzyl-protected 4-bromoin-
doles underwent decomposition of the heteroaryl electrophile
under the conditions for copper catalysis. Drawing on a single
report by Ishikawa and co-workers describing N-arylation of
valine with a derivatized 4-bromoindole,19 we found that tosyl
protection of the indole ring enabled successful copper-
catalyzed coupling. While successful, the protocol described
Figure 2. (A) Copper-catalyzed arylation of valine with 4-bromo-N-
tosylindole. (B) In situ oxidation of 5 results in N-arylimino acid
intermediate, which yields 6 upon hydrolysis.
investigation, we discovered that desired product 5 was prone
to oxidation under the reaction conditions,20 affording an N-
arylimino acid intermediate, which then yielded byproduct 6
upon hydrolysis (Figure 2B). Since C−N bond formation
occurred at a faster rate than the undesired oxidation, we
hypothesized that adjusting the reaction time as well as
concentration would prevent in situ degradation of 5 and
increase the isolated yield of the desired product. Accordingly,
shortening the reaction to 20 h provided 5:6 in a 14:1 ratio and
85% isolated yield of 5, with no epimerization of the α-carbon
(Figure 2A).
The substrate scope of this copper-catalyzed process was
examined with a range of hydrophobic amino acids. Similar to
valine, the reaction of 4 with isoleucine was effective, resulting
in a greater than 90% yield of the desired product 7 (Figure 3).
Less sterically hindered amino acids leucine and norvaline also
cleanly allowed for C−N bond formation in 83% and 88%
isolated yields, respectively (Figure 3). Further, a conforma-
tionally restricted amino acid, proline, was a suitable
nucleophile for this cross-coupling process. However, it should
be noted that the product of this transformation readily
degrades upon purification. Lastly, amino acids such as
phenylalanine and methionine, which possess hydrophobic
functional groups, were coupled to 4 in high yield. Importantly,
this initial sequence for installation of an amino acid-derived
subunit addresses a key practical limitation of previous
synthetic routes, which are typically low yielding and/or
require numerous steps to prepare suitable partners to
construct the hydrophobic amino acid motif.11 Therefore, this
copper-catalyzed arylation of diverse hydrophobic amino acids
circumvents lengthy manipulations and provides a general
strategy to introduce the desired substituents at the C12-
position.
B
DOI: 10.1021/acs.orglett.6b00614
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
addition from the 3-position of the free N−H indole.
Numerous protocols for N-tosyl protecting group removal,
including mildly basic conditions,21 fluoride reagents,22 and
single-electron-transfer processes,23 resulted only in decom-
position of 14. However, when 14 was subjected magnesium in
methanol, detosylation occurred with concurrent dehydration
of the C8-alcohol to provide a 12% yield of alkene 15 (Table
1). Importantly, 15 possesses the necessary structural proper-
a
Table 1. Deprotection−Dehydration of 14
a
entry
Mg (equiv)
temp (°C)
time (h)
yield (%)
1
2
3
4
5
6
7
5
10
20
20
20
40
80
rt
rt
7
7
12
27
50
22
<5
26
<5
rt
rt
7
24
7
50
rt
7
rt
7
Figure 3. Copper-catalyzed arylation of hydrophobic amino acids with
4-bromo-N-tosylindole. Reaction conditions: 0.50 mmol of 4, 0.60
mmol of amino acid, 0.05 mmol of CuI, and 0.75 mmol of Cs2CO3 in
0.50 mL of DMSO under argon atmosphere for 20 h. Isolated yield is
average of two experiments. (a) Product decomposes during isolation;
reported yield is based upon 1H NMR analysis of crude product
mixture with internal standard. (b) 0.10 mmol CuI used.
a
Isolated yields.
ties for cyclization: nucleophilic C3-position (deprotected
indole) and electrophilic C8-position (2-aminoacrylate moiety).
To capitalize on this result, we undertook further optimization
of the deprotection−dehydration reaction. Increasing the
equivalents of magnesium from 5 to 10 resulted in a higher
yield (27%), and 20 equiv proved to be ideal, providing a 50%
yield of 15. Additional magnesium beyond 20 equiv resulted in
reduction of the C8−C9 alkene of 15 and reduced overall
yields. Longer reaction times or higher temperatures were
likewise deleterious.
We next sought to establish conditions for the concise
assembly of macrocyclic indolactam structures, commencing
with synthesis of indolactam V (1) (Scheme 2). Upon tosyl
a
Scheme 2. Preparation of Intermediate 8
To close the indolactam V macrocycle we envisioned
activation of the C8−C9 enoate alkene with an oxophilic
Lewis acid and C3-alkylation of the pendant indole (Scheme
3). Garg and co-workers have previously studied the cyclization
Scheme 3. Total Synthesis of (−)-Indolactam V (1)
a
MeSer = L-serine methyl ester HCl.
properties of 15, which they prepared in 15 steps from
commercially available 5-benzyloxyindole.11k Accordingly,
application of the modified conditions for activation with
ZrCl4 reported by the Garg effectively promoted cyclization of
15 to the desired tricycle 16 in a 77% yield (13% recovered 15).
Epimerization at C9 to the natural stereoisomer with NaHCO3
(50% yield with 45% 16 recovered)11d followed by reduction of
the ester with LiBH4 (99% yield) provided indolactam V (1)
with an optical rotation identical to that of the reported value.24
In summary, we have developed a concise, eight-step
synthetic route of indolactam V that utilizes our modular
cross-coupling protocol. The development of an effective
protection of 4-bromoindole, 4 was subjected to three
consecutive steps: (1) C−N bond formation with valine to
furnish 5, (2) reductive amination of 5 with formaldehyde and
NaBH3CN to provide 13, and (3) EDC- promoted acid
coupling between 13 and serine methyl ester to afford 14.
Importantly, this sequence yielded 14 in a 55% yield over 3
steps and required only a single purification (column
chromatography purification of 14) to be performed.
Our macrolactamization strategy relied on electrophilic
activation of a C8-alcohol and ring closure via nucleophilic
C
DOI: 10.1021/acs.orglett.6b00614
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Masuda, T.; Sakai, K.; Goto, T. Tetrahedron Lett. 1986, 27, 5735.
(d) Masuda, T.; Nakatsuka, S.-I.; Goto, T. Agric. Biol. Chem. 1989, 53,
2257. (e) Kogan, T. P.; Somers, T. C.; Venuti, M. C. Tetrahedron
1990, 46, 6623. (f) Mascal, M.; Moody, C. J.; Slawin, A. M. Z.;
Williams, D. J. J. Chem. Soc., Perkin Trans. 1 1992, 823.
(g) Semmelhack, M. F.; Rhee, H. Tetrahedron Lett. 1993, 34, 1395.
(h) Quick, J.; Saha, B.; Driedger, P. E. Tetrahedron Lett. 1994, 35,
magnesium-promoted one-pot deprotection−dehydration re-
action further allows rapid access to an advanced precursor and
facile cyclization to the indolactam tricycle. A key advantage of
our strategy is the application of our optimized copper-
catalyzed amino acid arylation, which provides a high yielding
method for introduction of diverse hydrophobic subunits. We
anticipate that application of this protocol to acidic, basic, and
unnatural amino acids will serve as a versatile platform for
diversification of the indolactam scaffold. Ongoing studies are
focused on the synthesis and biological evaluation these
analogues as well as more elaborate members of the indolactam
alkaloid family.
́
́
8549. (i) Suarez, A. I.; Garcıa, M. C.; Compagnone, R. S. Synth.
Commun. 2004, 34, 523. (j) Xu, Z.; Zhang, F.; Zhang, L.; Jia, Y. Org.
Biomol. Chem. 2011, 9, 2512. (k) Bronner, S. M.; Goetz, A. E.; Garg,
N. K. J. Am. Chem. Soc. 2011, 133, 3832. (l) Mari, M.; Bartoccini, F.;
Piersanti, G. J. Org. Chem. 2013, 78, 7727. (m) Fine Nathel, N. F.;
Shah, T. K.; Bronner, S. M.; Garg, N. K. Chem. Sci. 2014, 5, 2184.
(n) Noji, T.; Okano, K.; Tokuyama, H. Tetrahedron 2015, 71, 3833.
(12) (a) Irie, K.; Nakagawa, Y.; Ohigashi, H. Curr. Pharm. Des. 2004,
10, 1371. (b) Irie, K.; Yanagita, R. C.; Torii, K.; Nakagawa, Y.
Heterocycles 2007, 73, 289.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b00614.
(13) (a) Nakagawa, Y. Biosci., Biotechnol., Biochem. 2012, 76, 1262.
(b) Irie, K.; Nakagawa, Y.; Ohigashi, H. Curr. Pharm. Des. 2004, 10,
1371.
(14) (a) Bogi, K.; Lorenzo, P.; Szallasi, Z.; Acs, P.; Wagner, G. S.;
̈
Experimental procedures and characterization of the
described compounds (PDF)
Blumber, P. M. Cancer Res. 1998, 58, 1423. (b) Yotti, L. P.; Chang, C.
C.; Trosko, J. E. Science 1979, 206, 1089. (c) Setterblad, N.; Onyango,
I.; Pihlgren, U.; Rask, L.; Andersson, G. J. Immunol. 1998, 161, 4819.
(d) Rohrschneider, L. R.; Boutwell, R. K. Cancer Res. 1973, 33, 1945.
(15) (a) Irie, K.; Yanagita, R. C.; Nakagawa, Y. Med. Res. Rev. 2012,
32, 518. (b) Kedei, N.; Lubart, E.; Lewin, N. E.; Telek, A.; Lim, L.;
Mannan, P.; Garfield, S. H.; Kraft, M. B.; Keck, G. E.; Kolusheva, S.;
Jelinek, R.; Blumberg, P. M. ChemBioChem 2011, 12, 1242.
(16) Nakagawa, Y.; Irie, K.; Komiya, Y.; Ohigashi, H.; Tsuda, K.-I.
Tetrahedron 2004, 60, 7077.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: klbillin@sfsu.edu.
Notes
The authors declare no competing financial interest.
(17) (a) Kajiyama, S.-I.; Irie, K.; Kido, T.; Koshimizu, K.; Hayashi,
H.; Arai, M. Tetrahedron 1991, 47, 5453. (b) Hirota, M.; Suganuma,
M.; Yoshizawa, S.; Horiuchi, T.; Nakayasu, M.; Hasegawa, M.; Endo,
Y.; Shudo, K.; Fujiki, H. Jpn. J. Cancer Res. 1987, 78, 577.
(18) (a) Sambiagio, C.; Marsden, S. P.; Blacker, A. J.; McGowan, P.
C. Chem. Soc. Rev. 2014, 43, 3525. (b) Ma, D.; Zhang, Y.; Yao, J.; Wu,
S.; Tao, F. J. Am. Chem. Soc. 1998, 120, 12459. (c) Okano, K.;
Tokuyama, H.; Fukuyama, T. Org. Lett. 2003, 5, 4987. (d) Kubo, T.;
Katoh, C.; Yamada, K.; Okano, K.; Tokuyama, H.; Fukuyama, T.
Tetrahedron 2008, 64, 11230.
(19) Ishikawa, T.; Kurokawa, M.; Nakanishi, W. Heterocycles 2007,
71, 847.
(20) Thomas, C.; Wu, M.; Billingsley, K. L. J. Org. Chem. 2016, 81,
330.
ACKNOWLEDGMENTS
■
This study was supported by generous startup funds from the
San Francisco State University College of Science and
Engineering.
REFERENCES
(1) Newton, A. C. J. Biol. Chem. 1995, 270, 28495.
■
(2) (a) Churchill, E.; Budas, G.; Vallentin, A.; Koyanagi, T.; Mochly-
Rosen, D. Annu. Rev. Pharmacol. Toxicol. 2008, 48, 569. (b) Masliah,
E.; Cole, G.; Shimohama, S.; Hansen, L.; DeTeresa, R.; Terry, R. D.;
Saitoh, T. J. Neurosci. 1990, 10, 2113. (c) Dempsey, E. C.; Newton, A.
C.; Mochly-Rosen, D.; Fields, A. P.; Reyland, M. E.; Insel, P. A.;
Messing, R. O. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000, 279, L429.
(3) (a) Mochly-Rosen, D.; Das, K.; Grimes, K. V. Nat. Rev. Drug
Discovery 2012, 11, 937. (b) Mackay, H. J.; Twelves, C. J. Nat. Rev.
Cancer 2007, 7, 554.
̌
(21) (a) Bajwa, J. S.; Chen, G.-P.; Prasad, K.; Repic, O.; Blacklock, T.
K. Tetrahedron Lett. 2006, 47, 6425. (b) Haskins, C. M.; Knight, D. W.
Tetrahedron Lett. 2004, 45, 599.
(22) Carato, P.; Yous, S.; Sellier, D.; Poupaert, J. H.; Lebegue, N.;
(4) Griner, E. M.; Kazanietz, M. G. Nat. Rev. Cancer 2007, 7, 281.
(5) (a) Chen, S.; Borowiak, M.; Fox, J. L.; Maehr, R.; Osafune, K.;
Davidow, L.; Lam, K.; Peng, L. F.; Schreiber, S. L.; Rubin, L. L.;
Melton, D. Nat. Chem. Biol. 2009, 5, 258. (b) Thatava, T.; Nelson, T.
J.; Edukulla, R.; Sakuma, T.; Ohmine, S.; Tonne, J. M.; Yamada, S.;
Kudva, Y.; Terzic, A.; Ikeda, Y. Gene Ther. 2011, 18, 283.
(6) (a) Basu, A. Mol. Pharmacol. 1998, 53, 105. (b) Troll, W.;
Wiesner, R. Annu. Rev. Pharmacol. Toxicol. 1985, 25, 509.
(7) (a) Vaughan, R. A.; Huff, R. A.; Uhl, G. R.; Kuhar, M. J. J. Biol.
Chem. 1997, 272, 15541. (b) Nelson, T. J.; Alkon, D. L. Trends
Biochem. Sci. 2009, 34, 136.
Berthelot, P. Tetrahedron 2004, 60, 10321.
(23) Ankner, T.; Hilmersson, G. Org. Lett. 2009, 11, 503.
(24) Irie, K.; Hirota, M.; Hagiwara, N.; Koshimizu, K.; Hayashi, H.;
Murao, S.; Tokuda, H.; Ito, Y. Agric. Biol. Chem. 1984, 48, 1269.
(8) 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.
(9) Fujiki, H.; Sugimura, T.; Moore, R. E. Environ. Health Perspect.
1983, 50, 85.
(10) Loy, B. A.; Lesser, A. B.; Staveness, D.; Billingsley, K. L.;
Cegelski, L.; Wender, P. A. J. Am. Chem. Soc. 2015, 137, 3678.
(11) (a) Endo, Y.; Shudo, K.; Itai, A.; Hasegawa, M.; Sakai, S.-I.
Tetrahedron 1986, 42, 5905. (b) de Laszlo, S. E.; Ley, S. V.; Porter, R.
A. J. Chem. Soc., Chem. Commun. 1986, 344. (c) Nakatsuka, S.-I.;
D
DOI: 10.1021/acs.orglett.6b00614
Org. Lett. XXXX, XXX, XXX−XXX