Total synthesis of ningalin D

Article abstract of DOI:10.1021/ja0526416

A concise (nine-step) and effective (19% overall yield) total synthesis of ningalin D (1a) is disclosed and is based on a key 1,2,4,5-tetrazine → 1,2-diazine → pyrrole Diels-Alder strategy to assemble a fully substituted pyrrole core central to its structure. Additional highlights of the synthesis include a double Dieckmann condensation to introduce the C and D aryl rings enlisting substituents judiciously placed on the dienophile and intrinsic to the widely used tetrazine 2, a highly effective Suzuki coupling of the resulting C and D phenol triflates for introduction of the sterically demanding F and G aryl rings, and an unusually effective formal oxidative decarboxylation reaction cascade initiated by a Curtius rearrangement to directly provide the biphenylene quinone methide found imbedded in the structure of ningalin D. The cytotoxic and multidrug resistance (MDR) reversal activity of ningalin D, its derivatives, and the key synthetic intermediates are detailed.

Full text of DOI:10.1021/ja0526416

Published on Web 07/07/2005
Total Synthesis of Ningalin D
Akiyuki Hamasaki, Jeffrey M. Zimpleman, Inkyu Hwang, and Dale L. Boger*
Contribution from the Department of Chemistry and the Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received April 22, 2005; E-mail: boger@scripps.edu
Abstract: A concise (nine-step) and effective (19% overall yield) total synthesis of ningalin D (1a) is disclosed
and is based on a key 1,2,4,5-tetrazine f 1,2-diazine f pyrrole Diels-Alder strategy to assemble a fully
substituted pyrrole core central to its structure. Additional highlights of the synthesis include a double
Dieckmann condensation to introduce the C and D aryl rings enlisting substituents judiciously placed on
the dienophile and intrinsic to the widely used tetrazine 2, a highly effective Suzuki coupling of the resulting
C and D phenol triflates for introduction of the sterically demanding F and G aryl rings, and an unusually
effective formal oxidative decarboxylation reaction cascade initiated by a Curtius rearrangement to directly
provide the biphenylene quinone methide found imbedded in the structure of ningalin D. The cytotoxic and
multidrug resistance (MDR) reversal activity of ningalin D, its derivatives, and the key synthetic intermediates
are detailed.
First isolated in 1997 by Fenical and Kang,1 the ningalins
constitute a family of structurally interesting and biologically
active marine natural products. By far, the most complex of
these is ningalin D (1a) incorporating a biphenylene quinone
methide superimposed on a now oxidized pentasubstituted
pyrrole core that characterizes this class of natural products
(Figure 1). To our knowledge, the only closely related natural
product disclosed to date is purpurone (1b),2 an inhibitor of
ATP-citrate lyase, whose name reflects the purple color of the
natural products and the sponges from which both 1a and 1b
were isolated (Didemnum sp. and Iotrochota sp., respectively).
In the course of developing total syntheses of the simpler
members of the ningalin family,3 we disclosed several deriva-
tives that possess potent P-gp inhibitory activity,3 effective
multidrug resistance (MDR) reversal properties in cellular
functional assays,3-5 and efficacious (potentiation/resensitiza-
tion) in vivo antitumor activity against sensitive and resistant
tumors upon coadministration with antitumor therapeutics
(vinblastine, taxol) in xenograph animal models.6 Although such
derivatives lack intrinsic cytotoxic activity themselves, they
resensitize MDR tumors and hypersensitize sensitive tumors to
(1) Kang, H.; Fenical, W. J. Org. Chem. 1997, 62, 3254.
(2) Chan, G. W.; Francis, T.; Thureen, D. R.; Offen, P. H.; Pierce, N. J.;
Westley, J. W.; Johnson, R. K.; Faulkner, D. J. J. Org. Chem. 1993, 58,
2544.
(3) (a) Boger, D. L.; Boyce, C. W.; Labroli, M. A.; Sehon, C. A.; Jin, Q. J.
Am. Chem. Soc. 1999, 121, 54. (b) Boger, D. L.; Soenen, D. R.; Boyce, C.
W.; Hedrick, M. P.; Jin, Q. J. Org. Chem. 2000, 65, 2479. For additional
disclosures of ningalin total syntheses, see the following. Ningalin B: (c)
Iwao, M.; Takeuchi, T.; Fujikawa, N.; Fukuda, T.; Ishibashi, F. Tetrahedron
Lett. 2003, 44, 4443. (d) Bullington, J. L.; Wolff, R.; Jackson, P. F. J.
Org. Chem. 2002, 67, 9439. Ningalin C: (e) Namsaaid, A.; Ruchirawat,
S. Org. Lett. 2002, 4, 2633 and ref 7.
(4) Soenen, D. R.; Hwang, I.; Hedrick, M. P.; Boger, D. L. Bioorg. Med. Chem.
Lett. 2003, 13, 1777.
(5) Tao, H.; Hwang, I.; Boger, D. L. Bioorg. Med. Chem. Lett. 2004, 14, 5979.
(6) Chou, T.-C.; Guan, Y.; Soenen, D. R.; Danishefsky, S. J.; Boger, D. L.
Cancer Chemother. Pharmacol. 2005, in press.
Figure 1. Ningalin D and retrosynthetic analysis.
conventional therapeutics through inhibition of the over-
expressed or constitutive drug effux pump P-gp.3-6
As a consequence of these biological observations with the
simpler ningalin derivatives, and because of the intrinsically
interesting structure of ningalin D, we have pursued and herein
detail the first total synthesis of ningalin D in studies that
complement the only other reported efforts in the area, a
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J. AM. CHEM. SOC. 2005, 127, 10767-10770
10767

A R T I C L E S
Hamasaki et al.
Scheme 1. Total Synthesis of Ningalin D
Scheme 2. Reductive Ring Contraction
biomimetic total synthesis of purpurone disclosed by Steglich.7
Key elements of the concise and nonobvious approach include
an inverse electron demand heterocyclic azadiene Diels-Alder
reaction (1,2,4,5-tetrazine f 1,2-diazine) followed by a reduc-
tive ring contraction of the resultant 1,2-diazine, affording the
fully substituted pyrrole core central to the structure of 1 (Figure
1). A double Dieckmann cyclization enlisting substituents
judiciously placed on the dienophile (-CH2CO2Me) and
intrinsic to the widely used tetrazine 2 (-CO2Me) was used to
close the C and D aryl rings; the sterically demanding F and G
aryl rings were introduced enlisting a highly effective Suzuki
coupling onto the corresponding C and D ring phenol triflates,
and an unusually effective formal oxidative decarboxylation
reaction cascade initiated by a Curtius rearrangement was
discovered to directly provide the biphenylene quinone methide
of ningalin D.
Preparation of the symmetrical alkyne 3, a suitably substituted
electron-rich dienophile for Diels-Alder cycloaddition with 2,
was accomplished in two steps from commercially available
2-bromo-4,5-dimethoxyacetonitrile.8a Conversion of the nitrile
to the known methyl ester9 (cat. H2SO4, MeOH, 90%) followed
by a double Stille coupling with 1,2-bis(tributylstannyl)acetylene
(0.05 equiv of (Ph3P)4Pd, toluene, 110 °C, 6 h, 71-80%)
provided ready access to 3. The key [4 + 2] cycloaddition of 3
with dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (2)10 proceeded
smoothly in refluxing toluene (110 °C) to provide the sym-
metrical 1,2-diazine 4 in superb conversions (87%) and was
followed by a subsequent reductive ring contraction reaction
effected by treatment with Zn/TFA (30 equiv of Zn, TFA,
25 °C, 7 h, 64%) to provide the pyrrole 5 (Scheme 1). Although
such reductive ring contraction reactions are typically conducted
with Zn/HOAc,11 we have disclosed that the overall reaction
cascade is much faster (4-7 vs 24 h for 4, 25 °C) with Zn/
TFA12 and often much cleaner (Scheme 2).
N-Alkylation of 5 with 68b (3 equiv, 3 equiv of CsCO3,13
DMF, 60 °C, 2 h, 92%) afforded 7 and set the stage for
introduction of the aryl C and D rings via a double Dieckmann
condensation, which was effected by treatment of 7 with NaH
(6 equiv, DMF, 25 °C, 15 h, 81%), cleanly providing 8. Notably,
the second of the two Dieckmann condensations proceeds more
slowly than the first, requiring adoption of a sterically encum-
bered coplanar arrangement of the A and B aryl rings.
Conversion of the bisphenol 8 to the corresponding bistriflate
9 (5 equiv of Tf2O, py-CH2Cl2 (1:1), 0-25 °C, 2 h, 92%)
preceded an unusually effective double Suzuki coupling with
10 (0.1 equiv of (Ph3P)4Pd, 2.2 equiv of LiCl, 1 M aq. K2-
CO3-DME, 80 °C, 15 h, 88%) to provide 11. Notably, the
(7) Peschko, C.; Steglich, W. Tetrahedron Lett. 2000, 41, 9477.
(8) (a) Trans-World Chemicals, Rockville, MD. (b) Sigma-Aldrich library of
rare chemicals (commercial) or prepared from the corresponding alcohol
(Aldrich): I2, Ph3P, imidazole, CH2Cl2, 96%.
(9) Ambrose, R.; von Angerer, S.; Weigrebe, W. Arch. Pharm. 1988, 321,
481.
(10) (a) Boger, D. L.; Panek, J. S.; Patel, M. Org. Synth. 1992, 70, 79. (b) Boger,
D. L.; Panek, J. S.; Coleman, R. S.; Sauer, J.; Huber, F. X. J. Org. Chem.
1985, 50, 5377.
(11) (a) Boger, D. L.; Coleman, R. S.; Panek, J. S.; Yohannes, D. J. Org. Chem.
1984, 49, 4405. Reviews: (b) Boger, D. L. Tetrahedron 1983, 39, 2869.
(c) Boger, D. L. Chem. ReV. 1986, 86, 781.
(12) Boger, D. L.; Patel, M. J. Org. Chem. 1988, 53, 1405.
(13) Bullington, J. L.; Wolff, R. R.; Jackson, P. F. J. Org. Chem. 2002, 67,
9439.
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Total Synthesis of Ningalin D
A R T I C L E S
inclusion of LiCl (2.2 equiv) in the reaction mixture14 was key
to the superb conversions, which in its absence proved much
lower (0-35%).
Scheme 3. Oxidative Decarboxylation Reaction
The final conversion of 11 to the penultimate ningalin D
precursor required conversion of the aryl esters to phenols and
was anticipated to arise from Baeyer-Villiger oxidation of the
corresponding ketones or a benzylic hydroperoxide rearrange-
ment of the corresponding secondary or tertiary alcohols.
Although not investigated in detail, each of these proved
problematic to implement due to the sterically hindered nature
of the site. However, hydrolysis of diester 11 to the dicarboxylic
acid 12 could be effected cleanly with anhydrous hydroxide15
(26 equiv of t-BuOK, 8 equiv of H2O, DMSO, 80 °C, 24 h,
84%), whereas typical saponification conditions (2 N aq. KOH,
reflux, 48 h) failed to touch 11. Characteristic of the congested
nature of the site, attempts to prepare the acid chloride of 12
(SOCl2-DMF, CH2Cl2, 25 °C, 4 h) resulted in clean conversion
to 15 (70%) derived from a facile double Friedel-Crafts
acylation of the proximal and activated F and G aryl rings (eq
1).
Table 1. Cytotoxic Activity
IC50 (µM)
compound
L1210
HCT116
1a
7
8
11
12
13
7
70
>100
>100
>100
>100
1
>100
>100
>100
>100
1
14
7
6
15
16
>100
>100
>100
>100
0.007
0.07
vinblastine
doxorubicin
0.007
0.06
Table 2. MDR Activity
Although several direct conversions of 12 to permethyl
ningalin D were examined, including a carboxy inversion
reaction and a Pb(OAc)4-catalyzed oxidative decarboxylation
reaction, the surprisingly most straightforward approach proved
to be initiated by Curtius rearrangement of the dicarboxylic acid
12, which was anticipated to provide the corresponding diamine.
Under the conditions of a modified Curtius rearrangement and
in situ hydrolysis of the resulting isocyanate (5 equiv of DPPA,16
5 equiv of i-Pr2NEt, CH2Cl2, 25 °C, 15-20 h; then H2O-THF,
air atmosphere, reflux, 90 h; 70% 13), we observed and
subsequently adopted conditions that optimized the conversion
directly to permethyl ningalin D (13). Thus, a remarkably facile
in situ oxidation of the expected diamine to the biphenylene
quinodiimide and subsequent imine hydrolysis were observed
to occur under the reaction conditions, providing 13 directly in
superb overall conversions (70%) for the seven transformations
from the dicarboxylic acid 12 (Scheme 3). Exhaustive depro-
tection of 13 (15 equiv of BBr3, CH2Cl2, -78 to 25 °C, 16 h,
96%) requiring the removal of 10 methyl ethers cleanly provided
ningalin D (1a) identical in all respects with authentic material.1
Acetylation of 1a (Ac2O, py, 25 °C, 3 h, 98%) provided ningalin
D decaacetate (14), which also displayed properties identical
in all respects with those reported for authentic material.1
With 1a, 13, and the precursor intermediates in hand, they
were examined alongside permethyl ningalin B (16)3b in assays
HCT116/VM46 IC50 (nM)
compound
(1
µM)
vinblastine (%)a
doxorubicin (%)a
none
1a
7
100
900
100 (0%)
100 (0%)
10 (70%)
8.5 (82%)
100 (0%)
8.5 (82%)
100 (0%)
7 (100%)
7 (100%)
900 (0%)
750 (8%)
330 (21%)
90 (80%)
1000 (0%)
100 (70%)
900 (0%)
90 (80%)
90 (80%)
8
11
12
13
14
15
16
a Reversion %, parental HCT116 IC50 for vinblastine ) 7 nM and
doxorubicin ) 70 nM.
to establish their intrinsic cytotoxic activity (Table 1, an
undesired property) and their ability to reverse multidrug
resistance,17 resensitizing a resistant cell line to front line
antitumor drugs (Table 2). Ningalin D (1a), permethyl ningalin
D (13), and ningalin D decaacetate (14) exhibited modest
cytotoxic activity against a murine leukemia cell line (L1210,
IC50 ) 1-7 µM) and a parental human colon cancer cell line
(HCT116, IC50 ) 1-70 µM). Each of the modestly active
compounds contains the embedded biphenylene quinone methide
characteristic of ningalin D, suggesting that their use as MDR
reversal agents, unlike 16,3b would be complicated by this
secondary cytotoxic activity.
(14) Huth, A.; Beetz, I.; Schumann, I. Tetrahedron 1989, 45, 6679.
(15) (a) Chang, F. C.; Wood, N. F. Tetrahedron Lett. 1964, 2969. (b) Gassman,
P. G.; Schenk, W. N. J. Org. Chem. 1977, 42, 918.
(17) Reviews: (a) Robert, J.; Jarry, C. J. Med. Chem. 2003, 46, 4805. (b) Wiese,
M.; Pajeva, I. K. Curr. Med. Chem. 2001, 8, 685.
(16) Ninomiya, K.; Shioiri, T.; Yamada, S. Tetrahedron 1974, 30, 2151.
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A R T I C L E S
Hamasaki et al.
accompanying cytotoxic activity, 11 and especially 15 were
found to be active, 8 exhibited an intermediate level of activity,
and both 7 and 12 were inactive. The relative inactivity of the
polar derivatives 8, 12, and 7 was not surprising, and the MDR
reversal activity of 11 expectedly approached that of 16. More
unexpectedly, the inadvertently prepared Friedel-Crafts acyl-
ation byproduct 15 proved to be a potent MDR reversal agent,
matching the efficacy of 16 at 1 µM.
Notably, each of the active compounds incorporates three
hydrophobic domains that characterize the P-gp binding phar-
macophore models,18 but unique in ningalin series of MDR
reversal agents is the lack of a basic amine central to the
structure. As such, they not only constitute efficacious com-
pounds exhibiting unusually potent MDR reversal activity for
a lead series, but they also depart from structural features
considered central to common pharmacophore models of P-gp
binding.
Figure 2. Permethyl ningalin B.
Pertinent to our interests, the compounds were examined
against a resistant HCT116 cell line that embodies the MDR
phenotype through overexpression of P-glycoprotein (P-gp). This
cell line, HCT116/VM46, which is resistant to both vinblastine
(IC50 ) 100 vs 7 nM for parental HCT116) and doxorubicin
(IC50 ) 900 vs 70 nM for parental HCT116), was treated with
each of the ningalin derivatives at 1 µM and the IC50 established
for coadministered vinblastine or doxorubicin (Table 2). Pro-
totypical MDR reversal agents are usually examined at higher
concentrations (e.g., 7.5-10 µM verapamil), and few lead series
exhibit effective properties when examined at 1 µM as detailed
herein.17 Under these stringent conditions, ningalin D (1a) as
well as ningalin D decaacetate (14) was inactive, whereas
permethyl ningalin D (13) effectively resensitized the resistant
HCT116/VM46 cell line to vinblastine (IC50 ) 8.5 nM, 80%
reversion) and doxorubicin (IC50 100 nM, 70% reversion). This
behavior of the hydrophobic permethyl ether derivative of the
natural product versus the natural product itself (13 vs 1a) is
analogous to distinctions we observed earlier with 16 (permethyl
ningalin B, Figure 2) versus the inactive natural product ningalin
B (free phenols).3b More significantly, and of the derivatives
that lack the biphenylene quinone methide and its undesired
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA42056) and The
Skaggs Institute for Chemical Biology. J.M.Z. was a Skaggs
predoctoral fellow.
Supporting Information Available: Full experimental details
are provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA0526416
(18) (a) Pearce, H. L.; Safa, A. R.; Bach, N. J.; Winter, M. A.; Cirtain, M. C.;
Beck, W. T. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5128. (b) Pearce, H.
L.; Winter, M. A.; Beck, W. T. AdV. Enz. Reg. 1990, 30, 357. (c) Garrigues,
A.; Loisau, N.; Delaforge, M.; Ferte, J.; Garrigos, M.; Andre, F.; Orlowski,
S. Mol. Pharmacol. 2002, 61, 1288. (d) Ekins, S.; Kim, R. B.; Leake, B.
F.; Dantzig, A. H.; Schuetz, E. G.; Lan, L.-B.; Yasuda, K.; Shepard, R. L.;
Winter, M. A.; Schuetz, J. D.; Wikel, J. H.; Wrighton, S. A. Mol.
Pharmacol. 2002, 61, 964 and 974. (e) Pajeva, I. K.; Wiese, M. J. Med.
Chem. 2002, 45, 5671.
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