Synthesis of the reported structures for kealiinines B and C

Article abstract of DOI:10.1021/ol3019242

Syntheses of the reported structures of kealiinines B and C have been executed. An intermolecular electrophile-induced cyclization of a pendant arene on an ene-guanidine affords the tetracyclic, oxidized naphthimidazole cores.

Full text of DOI:10.1021/ol3019242

ORGANIC
LETTERS
2012
Vol. 14, No. 18
4734–4737
Synthesis of the Reported Structures for
Kealiinines B and C
Joseph B. Gibbons,† Keith M. Gligorich,‡ Bryan E. Welm,‡ and Ryan E. Looper*,†
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City,
Utah 84112-0850, United States, and Department of Surgery, Huntsman Cancer
Institute, 2000 Circle of Hope, Salt Lake City, Utah 84112, United States
r.looper@utah.edu
Received July 11, 2012
ABSTRACT
Syntheses of the reported structures of kealiinines B and C have been executed. An intermolecular electrophile-induced cyclization of a pendant
arene on an eneꢀguanidine affords the tetracyclic, oxidized naphthimidazole cores.
Marine sponges of the class Calcarea provide a large number
of small molecule alkaloids containing a 2-aminoimidazole
core with interesting biological activities (Figure 1).1,2 For
example, naamine D has been shown to be a competetive
inhibitor of iNOS.3 Naamidine A has shown antimitogenic
effects by inhibiting EGF-stimulated DNA synthesis in the
growth of squamous tumor cells as well as cell cycle arrest
in the G1 phase.4,5 As a result, interest has emerged to in-
corporate this heterocyclic scaffold as a pharmacophore in
biomedical research.
Within this class of natural products are several oxidized
variants, including 14-hydroxynaamidines A and G6 which
are hydroxylated at C14 and naamidine F7 which is isolated
as the A-ring quinone. 2-Deoxy-2-aminokealiiquinone8 is
oxidized both in the A-ring and at C14.
† University of Utah.
‡ Huntsman Cancer Institute.
(1) Sullivan, J. D.; Giles, R. L.; Looper, R. E. Curr. Bioact. Compd.
Figure 1. Representative Calcarea alkaloids.
2009, 5, 39.
(2) Koswatta, P. B.; Lovely, C. J. Nat. Prod. Rep. 2011, 28, 511.
(3) Dunbar, D. C.; Rimoldi, J. M.; Clark, A. M.; Kelly, M.; Hamann,
M. T. Tetrahedron 2000, 56, 8795.
(4) Copp, B. R.; Fairchild, C. R.; Cornell, L.; Casazza, A. M.;
Robinson, S.; Ireland, C. M. J. Med. Chem. 1998, 41, 3909.
(5) James, R. D.; Jones, D. A.; Aalbersberg, W.; Ireland, C. M. Mol.
Can. Ther. 2003, 2, 747.
(6) Mancini, I.; Guella, G.; Debitus, C.; Pietra, F. Helv. Chim. Acta
1995, 78, 1178.
In 2004, a chemical investigation of the marine sponge
Leucetta chagosensis by Proksch and co-workers afforded
the new imidazole alkaloids kealiinines AꢀC.9 Related to
2-deoxy-2-aminokealiiquinone, these natural products are
oxidizedatC14 by virtue of a new CꢀC bond butremain at
the hydroquinone oxidation state in the A-ring. Although
their biosynthesis has not been studied, it is reasonable to
(7) Carroll, A. R.; Bowden, B. F.; Coll, J. C. Aust. J. Chem. 1993, 46,
1229.
(8) Fu, X.; Schmitz, F. J.; Tanner, R. S.; Kelly-Borges, M. J. Nat.
Prod. 1998, 61, 384.
(9) Hassan, W.; Edrada, R.; Ebel, R.; Wray, V.; Berg, A.; van Soest,
R.; Wiryowidagdo, S.; Proksch, P. J. Nat. Prod. 2004, 67, 817.
r
10.1021/ol3019242
Published on Web 09/11/2012
2012 American Chemical Society

Scheme 1. Plausible Biomimetic Strategy
Scheme 2. Substrate Preparation
N-methylallylamine, and the aryl acetaldehydes 1 or 2
(Scheme 2).15 Although this reaction required elevated
heating for extended reaction times due to the formation
of stable enamine intermediates, 3 and 4 could be obtained in
good yields on multigram scale. Deallylation of these inter-
mediates with Pd(0) gave the secondary propargylamines 5
and 6.16 Guanylation of these intermediates with S-Me-
N,N0-di-Boc-pseudothiourea and Hg(II) provided the pro-
pargylguanidines 7 and 8 in good yield.17
suggest that the kealiinines are intermediates between
kealiiquinones and naamine derivatives by ring closure
and aromatization.6,10 For example, naamines may be
hydroxylated at C14 (Scheme 1, A). These intermediates
may be subject to ionization to promote a FriedelꢀCrafts
reaction (pathway 1). Oxidation would then provide the
kealiinine skeleton. Alternatively, the hydroxylated inter-
mediate may be subject to elimination (pathway 2) to give
an intermediate which can undergo 6-π electrocyclization
and oxidation to give the kealiinines. This biosynthetic
strategy has also been explored in the laboratory by both
Ohta and Lovely to access these naphthimidazole cores.11
Our strategy for synthesizing kealiinines B and C was
also inspired by these hypothetical reactive intermediates
that can be generated from the oxidized naamine
(Scheme 1, B). If a cyclic eneꢀguanidine were reactive
toward an electrophile, the intermediate formed would be
reactive with a pendant nucleophile leading to Friedelꢀ
Crafts type alkylation or elimination/6-π electrocycliza-
tion. Access to these eneꢀguanidines was envisaged to be
straightforward with the development of 5-exo-dig selec-
tive propargylguanidine hydroaminations recently re-
portedby bothour group and Van derEycken’sgroup.12,13
Synthesis of the propargylguanidine precursors was
initiated with a Cu(I)-catalyzed three-component iminiumꢀ
acetylide addition (A3-coupling)14 of 4-ethynylanisole,
We initiated our cyclization studies with the trimethoxy-
substituted arene for kealiinine C as it is symmetric
(Scheme 2). To our surprise, cyclization of the propargyl-
guanidine 7 gave 9 as a single regioisomer in good yield.
Previous studies had shown that electron-rich alkynes
(e.g., pMeOPh-) typically gave poor 5-exo/6-endo selectivity.
Angle compression provided by substitution at the pro-
pargylic position presumably overrides this electronic
selectivity through a ThorpeꢀIngold effect. We next an-
ticipated that activation of the eneꢀguanidine with NBS
should generate the bromonium ion A. A key to this
synthetic plan was that this bromonium ion can be opened
via pathway a and not cyclization through pathway b to
give intermediate B. While at first one might expect the
formal products of a 5-exo cyclization (pathway b) to
outcompete a 6-endo cyclization, there are several examples
where the related electrophile-induced cyclizations are
successful.18 Further, donation from the p-methoxy substi-
tuent might make this a formal 6-exo cyclization.19 In our
experience, the eneꢀguanidines are not very nucleophilic,
suggesting that donation of the neighboring nitrogen lone
pair is hampered by steric compression. This gave us hope
that pathway a might be competitive.
(10) Akee, R.; Carroll, T.; Yoshida, W.; Scheuer, P.; Stout, T.;
Clardy, J. J. Org. Chem. 1990, 55, 1944.
(11) (a) Kawasaki, I.; Taguchi, N.; Yamashita, M.; Ohta, S. Chem.
Pharm. Bull. 1997, 45, 1393. (b) Koswatta, P. B.; Lovely, C. J. Chem.
Commun. 2010, 46, 2148. (c) Lima, H. M.; Sivappa, R.; Yousufuddin,
M.; Lovely, C. J. Org. Lett. 2012, 14, 2274.
(12) Gainer, M. J.; Newbold, N. R.; Takahashi, Y.; Looper, R. E.
Angew. Chem., Int. Ed. 2011, 50, 684.
(13) Ermolat’ev, D. S.; Bariwal, J. B.; Steenackers, H. P. L.; De
Keersmaecher, S. C. J.; Van der Eycken, E. V. Angew. Chem., Int. Ed.
2010, 49, 9465.
(14) For leading references, see: (a) Wei, C.; Li, Z.; Li, C.-J. Synlett
2004, 1472. (b) Zani, L.; Bolm, C. Chem. Commun. 2006, 4263. (c) Li,
C.-J. Acc. Chem. Res. 2010, 43, 581. (d) Koradin, C.; Polborn, K.;
Knochel, P. Angew. Chem., Int. Ed. 2002, 41, 2535. (e) Gommermann,
N.; Koradin, C.; Polborn, K.; Knochel, P. Angew. Chem., Int. Ed. 2003,
42, 5763.
(15) Both aryl acetaldehydes are commercially available or obtain-
able by the LiAlH4 reduction of the more readily available phenylacetic
acid to the alcohol, followed by IBX oxidation to the aldehyde. See:
Pelphrey, P. M.; Popov, V. M.; Joska, T. M.; Beierlein, J. M.; Bolstad,
E. S.; Fillingham, Y. A.; Wright, D. L.; Anderson, A. C. J. Med. Chem.
2007, 50, 940.
(16) Guibe, F.; Garro-Helion, F.; Merzouk, A. J. Org. Chem. 1993,
58, 6109.
(17) Horwell, D.; Guo, Z.; Cammidge, A. Synth. Commun. 2000, 40,
2933.
Org. Lett., Vol. 14, No. 18, 2012
4735

well in CD3OD when referenced to the observed proton
resonances for the natural product and not the solvent.
We next targeted kealiinine B to examine the regioselec-
tivity of the oxidative cyclization relative to the A ring. Treat-
ment of the propargyl guanidine 8 with AgOAc gave the
eneꢀguanidine 11, also as a single regioisomer (Scheme 4).
Exposure to NBS again delivered a single cyclized, fully
aromatic, and mono-Boc protected intermediate 12. This
was presumed to be the correct isomer for kealiinine B as H8
was a singlet. Presumably, attack of the bromonium ion is
preferred by rotamer A as rotamer B would encounter more
demanding steric interactions with the neighboring aromatic
ring. Deprotection of 12 gave kealiinine B in 82% yield.
Again, the 1H NMR data for synthetic kealiinine B did not
match that reported for the natural compound in DMSO-d6.
The free base did not match either, but it was a crystalline
solid which permitted us to unambiguously assign the
structure of our synthetic material via X-ray crystallo-
graphy.21 Having now realized that kealiinine C only
matched the natural material in CD3OD, we examined the
TFA salt of kealiinine B in CD3OD and when aligned to the
reported peaks there was excellent agreement for all proton
resonances except for that at H6 which differed by 0.27 ppm.
Still concerned about the spectroscopic data, the Proksch
laboratory was generous enough to compare our synthetic
sample with a small amount of natural material by HPLC.
This revealed two things: (1) that the natural sample was
actually an inseperable mixture of kealiinines B and C and
(2) the compounds did coelute, suggesting they were indeed
identical. We were also able to examine the original spec-
trum in CD3OD for the natural products (a mixture of B
and C) which revealed that H6 in kealiinine B is actually at
7.74 ppm not 7.90 ppm as originally reported.22 This is in
good agreement with our synthetic material, and gave us
confidence that the structures of the natural products are
correct and agree nicely with our synthetic materials.
The fact that the spectra were still not in agreement in
DMSO-d6 was puzzling (mostprotons differing by 0.1ꢀ0.2
ppm). We hypothesized that (1) this might be due to a
concentration effect; however; dilution experiments did
not reveal a change in the chemical shifts of the protons,
or (2) the presence of both natural products in the sample
might give rise to unique intermolecular interactions which
change the chemical shifts; however, titration of kealiinine
C into a solution of B also showed no obvious change in
chemical shifts. While we are confident, from the LC and
1H NMR data in CD3OD, that these are indeed the
structures corresponding to the natural products, we must
concede the possibility that alternate structures might exist
that fit the DMSO-d6 data with better agreement.
Scheme 3. Synthesis of Kealiinine C
To our delight, treatment of 9 with NBS in acetonitrile
provided the cyclized, fully aromatic, and mono-Boc-pro-
tected intermediate 10 in 56% isolated yield. The only other
compound isolated from this reaction was brominated at C8
(ca. 5%). While it does appear that the p-methoxyphenyl
substituent on the eneꢀguanidine directs cyclization to give
the 6-membered ring, we cannot rule out a 5-exo pathway
followed by a pinacol rearrangement or that an elimination
6-π elctrocyclization is operative. Either way, any potential
intermediates must funnel to intermediate C, which is
capable of losing a Boc group and undergoing a two-
electron oxidation to give 10. Deprotection of this inter-
mediate gave kealiinine C in good yield (Scheme 3), and the
structure was ultimately confirmed by X-ray crystallo-
graphy.20 In the isolation paper only H NMR data in
1
CD3OD and DMSO-d6 were reported for both kealiinines
B and C. Comparison of our spectra to that reported in
DMSO-d6 shared very little agreement. However, the TFA
salt of synthetic kealiinine C did match the natural product
In the original isolation of kealiinines AꢀC only biologi-
cal activity for kealiinine A was reported. This was reported
in a brine shrimp lethality assay with an IC50 = 20 μg/mL.9
The activity of kealiinines B and C was not reported, likely
due to the small amounts of isolated material and the fact
(18) (a) Padwa, A.; Lee, H.; Rashatasakhon, P.; Rose, M. J. Org.
Chem. 2004, 69, 8209. (b) Nicolaou, K. C.; Prasad, C. V. C.; Somers,
P. K.; Hwang, C. K. J. Am. Chem. Soc. 1989, 111, 5330–5334. (c)
Bedford, S. B.; Bell, K. E.; Bennett, F.; Hayes, C. J.; Knight, D. W.;
Shaw, D. E. J. Chem. Soc., Perkin Trans. 1 1999, 15, 2143.
(19) A similar donation strategy has been used to favor 5-endo-dig
over 5-exo-dig cyclizations. David, W. K.; Redfern, A. L.; Gilmore, J.
Tetrahedron Lett. 1998, 39, 8909.
(21) This structure has been deposited at the Cambridge Crysallo-
graphic Database (CCDC No. 887953).
(20) This structure has been deposited at the Cambridge Crysallo-
graphic Database (CCDC No. 887954).
(22) See “Spectral comparisons” in the Supporting Information for
more details.
4736
Org. Lett., Vol. 14, No. 18, 2012

Scheme 4. Synthesis of Kealiinine B
Figure 3. Interrogating caspase 3 activity.
however, did not appreciably inhibit proliferation at con-
centrations below 100 μM.
The antiproliferative effects of members of this family
have most extensively been studied for naamidine A, which
is known to cause apoptosis through a caspase-3 dependent
cascade after activation of the MAPK pathway.23 We
examined the similarity of kealiinines B and C’s activity in
this regard, and it appears that the antiproliferative effects
arise from a caspase independent pathway (Figure 3).
Treatment of MCF-10A or T47D cells, which are sensitive
to caspase activation,24 with 30 μM concentrations of
kealiinine B or C for 48 h showed no evidence of caspase-3
cleavage or PARP cleavage, known hallmarks of caspase
activation (lanes 2 and 3). Due to limited amounts of natural
naamidine A, staurosporine was used as a positive control25
and shows that indeed both cell lines are capable of under-
going caspase-3-dependent apoptosis (Lane 4).
In conclusion, we have demonstrated that eneꢀ
guanidines, derived from the hydroamination of propar-
gylguanidines, are useful intermediates for the synthesis of
the reported structures for kealiinines B and C via an
electrophilic activation/cyclization sequence. The ability
to prepare these compounds individually has allowed us to
provide independent and comprehensive characterization
for these purported natural products, as well as an in-
dependent evaluation of their antiproliferative activity.
Current efforts are directed to understand the mechanism
by which kealiinine B inhibits cell proliferation.
Acknowledgment. This research was supported by
grants from the National Institutes of Health ((R01-GM
090082 and R01-CA140296). K.M.G. thanks the DOD
Breast Cancer Research Program for a postdoctoral fel-
lowship (W81XWH-09-1-0431). We are very grateful to
Prof. Carl Lovely (UT-Arlington) for helpful discussions
and for sharing spectral and X-ray data to confirm the
identity of these products. We also thank Prof. Peter
Figure 2. Antiproliferative activities.
that they were isolated as an inseparable mixture. We
evaluated our synthetic kealiinines B and C against several
breast cancer cell lines, including a normal breast cell line
(MCF-10A), a luminal ERþ cell line (MCF-7), an aggres-
sive luminal ERþ line (T47D), and a “basal-like” triple
negative cell line (MDA-MB-231). Kealiinine B showed
antiproliferative activity against all of these cell lines with
IC50’s ranging from ∼10 to 13 μM (Figure 2). Kealiinine C,
€
Proksch (UNI-Dusseldorf) for assistance with compari-
sons of synthetic and natural samples.
Supporting Information Available. Experimental and
characterization details, including 1H and 13C NMR
spectra for all compounds. Biological protocols as well
as Western Blots. This material is available free of charge
via the Internet at http://pubs.acs.org
(23) LaBarbera, D. V.; Modzelewska, K.; Glazar, A. I.; Gray, P. D.;
Kaur, M.; Liu, T.; Grossman, D.; Harper, M. K.; Kuwada, S. K.;
Moghal, N.; Ireland, C. M. Anticancer Drugs 2009, 20, 425.
(24) Budihardjo, I.; Oliver, H.; Lutter, M.; Luo, X.; Wang, X. Annu.
Rev. Cell Dev. Biol. 1999, 15, 269.
(25) Chae, H. J.; Kang, J. S.; Byun, J. O.; Han, K. S.; Kim, D. U.; Oh,
S. M.; Kim, H. M.; Chae, S. W.; Kim, H. R. Pharmacol. Res. 2000, 42, 373.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 18, 2012
4737