VOLUME 122, NUMBER 21

MAY 31, 2000

© Copyright 2000 by the
American Chemical Society

Total Synthesis of (()-Cylindrospermopsin

Chaoyu Xie,† Maria T. C. Runnegar,‡ and Barry B. Snider*,†
Contribution from the Department of Chemistry, Brandeis UniVersity,
Waltham, Massachusetts 02454-9110, and Research Center for LiVer Diseases, Department of Medicine,
UniVersity of Southern California Medical Center, Los Angeles, California 90033
ReceiVed February 22, 2000

Abstract: The first total synthesis of the novel hepatotoxin (()-cylindrospermopson (1) has been accomplished
in 20 steps from 4-methoxy-3-methylpyridine (12) in 3.5% overall yield. The substituted piperidine A ring 19
was generated stereospecifically by a four-step sequence using the addition of trimethylsilylethynylmagnesium
bromide to 12 to give 16 and stereospecific addition of vinylcuprate to 16 to form 17. The reaction of diamine
26 with cyanogen bromide produced the cyclic guanidine C ring of 27. The key step in the synthesis was
bromination of ketone 31, followed by hydrogenation to liberate the free guanidine, which underwent an
intramolecular SN2 reaction to form the tetrahydropyrimidine ring B of 32. Further hydrogenation reduced the
ketone to yield 42% of 32 containing the fully functionalized tricyclic system and protected hydroxymethyluracil
side chain of cylindrospermopsin. Hydrolysis of the pyrimidine in concentrated hydrochloric acid and selective
monosulfation completed the synthesis of cylindrospermopsin.

Introduction as shown. Feeding studies indicated that cylindrospermopsin is

an acetogenin with guanidinoacetic acid serving as the starter
Cylindrospermopsin (1) was isolated from the cyanobacterium
unit of the polyketide chain.3
Cylindrospermopsis raciborskii by Moore and shown to be the
causative agent of a 1979 outbreak of hepatoenteritis in
Australia.1,2 The structure of cylindrospermopsin was determined
spectroscopically. The relative stereochemistry of the ring
carbons was assigned on the basis of analysis of the coupling
constants.1,2 The stereochemistry of the side chain alcohol was
tentatively assigned on the basis of the coupling constants,
NOEs, and the unusual behavior of the uracil carbons in the
13C NMR spectrum, which suggested that the guanidinium

hydrogen is hydrogen bonded to the imine tautomer of the uracil Cylindrospermopsin has also been isolated from Umezakia
natans and Aphanizomenon oValisporum.4-8 Algal blooms that
* To whom correspondence should be addressed. Telephone: 781-736-
2550. Fax: 781-736-2516. E-mail: [email protected]. (3) Burgoyne, D. L.; Hemscheidt, T. K.; Moore, R. E.; Runnegar, M. T.
† Brandeis University. C. J. Org. Chem. 2000, 65, 152.
‡ University of Southern California Medical Center. (4) Harada, K. I.; Ohtani, I.; Iwamoto, K.; Suzuki, M.; Watanabe, M.
(1) Ohtani, I.; Moore, R. E.; Runnegar, M. T. C. J. Am. Chem. Soc. F.; Watanabe, M.; Terao, K. Toxicon 1994, 32, 73.
1992, 114, 7941. (5) Terao, K.; Ohmori, S.; Igarashi, K.; Ohtani, I.; Watanabe, M. F.;
(2) Moore, R. E.; Ohtani, I.; Moore, B. S.; De Koning, C. B.; Yoshida, Harada, K. I.; Ito, E.; Watanabe, M. Toxicon 1994, 32, 833.
W. Y.; Runnegar, M. T. C.; Carmichael, W. W. Gazz. Chim. Ital. 1993, (6) Hawkins, P. R.; Chandrasena, N. R.; Jones, G. J.; Humpage, A. R.;
123, 329. Falconer, I. R. Toxicon 1997, 35, 341.

10.1021/ja000647j CCC: $19.00 © 2000 American Chemical Society

Published on Web 05/13/2000
5018 J. Am. Chem. Soc., Vol. 122, No. 21, 2000 Xie et al.

produce cylindrospermopsin are widespread in tropical waters cylindrospermopsin (1).33 Acetylide 2 was prepared by addition
where occurrences of gastrointestinal diseases of unknown origin of trimethylsilylethynylmagnesium bromide to pyridine and
are common.4-16 The toxicological effects of cylindrospermop- benzyl chloroformate.37 Methyl 2,6-dimethoxy-4-pyrimidin-
sin and analytical techniques for its detection in drinking water ecarboxylate was prepared by the procedure of Gershon.38
have been extensively studied.17-28 Reduction of the ester with LiBH4 in THF afforded 93% of the
Cylindrospermopsin is hepatotoxic in mice and appears to primary alcohol, which was oxidized to 90% of aldehyde 339
function by depletion of the cellular antioxidant glutathione with Dess-Martin reagent. Addition of acetylide 2 to aldehyde
(GSH) in hepatocytes.17,19 The renal toxicity of cylindrosper- 3 afforded 85% of coupled alcohol 4. Hydrogenation, guanidi-
mopsin in mice has also been demonstrated.23 7-Deoxycylin- nylation, and oxidation gave ketone 5 in 50% overall yield.
drospermopsin26 and 7-epicyclindrospermopsin27 have recently Bromination of 5 with CuBr240 in EtOAc for 15 min at 40 °C
been isolated. provided unstable bromo ketone 6. Hydrogenolysis of crude 6
The novel structure of cylindrospermopsin, with a guanidine over Pd/C in MeOH liberated the free guanidine, which
embedded in a tricyclic system, six chiral centers, and polar underwent an intramolecular SN2 reaction to form the second
sulfate, uracil, and guanidine functional groups, makes its ring. Under these conditions the ketone was hydrogenated41 so
synthesis challenging. Its potent toxicity makes the synthesis that we selectively obtained 66% of the desired hydroxy
of cylindrospermopsin an important problem that has been the guanidine 7 and 15% of the other three diastereomers. The
subject of intense interest.29-36 model study was completed by hydrolysis of the pyrimidine
We recently reported a short synthesis of model 8 that ring in concentrated hydrochloric acid at reflux42 for 6 h to afford
contains the AB rings and hydroxymethyluracil side chain of 8 quantitatively.

(7) Banker, R.; Carmeli, S.; Hadas, O.; Teltsch, B.; Porat, R.; Sukenik,
A. J. Phycol. 1997, 33, 613.
(8) Shaw, G. R.; Sukenik, A.; Livne, A.; Chiswell, R. K.; Smith, M. J.;
Seawright, A. A.; Norris, R. L.; Eaglesham, G. K.; Moore, M. R. EnViron.
Toxicol. 1999, 14, 167.
(9) Presing, M.; Herodek, S.; Voros, L.; Kobor, I. Arch. Hydrobiol. 1996,
136, 553.
(10) Fabbro, L. D.; Duivenvoorden, L. J. Mar. Freshwater Res. 1996,
47, 685.
(11) Chiswell, R.; Smith, M.; Norris, R.; Eaglesham, G.; Shaw, G.;
Seawright, A.; Moore, M. Australas. J. Ecotoxicol. 1997, 3, 7.
(12) Chapman, A. D.; Schelske, C. L. J. Phycol. 1997, 33, 191.
(13) Nagy, L.; Hiripi, L.; Kovacs, A.; Voros, L. Hidrol. Kozl. 1998, 78,
298.
(14) Sukenik, A.; Rosin, C.; Porat, R.; Teltsch, B.; Banker, R.; Carmeli,
S. Isr. J. Plant Sci. 1998, 46, 109.
(15) de Souza, R. C. R.; Carvalho, M. C.; Truzzi, A. C. EnViron. Toxicol.
Water Qual. 1998, 13, 73.
(16) Harada, K. J. Health Sci. 1999, 45, 150.
(17) Runnegar, M. T.; Kong, S.-M.; Zhong, Y.-Z.; Ge, J.-L.; Lu, S. C.
Biochem. Biophys. Res. Commun. 1994, 201, 235.
(18) Sadler, R. Proc. Int. Semin. Water EnViron. 1994, 88.
(19) Runnegar, M. T.; Kong, S.-M.; Zhong, Y.-Z.; Lu, S. C. Biochem.
Pharmacol. 1995, 49, 219.
(20) Kiss, T.; Vehovszky, A.; Laszlo, H.; Voros, L. Hidrol. Kozl. 1997, We envisioned that cylindrospermopsin (1) could be prepared
77, 69. analogously by bromination of ketone 9. Ketone 9 should be
(21) Kiss, T.; Vehovszky, A.; Laszlo, H.; Kovacs, A.; Voros, L. Hidrol.
Kozl. 1998, 78, 355. (36) Weinreb, S. M.; Keen, S. P.; Heintzelman, G. R.; Fang, W.-K.
(22) Seawright, A. A.; Nolan, C. C.; Shaw, G. R.; Chiswell, R. K.; Norris, Abstracts of Papers, 218th National Meeting of the American Chemical
R. L.; Moore, M. R.; Smith, M. J. EnViron. Toxicol. 1999, 14, 135. Society, New Orleans, LA, Aug 22-26, 1999; American Chemical
(23) Falconer, I. R.; Hardy, S. J.; Humpage, A. R.; Froscio, S. M.; Tozer, Society: Washington, DC, 1999; ORGN 586.
G. J.; Hawkins, P. R. EnViron. Toxicol. 1999, 14, 143. (37) (a) Yamaguchi, R.; Nakazono, Y.; Kawanisi, M. Tetrahedron Lett.
(24) Eaglesham, G. K.; Norris, R. L.; Shaw, G. R.; Smith, M. J.; Chiswell, 1983, 24, 1801. (b) Yamaguchi, R.; Nakazono, Y.; Matsuki, T.; Hata, E.-
R. K.; Davis, B. C.; Neville, G. R.; Seawright, A. A.; Moore, M. R. EnViron. I.; Kawanisi, M. Bull. Chem. Soc. Jpn. 1987, 60, 215.
Toxicol. 1999, 14, 151. (38) Gershon, H. J. Org. Chem. 1962, 27, 3507.
(25) Chiswell, R. K.; Shaw, G. R.; Eaglesham, G.; Smith, M. J.; Norris, (39) Stogryn, E. L. J. Heterocycl. Chem. 1974, 11, 251.
R. L.; Seawright, A. A.; Moore, M. R. EnViron. Toxicol. 1999, 14, 155. (40) Matsumoto, M.; Ishide, Y.; Watanabe N. Heterocycles 1985, 23,
(26) Norris, R. L.; Eaglesham, G. K.; Pierens, G.; Shaw, G. R.; Smith, 165.
M. J.; Chiswell, R. K.; Seawright, A. A.; Moore, M. R. EnViron. Toxicol. (41) (a) Bettoni, G.; Cellucci, C.; Ferorelli, S.; Perrone, R.; Tortorella,
1999, 14, 163. V. Tetrahedron 1986, 42, 2117. (b) Tramontini, M. Synthesis 1982, 605.
(27) Banker, R.; Teltsch, B.; Sukenik, A.; Carmeli, S. J. Nat. Prod. 2000, (42) (a) Takahashi, T.; Kotsubo, H.; Koizumi, T. J. Chem. Soc., Perkin
63, 387-389. Trans. 1 1991, 1677. (b) Wada, A.; Yamamoto, J.; Hase, T.; Nagai, S.;
(28) (a) Saker, M. L.; Thomas, A. D.; Norton, J. H. EnViron. Toxicol. Kanatomo, S. Synthesis 1986, 555. (c) Uno, H.; Terakawa, T.; Suzuki, H.
1999, 14, 179. (b) Saker, M. L.; Eaglesham, G. K. Toxicon 1999, 37, 1065. Synthesis 1989, 381. (d) Khan, M. W.; Kundu, N. G. J. Chem. Research
(29) Heintzelman, G. R.; Parvez, M.; Weinreb, S. M. Synlett 1993, 551. (s) 1999, 20. (e) Griengl, H.; Wanek, E.; Schwarz, W.; Streicher, W.;
(30) (a) Snider, B. B.; Harvey, T. C. Tetrahedron Lett. 1995, 36, 4587. Rosenwirth, B.; Clercq, E. De J. Med. Chem. 1987, 30, 1199. (f) Kundu,
(b) Harvey, T. C. Ph.D. Thesis, Brandeis University, 1996; Diss. Abstr. N. G.; Choudhuri, L. N.; Das, B. J. Chem. Soc., Perkin Trans. 1 1989,
1996, 57-04, 2569. 1165. (g) Maruyama, T.; Kimura, S.; Honjo, M. Org. Prep. Proced. Int.
(31) Leroy, V. Ph.D. Thesis, The Ohio State University, 1995; Diss. 1988, 20, 485.
Abstr. 1995, 56-06B, 3203. (43) (a) Comins, D. L.; Brown, J. D. Tetrahedron Lett. 1986, 27, 4549.
(32) (a) Heintzelman, G. R.; Weinreb, S. M.; Parvez, M. J. Org. Chem. (b) Comins, D. L.; Weglarz, M. A.; O’Connor, S. Tetrahedron Lett. 1988,
1996, 61, 4594. (b) Heintzelman, G. R. Ph.D. Thesis, Pennsylvania State 29, 1751. (c) Brown, J. D.; Foley, M. A.; Comins, D. L. J. Am. Chem. Soc.
University, 1996; Diss. Abstr. 1996, 57-12B, 7530. 1988, 110, 7445. (d) Comins, D. L.; Joseph, S. P.; Goehring, R. R. J. Am.
(33) Snider, B. B.; Xie, C. Tetrahedron Lett. 1998, 39, 7021. Chem. Soc. 1994, 116, 4719. (e) Comins, D. L.; Dehghani, A.; J. Org.
(34) McAlpine, I. J. Ph.D. Thesis, University of California, Los Angeles, Chem. 1995, 60, 794. (f) Comins, D. L.; Joseph, S. P.; Hong, H.; Al-awan,
1998; Diss. Abstr. 1998, 59-09B, 4818. R. S.; Foti, C. J.; Zhang, Y.-M.; Chen, X.; LaMunyon, D. H.; Weltzien, M.
(35) Du, X. Ph.D. Thesis, University of California, Los Angeles, 1998; G. Pure Appl. Chem. 1997, 69, 477. (g) Comins, D. L.; LaMunyon, D. H.;
Diss. Abstr. 1998, 58-12B, 6581. Chen, X. J. Org. Chem. 1997, 62, 8182.
Total Synthesis of (()-Cylindrospermopsin J. Am. Chem. Soc., Vol. 122, No. 21, 2000 5019

accessible by addition of acetylene 10, where X is a latent

aminomethyl group, to aldehyde 3. It should be possible to
prepare 10 by conjugate addition to vinylogous amide 11, as
extensively studied by Comins.43 Vinylogous amide 11 should
be available by addition of trimethylsilylethynylmagnesium
bromide to 4-methoxy-3-methylpyridine (12). We report here
the realization of this approach leading to the first synthesis of
(()-cylindrospermopsin in 20 steps from 12 and 3.5% overall
yield.

Results and Discussion

4-Methoxy-3-methylpyridine (12)44,45 was prepared by modi-
fications of the literature procedure. 3-Methyl-4-nitropyridine
N-oxide (14),46 was treated with K2CO3 in methanol at 70 °C generated in 66% yield by treatment of 16 with vinylmagnesium
to displace the nitro group providing 15.45 Hydrogenolysis of bromide and BF3‚Et2O in the presence of a catalytic amount of
crude 15 over 10% Pd/C under 45 psi H2 for a week reduced CuBr‚Me2S at -78 °C in THF. The yield of 17 was improved
the N-oxide to provide 12 in 83% yield from 14. to 92% by utilizing TMSCl47 instead of BF3‚Et2O to activate
the cuprate addition. Due to the strong A(1,3) strain between the
alkynyl and N-acyl group of 16, dihydropyridone 16 adopts the
chair conformation with an axial alkynyl group. Stereoelec-
tronically preferred axial attack by the organocuprate reagent
at C-2 led to the desired isomer 17 with cis axial alkynyl and
vinyl substituents as observed by Comins in related examples.43
The stereochemistry of the methyl group adjacent to the carbonyl
Addition of trimethylsilylethynylmagnesium bromide to 12 group was introduced by axial protonation of the enolate which
could occur at either C-2 or C-6. We anticipated that the gives the more stable equatorial methyl group. Attempted
3-methyl group would block C-2, so that addition should occur equilibration of 17 in K2CO3/MeOH gave only recovered
regioselectively at C-6 even with a sterically undemanding starting material confirming that 17 is the thermodynamic
acetylide nucleophile. Investigation of conjugate additions to product.
16 (see below) indicated that a vinyl group was a suitable latent Cleavage of the Troc group of 17 with zinc dust in acetic
aminomethyl group. Therefore, benzyl chloroformate could not acid for 30 min afforded the free piperidone, which flipped to
be used to activate the pyridine to nucleophilic attack as in the give the conformer with equatorial vinyl and alkynyl substituents
synthesis of 2 since the Cbz group could not be removed without and an axial methyl group. The methyl group adjacent to the
reduction of the vinyl group. Treatment of 12 with TrocCl and ketone equilibrated under the acidic reaction conditions over
then trimethylsilylethynylmagnesium bromide at -30 °C and an additional 5 h to give the thermodynamically more stable
hydrolysis of the dihydropyridine with hydrochloric acid af- isomer 18 with all three substituents equatorial. Reduction of
forded 49% (87% based on recovered 12) of 16 and 2-3% of 18 with L-Selectride in THF at -78 °C and basic hydrolysis
the regioisomer resulting from addition at C-2. Reaction of 16 afforded the axial alcohol and cleaved the alkynylsilane provid-
with Et2AlCN afforded <20% of the desired 1,4-adduct. Other ing the desired piperidine 19 in 90% yield from 17 with control
procedures for cyanide addition were even less successful. of all four chiral centers on the A ring. The stereochemistry of
We then examined the copper-catalyzed addition of vinyl- 19 was confirmed by analysis of the coupling constants. Large
magnesium bromide to 16 since Comins has shown that cuprates diaxial coupling constants, J6-5ax ) 10.6 Hz and J2-3 ) 10.0
add to C-2 cis to a substituent at C-6 in a wide variety of Hz, established that the methyl, ethynyl, and vinyl substituents
N-carboalkoxy-5,6-dihydro-4-pyridones.43 Piperidone 17 was are equatorial. The absence of large diaxial coupling constants
for H4, J ) 3.2, 2.8, 2.4 Hz, indicated that the hydroxy group
(44) Profft, E.; Schulz, G. Arch. Pharm. 1961, 294, 292. is axial.
(45) Profft, E.; Krueger, W.; Kuhn, P.; Lietz, W. Ger. Pat. (East) 69,126;
Chem. Abstr. 1970, 72, 90309w. Conversion of 19 to ketone 9 required guanidinylation,
(46) Taylor, E. C., Jr.; Crovetti A. J. In Organic Syntheses; Rabjohn, elaboration of the vinyl group to an aminomethyl group, and
N., Ed.; Wiley: New York, 1963; Collect. Vol. IV, pp 654-656. addition of the acetylene to aldehyde 3. We were unable to
(47) (a) Corey, E. J.; Boaz, N. W. Tetrahedron Lett. 1985, 26, 6015. (b) convert the vinyl group to an aminomethyl group after introduc-
Alexakis, A.; Berlan, J.; Besace, Y. Tetrahedron Lett. 1986, 27, 1047. (c)
Bertz, S. H.; Miao, G.; Rossiter, B. E.; Snyder, J. P. J. Am. Chem. Soc. tion of the guanidine, and could not add the acetylene to
1995, 117, 11023. aldehyde 3 after introduction of the aminomethyl group.
5020 J. Am. Chem. Soc., Vol. 122, No. 21, 2000 Xie et al.

Therefore, we protected the amino and hydroxy groups of 19 NaCNBH3,48 afforded 68% of benzylamine 25. Hydrogenation
to give alkyne 21, which was coupled with aldehyde 3, even (1 atm) of 25 over 5% Pd/C in MeOH reduced the triple bond
though this generated a mixture of stereoisomers that had to be and hydrogenolyzed the benzyl and Cbz groups to afford 65-
carried through several steps before they reconverged in the 75% of crude diamine 26.
oxidation of alcohol 29 to ketone 30. Conversion of the diamine of 26 to a guanidine proved very
challenging. Eventually we found that slow addition of 1 equiv
Treatment of 19 with CbzCl and Na2CO3 in THF afforded
of cyanogen bromide49 to 26 in dilute toluene solution gave
96% of carbamate 20. Protection of the alcohol of 20 with
the primary cyanamide which cyclized to form guanidine 27.
TBSCl, imidazole and a catalytic amount of DMAP in CH2Cl2 The use of excess cyanogen bromide led to the bis-cyanamide.
provided 89% of 21. Treatment of 21 with ethylmagnesium Protection of the guanidine with CbzCl and NaH in THF at
bromide formed the alkynylmagnesium bromide, which was room temperature for 8 h afforded 28 in 45% overall yield from
treated with 3 to yield 83% of alcohol 22 as a 1:1 mixture of 25 and 10% of a byproduct in which one Cbz group and one
diastereomers. Protection of the alcohol of 22 with TBSCl, benzyl group are attached to the guanidine. Presumably, reaction
imidazole, and a catalytic amount of DMAP in CH2Cl2 gave of chloride ion with benzyl chloroformate formed benzyl
88% of 23. chloride.
Desilylation of 28 with TBAF in THF at room temperature
overnight gave 83% of 29, which was oxidized with MnO2 in
CH2Cl2 to form 87% of ketone 30. Since the eight steps from
22 to 30 were carried out with a mixture of diastereomers, the
structure and stereochemistry of ketone 30 was carefully
confirmed spectroscopically. The coupling constant between H13
and H14, J ) 11.0 Hz, and H10 and H11ax, J ) 11.6 Hz,
demonstrated that H10, H13 and H14 are axial. H12, with a very
narrow W1/2, is equatorial. The small geminal coupling constant
for H15, J ) 10.0 Hz, established that the five-membered C
ring had been formed.
Bromination of ketone 30 could not be accomplished. The
alcohol was therefore acetylated with acetic anhydride in
pyridine at room temperature, which gave 87% of a 9:1 mixture
of 31 and the enol acetate. The enol acetate was the only product
when DMAP was added to the reaction mixture. Basic hydroly-
sis of the enol acetate with KHCO3 in methanol also cleaved
one of the Cbz groups; thus, the 9:1 mixture was used since
both the ketone and enol acetate should form the same bromo
ketone.
Bromination of 31 with CuBr2 in EtOAc at room temperature
for 30 min gave an unstable mixture of bromo ketones and a
tricyclic compound in which one of the Cbz groups had been
lost and the SN2 reaction had taken place. Immediate hydro-
genation of this mixture over 5% Pd/C in methanol provided a
1:3 mixture of the desired product 32 and stereoisomer 33.
Fortunately, hydrogenation over 20% Pd(OH)2/C gave an easily
separable 3:2 mixture of 32 and 33 in 71% yield from the
mixture of acetoxy ketone 31 and the enol acetate. Hydro-
genolysis of the Cbz groups afforded the free guanidine, which
underwent an intramolecular SN2 reaction to form the third ring.
We expected that hydrogenation of the ketone should be the
slowest step. Equilibration of the ketone side chain before
hydrogenation should favor the desired isomer 32 with an
equatorial side chain. We therefore carried out the hydrogenation
in the presence of bases such as K2CO3, Et3N, and KHCO3 and
acids such as formic acid and HCl. Unfortunately, none of the
acids improved the ratio of 32 to 33. Much lower yields were
obtained in the presence of the bases.
The stereochemistry of the newly formed six-membered ring
of 32 was assigned on the basis of the coupling constant, J8,9ax
) 11.6 Hz, which is very similar to that of 1 (J8,9ax ) 11.4
Hz). This established that H8 is axial as in 1. The absorption
for H7 in 32, δ 4.68 (d, J ) 3.7 Hz) corresponds closely to that
(48) Borch, R. F.; Bernstein, M. D.; Durst, H. D. J. Am. Chem. Soc.
Ozonolysis of 23 for 20 min cleaved the double bond, 1971, 93, 2897.
providing 72% of aldehyde 24 after reductive workup with (49) (a) Siddiqui, S.; Begum, S.; Siddiqui, B. S. Z. Naturforsch. 1982,
37b, 1481. (b) Siddiqui, S.; Haider, S. I.; Ahmad, S. S.; Siddiqui, B. S. Z.
Me2S. Condensation of aldehyde 24 with benzylamine in Naturforsch. 1985, 40b, 546. (c) Ahmad, S. S.; Haider, S. I.; Fatima, I.
benzene, followed by reduction of the resulting imine with Synth. Commun. 1987, 17, 1861.
Total Synthesis of (()-Cylindrospermopsin J. Am. Chem. Soc., Vol. 122, No. 21, 2000 5021

Table 1. Cell Glutathione (GSH) as Percentage of Control in Rat

Hepatocytes Incubated with Natural (-)-1, Synthetic (()-1, and
Diol 34
concentration (µM)
compound 0.32 0.63 1.25 2.50 7.50 10.0
natural (-)-1 109 91 54 15 12 12
synthetic (()-1 -- 93 109 104 36 25
diol 34 110 61 30 19 14 9

(GSH) in heptatocytes. This loss always precedes cell death.

Cell GSH levels of rat hepatocytes incubated for 19 h with
natural (-)-1, synthetic (()-1, and diol 34 at the indicated
concentrations are shown in Table 1. Model guanidine 8 showed
no reduction of GSH levels at 100-500 µM. These results
indicate that synthetic (()-1 is active, confirming the chemical
identity of the natural and synthetic material. Racemic diol 34
is more potent than racemic 1 and as least as potent as the natural
toxin, clearly demonstrating that the sulfate group is not
necessary for either biological activity or entry into the cell.
In conclusion, the first total synthesis of the novel hepatotoxin
(()-cylindrospermopsin (1) has been accomplished in 20 steps
from 4-methoxy-3-methylpyridine (12) in 3.5% overall yield.
The substituted piperidine A ring 19 was generated stereospe-
cifically by a four-step sequence using the addition of trimeth-
ylsilylethynylmagnesium bromide to 12 to give 16 and ste-
of cylindrospermopsin, δ 4.70 (d, J ) 3.9 Hz), suggesting that reospecific addition of vinylcuprate 16 to form 17. The reaction
hydrogenation of the ketone afforded the correct stereochemistry of diamine 26 with cyanogen bromide produced the cyclic
at C-7. Stereoisomer 33 has the opposite ring fusion stereo- guanidine C ring of 27. The key step in the synthesis was
chemistry since the coupling constant, J8,9ax ) 5.5 Hz, indicates bromination of ketone 31, followed by hydrogenation to liberate
that H8 is equatorial. Only a single isomer was formed, but the the free guanidine, which underwent an intramolecular SN2
stereochemistry at C-7 in 33 could not be assigned. reaction to form the tetrahydropyrimidine ring B. Further
Hydrolysis of 32 in concentrated hydrochloric acid at 100 hydrogenation reduced the ketone to yield 42% of 32 containing
°C for 6 h afforded 95% of uracil diol 34 with 1H and 13C NMR the fully functionalized tricyclic system and protected hy-
spectral data virtually identical to those of cylindrospermopsin droxymethyluracil side chain of cylindrospermopsin. Hydrolysis
except for the protons and carbons close to C12. A similar of the pyrimidine in concentrated hydrochloric acid and selective
hydrolysis of 33 provided 95% of 35. monosulfation completed the synthesis of cylindrospermopsin.
Monosulfation50-52 of the ring alcohol of 34 was needed to
complete the synthesis. Since both alcohols are secondary, it Experimental Section
was not obvious which would be more reactive. Reaction of 34 General. NMR spectra were recorded at 400 MHz in CDCl3 unless
with 10 equiv of SO3‚DMF in anhydrous pyridine and DMF otherwise indicated. Chemical shifts are reported in δ and coupling
overnight and concentration in a vacuum gave a mixture of constants in Hz. IR spectra are reported in cm-1.
cylindrospermopsin (1) and the bis-sulfate ester containing some Formation of Dihydropyridine 16. A solution of trimethylsilyl-
DMF and pyridine, indicating that the ring alcohol is more ethynylmagnesium bromide in THF was prepared from ethynyltri-
reactive. We were delighted to find that reverse phase chroma- methylsilane (3.6 mL, 25.1 mmol) and EtMgBr (3 M in diethyl ether,
tography on C18-silica gel using D2O as the eluent and NMR 8.4 mL, 25.1 mmol) under nitrogen. TrocCl (3.9 mL, 20.9 mmol) was
added dropwise at -30 °C to a solution of 4-methoxy-3-methylpyridine
spectroscopy to monitor fractions gave pure bis-sulfate ester
(12) (2.57 g, 20.9 mmol) in 50 mL of THF. The resulting white
36 followed by pure 1. Sulfation of 34 with 6 equiv of SO3‚ suspension was stirred for 30 min at -30 °C, and the above
DMF gave 60-80% of cylindrospermopsin after purification. trimethylsilylethynylmagnesium bromide solution was transferred by
The 1H and 13C NMR spectral data of 1 are identical to those cannula to the suspension. The mixture was stirred at -30 °C for 2 h.
reported for the natural product.1 The absorption for H12 shifts Hydrochloric acid (1 M, 20 mL) was then added, and the mixture was
downfield to δ 4.60 in 1 from δ 4.03 in 32, while the absorption stirred at room temperature for 30 min and diluted with H2O (60 mL).
for H7 at δ 4.72 in 1 is virtually identical to that of 32 at δ The mixture was extracted with EtOAc (2 × 80 mL). The combined
4.78. H12 of bis-sulfate ester 36 also absorbs at δ 4.61, while organic layers were washed with water (50 mL) and brine (80 mL),
H7 is now also shifted downfield to δ 5.20, and H8 is shifted dried (Na2SO4), and concentrated. Flash chromatography of the residue
downfield from δ 3.87 in 1 to δ 3.99 in 36. on silica gel (25:1 hexane/EtOAc, 5:1 hexane/EtOAc) gave 520 mg
(7%) of a 1:2 mixture of 16 and the regioisomer resulting from addition
The toxicities of 8, diol 34, and synthetic cylindrospermopsin to the carbon adjacent to the methyl group, followed by 3.99 g (49%,
((()-1) were compared to that of natura (-)-13 in the in vitro 87% based on recovered 12) of 16 as a white crystalline solid. The
hepatocyte assay previously described.17 Natural 1 has been combined aqueous layers after workup were treated with 2 N NaOH
shown to cause depletion of the cellular antioxidant glutathione solution (70 mL), and extracted with EtOAc (2 × 60 mL). The EtOAc
layers were washed with brine (70 mL), dried (Na2SO4), and concen-
(50) Sulfation of Drugs and Related Compounds; Mulder, G. R., Ed.; trated to provide 900 mg (35%) of recovered 12.
CRC Press: Boca Raton, FL, 1981; p 51.
(51) Lubineau, A.; Lemoine, R. Tetrahedron Lett. 1994, 35, 8795. The data for 16: mp 119.8-120.2 °C; 1H NMR 7.66-7.58 (br, 1),
(52) Futaki, S.; Taike, T.; Yagami, T.; Ogawa, T.; Akita, T.; Kitagawa, 5.41 (ddd, 1, J ) 6.8, 1.5, 1.5), 5.00-4.80 (br, 2), 2.87 (dd, 1, J )
K. J. Chem. Soc., Perkin Trans. 1 1990, 1739. 16.4, 6.8), 2.70 (dd, 1, J ) 16.4, 1.5), 1.83 (d, 1, J ) 1.2), 0.11 (s, 9);
5022 J. Am. Chem. Soc., Vol. 122, No. 21, 2000 Xie et al.
13C NMR (25 °C) 191.3, 150.9 (br), 137.0 and 135.7 (br), 116.6 (br), 12.8, 11.3, 6.1), 1.89 (dddd, 1, J ) 12.8, 4.2, 2.1, 1.0), 1.01 (d, 3, J )
100.3, 94.4, 89.7, 75.8, 46.1, 41.3, 13.0, -0.4 (3 C) (at 35 °C the peaks 6.8); 13C NMR 156.2, 137.8, 136.6, 128.7 (2 C), 128.2, 127.9 (2 C),
at 150.9 and 116.6 became sharper, and the two peaks at 137.0 and 116.6, 84.4, 72.0, 67.9, 64.0, 61.4, 42.5, 37.9, 32.8, 11.5; IR (neat)
135.7 collapsed to a broad peak at 136.6); IR (KBr) 3062, 2175, 1754, 3448, 3295, 2106, 1685, 1406, 1326, 1262; HRMS (GC/MS EI 20 eV)
1660. Anal. Calcd for C14H18NO3SiCl3: C, 43.93; H, 4.74; N, 3.66. calcd for C18H21NO3 299.1521, found 299.1507.
Found: C, 43.76; H, 4.74; N, 3.58. Silyl Ether 21. To a solution of 20 (2.84 g, 9.47 mmol) in dry CH2-
The data for the regioisomer of 16 were determined from the Cl2 (20 mL) was added imidazole (2.84 g, 47.4 mmol), TBSCl (1.56
mixture: 1H NMR 7.72 (br d, 1, J ) 8), 5.52-5.48 (br, 1), 5.26-5.22 g, 10.4 mmol), and a catalytic amount of DMAP (50 mg). The mixture
(br, 1), 5.00-4.58 (br, 2), 2.81 (dq, 1, J ) 6.4, 6.4), 1.26 (d, 3, J ) was stirred at room temperature overnight and added to saturated NH4-
6.4), 0.12 (s, 9). Cl solution. The organic layer was separated, and the aqueous layer
Formation of 17. To a solution of CuBr‚SMe2 (486 mg, 2.36 mmol) was extracted with CH2Cl2 (2 × 90 mL). The combined organic layers
in 80 mL of THF was added vinylmagnesium bromide (1 M in THF, were washed with water (100 mL) and brine (100 mL), dried (Na2-
17.7 mL, 17.7 mmol), and then a solution of 16 (4.51 g, 11.8 mmol) SO4), and concentrated. Flash chromatography of the residue on silica
and TMSCl (7.5 mL, 59.0 mmol) in 40 mL of THF at -78 °C. The gel (10:1 hexane/EtOAc) gave 3.49 g (89%) of 21 as an oil: 1H NMR
mixture was stirred at -78 °C for 2 h, quenched by 10 mL of 1 N HCl 7.38-7.28 (m, 5), 6.38 (ddd, 1, J ) 17.2, 10.4, 7.0), 5.22 (ddd, 1, J )
solution, diluted with H2O (40 mL), and extracted with EtOAc (3 × 6.2, 2.4, 2.0), 5.19 (ddd, 1, J ) 17.2, 1.2, 1.2), 5.17 (s, 2), 5.11 (ddd,
80 mL). The combined organic layers were washed with water (120 1, J ) 10.4, 1.2, 1.2), 4.61 (br d, 1, J ) 7.0), 4.38 (ddd, 1, J ) 11.4,
mL) and brine (100 mL), dried (Na2SO4), and concentrated. Flash 4.2, 4.2), 2.28 (d, 1, J ) 2.4), 2.10 (ddq, 1, J ) 4.2, 2.8, 7.2), 1.97
chromatography of the residue on silica gel (10:1 hexane/EtOAc) gave (ddd, 1, J ) 13.2, 11.4, 6.2), 1.75 (ddd, 1, J ) 13.2, 4.2, 2.0), 0.98 (d,
4.47 g (92%) of 17 as a white crystalline solid: mp 91.4-91.8 °C; 1H 3, J ) 7.2), 0.89 (s, 9), 0.07 (s, 6); 13C NMR 155.8, 137.8, 136.4,
NMR 6.15-6.05 (br, 1), 5.53 (br s, 1), 5.33 (br d, 1, J ) 17.2), 5.18 128.3 (2 C), 127.6, 127.0 (2 C), 116.0, 84.3, 71.4, 67.4, 64.4, 61.0,
(dd, 1, J ) 10.0, 1.6), 5.13-5.05 (br, 1), 4.85-4.79 (br, 2), 2.90-2.81 42.2, 38.3, 33.2, 25.7 (3 C), 17.9, 11.4, -4.8, -4.9; IR (neat) 1702,
(m, 2), 2.62 (dd, 1, J ) 14.0, 2.4), 0.97 (d, 3, J ) 6.8), 0.10 (s, 9); 13C 1404, 1109; HRMS (GC/MS CI/CH4) calcd for C24H36NO3Si (MH+)
NMR 205.9, 153.0, 132.6, 120.3, 103.8, 95.1, 91.1, 75.4, 63.2, 46.4, 414.2464, found 414.2453.
45.6, 45.1 (br), 10.9, -0.6 (3 C); IR (KBr) 3081, 2174, 1724, 1642, Formation of 22. To a solution of 21 (3.49 g, 8.44 mmol) in THF
1456. Anal. Calcd for C16H22NO3SiCl3: C, 46.78; H, 5.40; N, 3.41. (60 mL) was added EtMgBr (1 M in THF, 9.3 mL, 9.3 mmol) at 0 °C
Found: C, 46.72; H, 5.42; N, 3.32. under N2. The reaction mixture was warmed to room temperature and
Formation of 18. A suspension of 17 (4.47 g, 10.9 mmol) and zinc stirred at room temperature for 2 h. A solution of 3 (1.56 g, 9.28 mmol)
dust (5.0 g) in CH2Cl2/HOAc (1/3, 40 mL) was stirred vigorously at in THF (40 mL) was then added dropwise to the mixture. The reaction
room temperature for 5 h. The mixture was filtered, and the filtrate was stirred at 0 °C for 2 h and poured into a saturated NH4Cl solution
was concentrated, neutralized with Na2CO3 solution, and extracted with (60 mL), the organic layer was separated, and the water layer was
EtOAc (3 × 70 mL). The combined organic layers were washed with extracted with EtOAc (2 × 80 mL). The combined organic layers were
brine (100 mL), dried (Na2SO4), and concentrated. Flash chromatog- washed with water (70 mL) and brine (70 mL), dried (Na2SO4), and
raphy of the residue on silica gel (3:1 hexane/EtOAc) gave 2.48 g (96%) concentrated. Flash chromatography of the residue on silica gel (10:1
of 18: 1H NMR 5.74 (ddd, 1, J ) 16.8, 10.0, 8.0), 5.16 (d, 1, J ) hexane/EtOAc, 3:1 hexane/EtOAc, then 1:1 hexane/EtOAc) gave 569
16.8), 5.13 (dd, 1, J ) 10.0, 1.2), 3.70 (dd, 1, J ) 9.6, 5.2), 2.88 (dd, mg (16%) of recovered 21, followed by 3.39 g (69%, 83% based on
1, J ) 10.4, 8.0), 2.58-2.49 (m, 2), 2.20 (dq, 1, J ) 10.4, 6.8), 2.01 recovered 21) of 22 as a 1:1 mixture of diastereomers: 1H NMR 7.37-
(br s, NH), 0.87 (d, 3, J ) 6.8), -0.075 (s, 9); 13C NMR 207.2, 138.1, 7.27 (m, 5), 6.483 (s, 1 × 0.5), 6.480 (s, 1 × 0.5), 6.22 (ddd, 1 × 0.5,
118.1, 104.3, 88.5, 66.1, 49.4, 48.2, 47.7, 9.9, -0.3 (3 C); IR (neat) J ) 17.0, 10.4, 5.8), 6.20 (ddd, 1 × 0.5, J ) 17.2, 10.4, 5.8), 5.24-
3316, 3079, 2178, 1715; HRMS (GC/MS,EI 20 eV) calcd for C13H22- 5.27 (m, 1), 5.15 (s, 2 × 0.5), 5.14 (s, 2 × 0.5), 5.13 (br d, 1, J )
NOSi (MH+) 234.1314, found 234.1316. 17.0), 5.02 (ddd, 1 × 0.5, J ) 10.4, 1.0, 1.0), 5.00 (ddd, 1 × 0.5, J )
Reduction of 18 to 19. L-Selectride (21.0 mL, 1.0 M in THF, 21.0 10.4, 1.0, 1.0), 4.58 (br s, 1, W1/2 ) 14.0), 4.30 (ddd, 1 × 0.5, J )
mmol) was added to a solution of 18 (2.48 g, 10.5 mmol) in 100 mL 10.8, 4.2, 4.2), 4.26 (ddd, 1 × 0.5, J ) 10.8, 4.2, 4.2), 4.00 (s, 3), 3.97
of THF at -78 °C. The solution was stirred 2 h, and the reaction was (s, 3), 3.91 (d, 1 × 0.5, J ) 5.2), 3.86 (d, 1 × 0.5, J ) 4.8), 2.07 (m,
quenched with saturated aqueous NaHCO3 (50 mL). The mixture was 1), 1.94 (ddd, 1 × 0.5, J ) 13.2, 11.6, 6.4), 1.92 (ddd, 1 × 0.5, J )
extracted with EtOAc (3 × 60 mL). The combined organic layers were 13.2, 11.6, 6.4), 1.74-1.67 (m, 1), 0.95 (d, 3, J ) 6.8), 0.86 (s, 9 ×
concentrated to provide a wet oil. The oil was then taken up in EtOH 0.5), 0.84 (s, 9 × 0.5), 0.02 (s, 6); 13C NMR 172.3, 168.5, 165.0, 155.8
(50 mL) and 1 N NaOH solution (7 mL). The solution was refluxed (2 C), (138.18, 138.06), 136.4, 128.4, (127.91, 127.89), (127.63, 127.61)
for 2 h, and poured onto saturated aqueous NaHCO3 solution, which (2 C), (115.79, 115.70), (97.90, 97.88), (86.71, 86.63), 81.1, (67.49,
was extracted with EtOAc (3 × 60 mL). The combined organic layers 67.46), (64.52, 64.48), (63.14, 63.05), (60.63, 60.59), 54.9, 54.1, (42.57,
were washed with brine (100 mL), dried (Na2SO4), and concentrated. 42.52), (37.93, 37.84), (33.32, 33.24), (25.69, 25.67) (3 C), (17.97,
Flash chromatography of the residue on silica gel (3:1 hexane/EtOAc, 17.96), (11.44, 11.39), (-4.86, -4.96) (2 C); IR (neat) 3429 (br), 1701,
then 1:1 hexane/EtOAc) gave 1.63 g (94%) of 19 as a white crystalline 1596, 1571, 1356; HRMS (DCI/NH3) calcd for C31H44N3O6Si (MH+)
solid: mp 107.0-107.2 °C; 1H NMR 5.70 (ddd, 1, J ) 16.4, 10.4, 582.2999, found 582.3003.
8.8), 5.19 (dd, 1, J ) 16.4, 2.0), 5.11 (dd, 1, J ) 10.4, 2.0), 3.96 (ddd, Bis-silyl Ether 23. To a solution of 22 (3.39 g, 5.83 mmol) in dry
1, J ) 10.6, 2.6, 2.6), 3.94 (ddd, 1, J ) 3.2, 2.8, 2.4), 3.18 (dd, 1, J ) CH2Cl2 (20 mL) was added imidazole (1.75 g, 29.2 mmol), and TBSCl
10.0, 8.8), 2.24 (d, 1, J ) 2.6), 2.03 (ddd, 1, J ) 14.0, 2.6, 3.2), 1.84 (962 mg, 6.41 mmol). The above mixture was stirred at room
(ddd, 1, J ) 14.0, 10.6, 2.8), 1.49 (ddq, 1, J ) 10.0, 2.4, 6.8), 0.89 (d, temperature for 5 h and poured into a saturated NH4Cl solution. The
3, J ) 6.8); 13C NMR 139.6, 117.1, 85.3, 70.5, 68.6, 60.0, 41.7, 40.1, organic layer was separated, and the aqueous layer was extracted with
39.0, 14.5; IR (KBr) 3288, 3179, 2119; HRMS (GC/MS EI 20 eV) CH2Cl2 (2 × 80 mL). The combined organic layers were washed with
calcd for C10H16NO (MH+) 164.1075, found 164.1071. water (120 mL) and brine (100 mL), dried (Na2SO4), and concentrated.
Formation of 20. To a solution of 19 (1.63 g, 9.87 mmol) in THF Flash chromatography of the residue on silica gel (10:1 hexane/EtOAc)
(40 mL) was added solid Na2CO3 (3.0 g) and CbzCl (1.7 mL, 11.8 gave 3.56 g (88%) of 23 as a 1:1 mixture of diastereomers: 1H NMR
mmol). The suspension was stirred vigorously overnight at room 7.35-7.28 (m, 5), 6.62 (s, 1), 6.28 (ddd, 1, J ) 17.2, 10.4, 7.4), 5.26-
temperature, and the Na2CO3 was filtered off. The filtrate was 5.22 (m, 2), 5.17-5.08 (m, 3), 4.98-4.95 (m, 1), 4.56 (br d, 1, J )
concentrated and purified on silica gel (10:1 hexane/EtOAc, then 1:1 6.7), 4.29-4.21 (m, 1), 3.97 (s, 3), 3.95 (s, 3), 2.07-2.02 (m, 1), 1.96-
hexane/EtOAc) to give 2.84 g (96%) of 20 as an oil: 1H NMR 7.37- 1.87 (m, 1), 1.71-1.65 (m, 1), 0.949 (d, 3 × 0.5, J ) 6.7), 0.945 (d,
7.30 (m, 5), 6.37 (ddd, 1, J ) 17.1, 10.4, 7.3), 5.26 (ddd, 1, J ) 6.1, 3 × 0.5, J ) 6.7), 0.92 (s, 9), 0.85 (s, 9 × 0.5), 0.84 (s, 9 × 0.5), 0.16
2.1, 2.1), 5.21 (ddd, 1, J ) 17.1, 1.4, 1.4), 5.17 (s, 2), 5.11 (ddd, 1, J (s, 6 × 0.5), 0.15 (s, 6 × 0.5), 0.12 (s, 6 × 0.5), 0.11 (s, 6 × 0.5); 13C
) 10.4, 1.4, 1.4), 4.63 (br d, 1, J ) 7.3), 4.44 (dddd, 1, J ) 11.3, 4.9, NMR (172.41, 172.43), 171.1, 165.0, 155.8, 138.1, 136.5, 128.4 (2
4.8, 4.2), 2.28 (d, 1, J ) 2.1), 2.26-2.18 (m, 1), 1.98 (ddd, 1, J ) C), 127.8, 127.6 (2 C), (115.8, 115.7), 97.3, 85.8, 81.9, 67.4, (65.33,
Total Synthesis of (()-Cylindrospermopsin J. Am. Chem. Soc., Vol. 122, No. 21, 2000 5023

65.29), 64.5, (60.95, 60.89), (54.68, 54.67), 53.8, (42.54, 42.53), (38.45, (125.4-127.4) (10 C), 97.2, (74.5, 74.3), (68.9, 68.8), 67.9, 67.0, 56.7,
38.34), (33.35, 33.24), 25.7 (6 C), (18.2, 17.9) (2 C), (11.42, 11.40), 54.5, 53.5, (51.89, 51.82), (47.77, 47.69), (39.00, 38.97), 38.2, (34.28,
-4.68, -4.70, -5.04, -5.18; IR (neat) 1706, 1696, 1573, 1461, 1357, 34.12), (27.47, 27.37), (25.7-25.6) (6 C), (17.95, 17.85) (2 C), (13.55,
HRMS (FAB) calcd for C37H58N3O6Si2 (MH+) 696.3864, found 13.50), (-4.5, -5.2) (4 C); IR (neat) 1758, 1594, 1355; HRMS (FAB)
696.3850. calcd for C45H68N5O8Si2 (MH+) 682.4606, found 682.4603.
Formation of Aldehyde 24. A stream of ozone was introduced into Data for the benzyl methylene groups of the byproduct: 5.04 (d, 1,
a solution of 23 (3.56 g, 5.11 mmol) in CH2Cl2 (200 mL) at -78 °C J ) 12.4), 4.98 (d, 1, J ) 12.4), 4.24 (d, 1, J ) 14.8), 4.19 (d, 1, J )
for about 20 min until the solution turned bluish. Nitrogen was bubbled 14.8).
through the solution for 10 min to purge the excess ozone. Me2S (1.50 Formation of Diol 29. A solution of 28 (370 mg, 0.429 mmol) and
mL, 20.5 mmol) was added, and the mixture was then stirred at -78 TBAF (1 M in THF, 900 µL, 0.90 mmol) in THF (10 mL) was stirred
°C for 20 min. The solution was concentrated, and the residue was at room temperature overnight. The solution was concentrated, and the
purified by flash chromatography on silica gel (5:1 hexane/EtOAc) to residue was purified by flash chromatography on silica gel (10:1 CH2-
provide 2.56 g (72%) of 24 as a 1:1 mixture of diastereomers: 1H Cl2/MeOH) to give 226 mg (83%) of 29 as a 1:1 mixture of
NMR 9.77 (s, 1 × 0.5), 9.67 (s, 1 × 0.5), 7.36-7.26 (m, 5), 6.57 (s, diastereomers: 1H NMR 7.44-7.22 (m, 10), 6.44 (s, 1 × 0.5), 6.42 (s,
1 × 0.5), 6.55 (s, 1 × 0.5), 5.35-5.13 (m, 5), 4.60-4.38 (m, 1), 3.96- 1 × 0.5), 5.13 (s, 2), 5.05 (br s, 2), 4.56-4.48 (m, 1), 3.958 (s, 3 ×
3.92 (m, 6), 2.72-2.53 (m, 1), 1.95-1.52 (m, 2), 0.90 (d, 3, J ) 6.8), 0.5), 3.955 (s, 3 × 0.5), 3.924 (s, 3 × 0.5), 3.920 (s, 3 × 0.5), 3.96-
0.89 (s, 9), 0.83 (s, 9 × 0.5), 0.82 (s, 9 × 0.5), 0.15-0.05 (m, 12); 13C 3.92 (m, 1), 3.77-3.67 (m, 1), 3.58-3.48 (m, 3), 2.55-2.30 (m, 1),
NMR (201.1, 200.7), (172.5, 172.4), (170.6, 170.5), 165.1, (159.80, 2.20-1.50 (m, 5), 0.94 (d, 3, J ) 6.8); 13C NMR (174.05, 173.94),
159.78), (135.9, 135.8), (128.6, 128.5) (2 C), (128.3, 128.2), (127.86, 172.2, 164.8, 159.0, 151.5, (149.9, 149.8), 137.1, 135.1, 128.6-127.6
127.82) (2 C), (97.24, 97.18), (84.1, 83.9), 83.3, (68.23, 68.15), 66.7 (10 C), (97.30, 97.29), (72.53, 72.36), (68.29, 68.31), (67.94, 67.91),
(br), (65.14, 65.11), 64.5, (54.8, 54.7), (53.89, 53.87), (43.9, 42.8), 38.4, 67.3, (56.4, 56.3), 54.7, 53.8, (51.9, 51.7), (48.1, 47.9), (39.1, 38.9),
(32.8, 32.3), (25.67, 25.64) (6 C), (18.2, 17.9) (2 C), (10.0, 9.1), (-4.8, (38.45, 38.37), (33.7, 33.5), (27.7, 27.6), (13.00, 12.93); IR (neat) 1752,
-5.1) (2 C), (-5.1, -5.2) (2 C); IR (neat) 1709, 1595, 1574, 1358; 1596, 1570, 1355; HRMS (DCI/NH3) calcd for C33H40N5O8 (MH+)
HRMS (DCI/NH3) calcd for C36H56N3O7Si2 (MH+) 698.3657, found 634.2877, found 634.2874.
698.3698. Formation of Ketone 30. Activated MnO2 (200 mg, 2.00 mmol)
Benzylamine 25. To a solution of 24 (2.56 g, 3.67 mmol) in toluene was added to a solution of 29 (160 mg, 0.253 mmol) in 6 mL of CH2-
(40 mL) containing Na2SO4 (5 g) was added benzylamine (2.0 mL, Cl2 at room temperature. The reaction mixture was stirred at room
18.4 mmol) and HOAc (1.3 mL, 22.0 mmol) under nitrogen. The temperature for 1 h and filtered through a plug of Celite. The filtrate
solution was stirred at room temperature for 2 h. MeOH (50 mL) and was concentrated and purified on silica gel (1:1 hexane/EtOAc, EtOAc)
NaCNBH3 (1.20 g, 18.4 mmol) were then added and the resulting to give 138 mg (87%) of 30 as an oil: 1H NMR 7.37-7.20 (m, 10),
mixture was stirred at room temperature for another 2 h. Saturated 6.89 (s, 1), 5.13 (s, 2), 4.97 (s, 2), 3.99 (s, 3), 3.98 (s, 3), 3.99-3.97
NaHCO3 solution (60 mL) was added, and the solution was extracted (m, 1), 3.96-3.90 (m, 1), 3.82-3.74 (m, 1), 3.58-3.55 (m, 1), 3.55
with EtOAc (3 × 80 mL). The combined organic layers were washed (ddd, 1, J ) 10.8, 8.0, 5.6), 3.27 (br t, 2, J ) 7.0, 7.0, 1.2), 2.78-2.69
with brine (100 mL), dried (Na2SO4), and concentrated. Flash chro- (m, 1), 2.26-2.17 (m, 1), 1.96 (ddd, 1, J ) 13.0, 11.0, 3.1), 1.82 (ddd,
matography of the residue on silica gel (3:1 hexane/EtOAc) gave 1.97 1, J ) 13.0, 3.2, 3.2), 1.71-1.64 (m, 1), 0.95 (d, 3, J ) 6.7); 1H NMR
g (68%) of 25 as a 1:1 mixture of diastereomers: 1H NMR 7.40-7.23 (C6D6) 7.40-7.00 (m, 11), 5.33 (s, 2), 5.02 (d, 1, J ) 12.2), 4.96 (d,
(m, 10), 6.65 (s, 1 × 0.5), 6.47 (s, 1 × 0.5), 5.30-5.13 (m, 4), 4.34- 1, J ) 12.2), 3.78 (s, 3), 3.50 (s, 3), 3.55-3.40 (m, 2), 3.20 (dd, 1, J
4.16 (m, 2), 3.97 (s, 3), 3.94 (s, 3), 3.82-3.60 (m, 2), 3.30-3.08 (m, ) 10.0, 8.0), 3.18-3.12 (m, 1), 2.99 (dd, 1, J ) 10.0, 5.6), 2.98-2.91
1), 2.94-2.82 (m, 1), 2.20-2.07 (m, 1), 1.97-1.80 (m, 1), 1.75-1.60 (m, 1), 2.84 (ddd, 1, J ) 12.8, 7.2, 5.6), 2.38-2.32 (m, 2), 1.5-1.2
(m, 1), 1.00-0.80 (m, 18), 0.20-0.00 (m, 12); 13C NMR 172.4, (171.11, (m, 3), 0.34 (d, 3, J ) 7.3); 13C NMR 200.4, 172.9, 165.6, 161.5, 158.9,
171.06), (165.1, 165.0), (140.6), 139.0, (136.49, 136.46), (128.6-126.6), 151.5, 149.2, 137.1, 135.2, 128.6 (2 C), 128.49, 128.47, 128.1 (2 C),
(10 C), 97.3, (85.7, 85.6), 81.3, 67.5, (65.31, 65.25), 64.6, (58.6, 58.5), 127.9 (2 C), 127.4 (2 C), 99.1, 68.3, 68.1, 67.2, 56.5, 55.1, 54.3, 51.4,
51.2, 51.1, 49.5, 42.5, 35.3, 33.1, 27.4, (25.71, 25.68) (6 C), (18.2, 47.8, 38.5, 38.1, 35.2, 25.8, 12.9; IR (neat) 1752, 1707, 1592, 1566,
18.0) (2 C), (11.6, 11.5), (-4.7, -4.9) (2 C), (-5.0, -5.1) (2 C); IR 1387, 1357, 1254; HRMS (FAB) calcd for C33H38N5O8 (MH+)
(neat) 3320, 1946, 1700, 1574, HRMS (FAB) calcd for C43H65N4O6Si2 632.2720, found 632.2718.
(MH+) 789.4443, found 789.4475. Acetate 31. To a solution of 30 (138 mg, 0.218 mmol) in pyridine
Bis-Cbz-Protected Guanidine 28. A suspension of 25 (1.97 g, 2.49 (5 mL) was added acetic anhydride (62 µL, 0.656 mmol). The mixture
mmol) and 5% Pd/C (1.00 g) in MeOH (70 mL) was stirred at room was stirred at room temperature overnight and concentrated under
temperature under H2 (1 atm) overnight. The Pd catalyst was filtered reduced pressure. Purification of the residue on silica gel (1:1 hexane/
off, and the filtrate was concentrated to provide 1.02 g of crude 26. EtOAc) provided 130 mg of a 5:1 mixture of acetate 31 and enol acetate
To a solution of crude 26 (1.02 g) in toluene (80 mL) was added of 31 (87%) as an oil that was used for the next step.
slowly a solution of CNBr (170 mg, 1.61 mmol) in toluene (40 mL) The data for 31 determined from the mixture: 1H NMR 7.36-7.21
over 3 h. The mixture was stirred at room temperature for 2 h and (m, 10), 6.90 (s, 1), 5.15 (br s, 1), 5.13 (s, 2), 4.99 (s, 2), 3.99 (s, 3),
concentrated to provide crude 27. 3.98 (s, 3), 3.95-3.92 (m, 1), 3.71-3.63 (m, 1), 3.56 (dd, 1, J ) 10.0,
To a solution of crude 27 in THF (20 mL) was added excess NaH 5.5), 3.48 (ddd, 1, J ) 11.0, 8.8, 6.1), 3.27 (dd, 2, J ) 7.0, 7.0), 2.71
(400 mg, 9.60 mmol) and CbzCl (914 µL, 6.40 mmol) at 0 °C. The (dddd, 1, J ) 13.4, 7.0, 7.0, 6.7), 2.22 (dddd, 1, J ) 13.4, 8.0, 7.0,
mixture was stirred at room temperature for 8 h. Unreacted NaH was 7.0), 2.08 (s, 3), 1.96 (ddd, 1, J ) 14.6, 3.7, 3.7), 1.88 (ddd, 1, J )
filtered off and quenched carefully with 2-propanol. The filtrate was 14.6, 11.0, 3.1), 1.80 (ddq, 1, J ) 11.0, 3.7, 6.8), 0.88 (d, 3, J ) 6.8);
concentrated and purified by flash chromatography on silica gel (10:1 13C NMR 200.2, 172.9, 170.1, 165.6, 161.4, 158.8, 151.5, 148.8, 137.1,

hexane/EtOAc, then 2:1 hexane/EtOAc) to give 880 mg (45% from 135.1, 128.6-127.5 (10 C), 99.1, 70.4, 68.4, 67.2, 57.1, 55.1, 54.3,
25) of 28 as a 1:1 mixture of diastereomers followed by 190 mg (10%) 51.9, 47.9, 37.0, 34.9, 34.8, 25.8, 21.0, 12.6; IR (neat) 1752, 1798,
of a byproduct with one Cbz group and one benzyl group on the 1590, 1566, 1387, 1358; HRMS (DCI/NH3) calcd for C35H40N5O9
guanidine. (MH+) 674.2826, found 674.2807.
The data for 28: 1H NMR 7.45-7.30 (m, 10), 6.58 (s, 1 × 0.5), The data for the enol acetate were determined from the mixture:
6.56 (s, 1 × 0.5), 5.19-5.05 (m, 4), 4.65 (dd, 1 × 0.5, J ) 7.2, 3.6), 1H NMR 7.36-7.20 (m, 10), 6.72 (dd, 1, J ) 7.7, 7.3), 6.24 (s, 1),

4.64 (dd, 1 × 0.5, J ) 7.2, 3.6), 3.99 (s, 3 × 0.5), 3.98 (s, 3), 3.97 (s, 5.15 (br s, 1), 5.14 (s, 2), 5.04 (s, 2), 4.06 (m, 1), 3.94 (s, 6), 3.67 (m,
3 × 0.5), 3.94-3.91 (m, 1), 3.70-3.40 (m, 4), 2.62-2.54 (m, 1), 2.40- 1), 3.54-3.44 (m, 2), 3.26 (ddd, 1, J ) 14.4, 7.7, 4.8), 2.84 (ddd, 1, J
2.30 (m, 1), 2.22-2.10 (m, 1), 2.05-1.54 (m, 4), 0.95 (s, 9), 0.94 (s, ) 14.4, 7.3, 7.0), 2.25 (s, 3), 2.07 (s, 3), 1.94 (ddd, 1, J ) 14.8, 4.0,
9), 0.91 (d, 3, J ) 6.8), 0.11 (s, 6 × 0.5), 0.09 (s, 6 × 0.5), 0.084 (s, 4.0), 1.86 (ddd, 1, J ) 14.8, 10.4, 3.2), 1.78 (ddq, 1, J ) 10.4, 3.1,
6 × 0.5), 0.075 (s, 6 × 0.5); 13C NMR (175.57, 175.46), (172.00, 6.7), 0.88 (d, 3, J ) 6.7); 13C NMR 120.9, 96.3, 68.7, 68.1, 67.5, 56.9,
171.97), (164.69, 164.65), 158.8, 151.4, (149.15, 149.12), 137.1, 135.1, 54.9, 54.3, 51.1, 48.7, 37.4, 35.0, 29.5, 21.0, 20.6, 12.7 (the carbons
5024 J. Am. Chem. Soc., Vol. 122, No. 21, 2000 Xie et al.

were assigned from an HMQC experiment; the quaternary carbons were 71.8, 70.6, 59.2, 55.2, 50.0, 46.3, 41.9, 40.1, 30.2, 15.4; HRMS (FAB)
not observed). calcd for C15H22N5O4 (MH+) 336.1672, found 336.1687.
Formation of Tricycles 32 and 33. A suspension of CuBr2 (100 Formation of 35. A solution of 33 (2.0 mg, 0.032 mmol) in
mg, 0.452 mmol) in EtOAc (30 mL) was stirred vigorously at 35 °C concentrated HCl (1 mL) was refluxed at 100 °C for 6 h. The mixture
(oil bath temperature) for 20 min. A solution of 31 and the enol acetate was evaporated under reduced pressure to give 1.8 mg (90%) of 35 as
mixture (65 mg, 0.089 mmol) in EtOAc (5 mL) was then added by the hydrochloride salt: 1H NMR (D2O) 5.86 (s, 1), 4.57 (d, 1, J )
syringe. The mixture was stirred at 35 °C for 20 min, diluted with 7.2), 4.07 (br s, 1), 3.87 (ddd, 1, J ) 8.4, 7.2, 4.3), 3.80 (dd, 1, J )
EtOAc (10 mL), and filtered through Celite. The filtrate was washed 9.2, 8.8), 3.82-3.75 (m, 1), 3.68-3.54 (m, 1), 3.32 (dd, 1, J ) 9.6,
with brine (10 mL), dried (Na2SO4), and concentrated to give 72 mg 9.2), 2.44 (ddd, 1, J ) 14.0, 4.3, 3.0), 2.12 (ddd, 1, J ) 14.0, 3.7, 3.7),
of crude bromination product that was used immediately for the 1.98-1.90 (m, 1), 1.83-1.73 (m, 1), 1.58 (ddd, 1, J ) 14.0, 11.6,
hydrogenation step. 2.5), 0.97 (d, 3, J ) 7.2).
A solution of the crude bromo ketone (72 mg) in methanol (10 mL) Formation of Cylindrospermopsin (1). A solution of 34 (1.2 mg,
was stirred over 20% Pd(OH)2/C (25 mg) at room temperature under 0.0036 mmol) in dry pyridine (0.15 mL) containing Na2SO4 (20 mg)
a hydrogen-filled balloon for 8 h, and filtered through Celite. The filtrate was stirred at room temperature for 30 min. A solution of SO3‚DMF
was evaporated under reduced pressure, and the residue was purified in dry DMF (0.1 M, 216 µL, 0.0216 mmol, 6 equiv) was added to the
on silica gel (80:20:1 CH2Cl2/MeOH/HCO2H) to give 31 mg of a 3:2 mixture, which was stirred at room temperature overnight. Pyridine
mixture of 32 and 33. Careful flash chromatography on silica gel (96: and DMF were evaporated, and the residue was taken up in MeOH to
4:1 CH2Cl2/MeOH/HCO2H) gave 15 mg (37%) of 32, followed by 7 remove Na2SO4. The methanol solution was concentrated, and the
mg (17%) of a 1:2 mixture of 32 and 33, and 7 mg (17%) of a 8:1 residue was purified by flash chromatography on Bakerbond C18 (40
mixture of 33 and an unknown impurity. µm) prep LC packing using D2O as the eluent. Pyridinium salts eluted
The data for 32: 1H NMR (CD3OD) 6.69 (s, 1), 5.12 (ddd, 1, J ) in the earlier fractions, followed by 1.1 mg (50-70%) of cylindrosper-
3.6, 3.6, 2.4), 4.68 (d, 1, J ) 3.7), 3.99 (s, 3), 3.98 (s, 3), 3.95 (ddd, mopsin (1): 1H NMR (D2O) (600 MHz) 5.83 (s, 1), 4.72 (d, 1, J )
1, J ) 11.0, 3.7, 3.6), 3.85 (dd, 1, J ) 8.6, 8.6), 3.80 (ddd, 1, J ) 3.7), 4.63-4.60 (m, 1), 3.86 (ddd, 1, J ) 11.2, 3.7, 3.5), 3.84 (dd, 1,
10.4, 10.4, 8.6), 3.55 (dddd, 1, J ) 11.6, 11.0, 3.6, 3.6), 3.25 (dd, 1, J J ) 9.2, 9.0), 3.73 (ddd, 1, J ) 11.4, 10.8, 9.0), 3.65 (dddd, 1, J )
) 10.4, 8.6), 2.15 (ddd, 1, J ) 14.0, 3.6, 3.6), 2.07 (s, 3), 1.95 (ddd, 11.7, 11.7, 3.5, 3.5), 3.24 (dd, 1, J ) 10.8, 9.2), 2.44 (ddd, 1, J )
1, J ) 13.4, 3.6, 3.6), 1.85 (ddq, 1, J ) 10.4, 2.4, 6.7), 1.62 (ddd, 1, 14.2, 3.5, 2.8), 2.15 (ddd, 1, J ) 13.2, 3.5, 3.5), 1.85 (ddq, 1, J )
J ) 13.4, 11.6, 11.0), 1.50 (ddd, 1, J ) 14.0, 11.6, 3.6), 0.95 (d, 3, J 11.4, 2.6, 6.8), 1.57 (ddd, 1, J ) 13.2, 11.7, 11.2), 1.54 (ddd, 1, J )
) 6.7); 13C NMR (CD3OD) 174.3, 172.8, 172.0, 166.7, 157.0, 99.8, 14.2, 11.7, 3.0), 0.98 (d, 3, J ) 6.8); 13C NMR (D2O) (600 MHz) (from
73.8, 71.9, 58.9, 55.5, 55.2, 54.8, 48.9 (obscured by CD3OD, the an HSQC experiment) 99.6, 78.2, 70.8, 57.9, 53.7, 48.3, 45.0, 39.7,
assignment was based on HMQC and HMBC experiments), 46.1, 40.0, 36.3, 28.5, 13.6; HRMS (FAB) calcd for C15H22N5O7S (MH+) 416.1240,
36.6, 29.2, 20.9, 13.7; HRMS (FAB) calcd for C19H28N5O5 (MH+) found 416.1252. The data are identical to those previously reported.1,2
406.2090, found 406.2082.
A similar reaction with 10 equiv of SO3‚DMF gave a mixture of
The data for 33 were determined from the mixture: 1H NMR (CD3-
bis-sulfate ester 36 and 1. Reverse phase flash chromatography as above
OD) 6.67 (s, 1), 5.14 (br s, 1), 4.60 (d, 1, J ) 4.9), 3.99 (s, 3), 3.98 (s,
gave 1 preceded by bis-sulfate ester 36 as the pyridinium salt: 1H NMR
3), 4.08-4.00 (m, 1), 3.86 (dd, 1, J ) 8.5, 8.5), 3.81 (ddd, 1, J )
(D2O) (600 MHz) 5.83 (s, 1), 5.20 (d, 1, J ) 4.3), 4.63-4.60 (m, 1),
10.0, 10.0, 8.5), 3.80-3.70 (m, 1), 3.26 (dd, 1, J ) 10.0, 8.5), 2.34-
3.99 (ddd, 1, J ) 12.2, 4.3, 4.0), 3.85 (dd, 1, J ) 9.2, 8.5), 3.73 (ddd,
2.26 (m, 1), 2.20 (ddd, 1, J ) 14.0, 3.6, 3.6), 2.13 (s, 3), 1.90-1.83
1, J ) 11.0, 10.8, 8.5), 3.72-3.64 (m, 1), 3.27 (dd, 1, J ) 11.0, 9.2),
(m, 1), 1.62 (ddd, 1, J ) 14.0, 10.4, 5.5), 1.48 (ddd, 1, J ) 12.2, 12.2,
2.46 (ddd, 1, J ) 14.7, 2.5, 2.5), 2.28 (ddd, 1, J ) 13.4, 4.0, 3.5),
2.4), 0.96 (d, 3, J ) 6.8); 13C NMR (CD3OD) (from an HMQC
1.90-1.84 (m, 1), 1.64 (ddd, 1, J ) 13.4, 12.2, 11.6), 1.56 (ddd, 1, J
experiment) 99.8, 75.7, 71.8, 59.1, 54.9, 54.8, 53.1, 49.0, 48.1, 38.9,
) 14.7, 12.0, 3.0), 0.99 (d, 3, J ) 6.8).
36.5, 27.9, 20.7, 13.3.
Formation of 34. A solution of 32 (2.5 mg, 0.004 mmol) in
concentrated HCl (1 mL) was refluxed at 100 °C for 6 h. The mixture Acknowledgment. We are grateful to the National Institutes
was evaporated under reduced pressure to give 2.4 mg (95%) of 34 as of Health (GM-46470) for financial support. We thank Ms.
the hydrochloride salt: 1H NMR (D2O) 5.83 (s, 1), 4.78 (d, 1, J ) Christine Hofstetter for assistance in obtaining NMR data.
3.6), 4.03 (br s, 1), 3.88 (ddd, 1, J ) 11.6, 3.7, 3.6), 3.86 (dd, 1, J )
9.2, 8.8), 3.76 (ddd, 1, J ) 11.0, 11.0, 8.8), 3.62 (dddd, 1, J ) 11.8, Supporting Information Available: Experimental proce-
11.6, 3.7, 3.6), 3.27 (dd, 1, J ) 11.0, 9.2), 2.15 (ddd, 1, J ) 13.4, 3.7, dures for preparation of 8 and 12 and copies of 1H and 13C
3.7), 2.10 (ddd, 1, J ) 14.7, 3.6, 3.0), 1.73 (ddq, 1, J ) 11.0, 1.9, 7.2), NMR spectra of key intermediates (PDF). This material is
1.59 (ddd, 1, J ) 13.4, 11.6, 11.6), 1.54 (ddd, 1, J ) 14.7, 11.8, 2.5), available free of charge via the Internet at http://pubs.acs.org.
0.97 (d, 3, J ) 7.2); 13C NMR (D2O) 169.1 (assignment was based on
HMBC), 158.0, 157.8, 153.3 (assignment was based on HMBC), 101.5, JA000647J