Total synthesis of Amaryllidaceae alkaloids utilizing sequential intramolecular heterocyclic azadiene Diels-Alder reactions of an unsymmetrical 1,2,4,5-tetrazine

Article abstract of DOI:10.1021/jo0012546

Convergent total syntheses of anhydrolycorinone, hippadine, and anhydrolycorinium chloride are detailed, enlisting sequential inverse electron demand Diels-Alder reactions of an unsymmetrical N-acyl-6-amino-1,2,4,5-tetrazine.

Full text of DOI:10.1021/jo0012546

9
120
J . Org. Chem. 2000, 65, 9120-9124
Tota l Syn th esis of Am a r yllid a cea e Alk a loid s Utilizin g Sequ en tia l
In tr a m olecu la r Heter ocyclic Aza d ien e Diels-Ald er Rea ction s of a n
Un sym m etr ica l 1,2,4,5-Tetr a zin e
Dale L. Boger* and Scott E. Wolkenberg
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
1
0550 North Torrey Pines Road, La J olla, California 92037
boger@scripps.edu
Received August 17, 2000
Convergent total syntheses of anhydrolycorinone, hippadine, and anhydrolycorinium chloride are
detailed, enlisting sequential inverse electron demand Diels-Alder reactions of an unsymmetrical
N-acyl-6-amino-1,2,4,5-tetrazine.
Lycorine alkaloids, isolated from Amaryllidaceae plants,
are characterized by their pyrrolophenathridine skeleton
1
and potent biological activity. For instance, hippadine
2
(
1) inhibits fertility in male mice, while kalbretorine (2),
ungeremine (3), and anhydrolycorinium chloride (4)
3
4
exhibit cytotoxic activity against a number of tumor cell
5
lines (Figure 1). Anhydrolycorinone (5), itself a natural
product, serves as an advanced intermediate for the total
synthesis of hippadine and anhydrolycorinium chloride,
both of which can be generated from anhydrolycorinone
in one or two steps.5
F igu r e 1.
reactions of an unsymmetrical 1,2,4,5-tetrazine in a
tetrazine f diazine f benzene strategy. The intramo-
,6
8
Herein, we describe a concise synthesis of 1, 4, and 5
complementary to efforts described to date.6 Central to
the approach is the implementation of two sequential
intramolecular inverse electron demand Diels-Alder
lecular Diels-Alder reactions of N-tethered dienophiles
,7
9
with both 6-acyl(amino)-1,2,4,5-tetrazines and 6-acyl-
(amino)-1,2-diazines8 are well-precedented, and it has
,10
been shown that N-acylation of 6-amino-3-(methylthio)-
1
,2,4,5-tetrazine accelerates its rate of participation in a
(
1) Chattopadhyay, S.; Chattopadhyay, U.; Marthur, P. P.; Saini,
K. S.; Ghosal, S. Planta Med. 1983, 49, 252.
2) Ghosal, S.; Lochan, R.; Ashutosh, R.; Kumar, Y.; Srivastara, R.
S. Phytochemistry 1985, 24, 1825.
3) Zee-Cheng, R. K. Y.; Yan, S.-J .; Cheng, C. C. J . Med. Chem. 1978,
1, 199.
4) Pettit, G. R.; Gaddamidi, V.; Goswami, A.; Cragg, G. M. J . Nat.
Prod. 1984, 47, 796.
5) Goshal, S; Rao, P. H.; J aiswal, D. K.; Kumar, Y.; Frahm, A. W.
Phytochemistry 1981, 20, 2003.
6) Humber, L. C.; Kondo, H.; Kotera, K.; Takagi, S.; Takeda, K.;
LUMOdiene-controlled Diels-Alder reaction to the extent
that cycloaddition can be confidently expected to occur
at 25 °C.9
(
(
The approach enabled the exploration of two possible
orders for implementation of the key heteroaromatic
azadiene Diels-Alder reactions (Scheme 1). Both ap-
proaches in which either the fused five-membered or six-
membered ring may be introduced first through use of
an intramolecular tetrazine Diels-Alder reaction were
examined. The former proved straightforward, and prepa-
ration of suitably substituted unsymmetrical 1,2,4,5-
tetrazines from 3,6-bis(methylthio)-1,2,4,5-tetrazine (6),
followed by a tandem N-acylation/intramolecular Diels-
Alder reaction, acylation with a substituted piperonylic
acid, and a second intramolecular Diels-Alder reaction
furnished the pyrrolophenanthridine core.
2
(
(
(
Taylor, W. I.; Thomas, B. R.; Tsuda, Y.; Tsukamoto, K.; Uyeo, H.;
Yajima, H.; Yanaihara, N. J . J . Chem. Soc. 1954, 4622.
(7) Fales, H. M.; Guiffrida, L. D.; Wildman, W. C. J . Am. Chem.
Soc. 1956, 78, 4145. Cook, J . W.; Loudon, J . D.; McCloskey, P. J . J .
Chem. Soc. 1954, 4176. Bennington, F.; Morin, R. D. J . Org. Chem.
1
1
1
962, 27, 142. Olson, D. R.; Wheeler, W. J .; Wells, J . N. J . Med. Chem.
974, 17, 167. Ghosal, S.; Saini, K. S.; Frahm, A. W. Phytochemistry
983, 22, 2305. Carruthers, W.; Evans, N. J . Chem. Soc. 1974, 1523.
Onaka, T.; Kanda, Y.; Natsume, M. Tetrahedron Lett. 1974, 1179.
Black, D. St. C.; Keller, P. A.; Kumar, N. Tetrahedron 1993, 49, 151.
Black, D. St. C.; Keller, P. A.; Kumar, N. Tetrahedron Lett. 1989, 30,
5
807. Snieckus, V.; Siddiqui, M. A. Tetrahedron Lett. 1990, 31, 1523.
Tota l Syn th esis of An h yd r olycor in on e, An h yd r o-
lycor in iu m Ch lor id e, a n d H ip p a d in e. 2-Bromo-
piperonal was protected as its diethyl ketal [HC(OEt) ,
3
Grigg, R.; Teasdale, A.; Sridharan, V. Tetrahedron Lett. 1991, 32, 3859.
Sakamoto, T.; Yasuhara, A.; Kondo, Y.; Yamanaka, H. Heterocycles
1
1
Lett. 1990, 31, 2331. Kanematsu, K.; Yasukouchi, T.; Hayakawa, K.
Tetrahedron Lett. 1987, 28, 5895. Prabhakar, S.; Lobo, A. M.; Marques,
M. M. J . Chem. Res. 1987, 167. Boeckman, R. K., J r.; Goldstein, S.
W.; Walters, M. A. J . Am. Chem. Soc. 1988, 110, 8250. Parnes, J . S.;
Carter, D. S.; Kurz, L. J .; Flippin, L. A. J . Org. Chem. 1994, 59, 3497.
Iwao, M.; Takehara, H.; Obata, S.; Watanabe, M. Heterocycles 1994,
993, 36, 2597. Castedo, L.; Guitian, E.; Perez, D. Tetrahedron Lett.
992, 33, 2407. Castedo, L.; Guitian, E.; Meiras, D. P. Tetrahedron
p-TsOH, EtOH, 80 °C, 24 h] and converted to the known
11
aldehyde 7 via lithium-halogen exchange (n-BuLi,
(8) Boger, D. L.; Coleman, R. S. J . Org. Chem. 1986, 51, 3250. Boger,
D. L.; Coleman, R. S. J . Am. Chem. Soc. 1987, 109, 2717. Boger, D. L.;
Zhang, M. J . Am. Chem. Soc. 1991, 113, 4230.
3
8, 1717. Hutchings, R.; Meyers, A. I. J . Org. Chem. 1996, 61, 1004.
(9) Boger, D. L.; Schaum, R. P.; Garbaccio, R. M. J . Org. Chem. 1998,
63, 6329.
(10) Boger, D. L.; Coleman, R. S. J . Org. Chem. 1984, 49, 2240.
Boger, D. L.; Sakya, S. M. J . Org. Chem. 1988, 53, 1415.
(11) Dallacker, F.; Schleuter, H. J .; Schneider, P. Z. Naturforsch.,
B. 1986, 41B, 1273.
Shao, H. W.; Cai, J . C. Chin. Chem. Lett. 1996, 7, 13. Perez, D.; Bures,
G.; Guitian, E.; Castedo, L. J . Org. Chem. 1996, 61, 1650. Tsuge, O.;
Hatta, T.; Tsuchiyama, H. Chem. Lett. 1998, 2, 155. Padwa, A.;
Brodney, M. A.; Liu, B.; Satake, K.; Wu, T. J . Org. Chem. 1999, 64,
3
595.
1
0.1021/jo0012546 CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/28/2000

Total Synthesis of Amaryllidaceae Alkaloids
J . Org. Chem., Vol. 65, No. 26, 2000 9121
Sch em e 1
(98%), conversion of 9 to the carboxylic acid 11 was
accomplished via Wittig reaction (2.0 equiv of CH
PPh , THF, -78 to 25 °C, 3.5 h, 63%, 1:1 cis:trans) and
subsequent ester hydrolysis (LiOH, THF/CH OH/H
:1:1, 55 °C, 18 h, 100%). Acidification of the hydrolysis
3
OCHd
3
3
2
O
3
reaction was carried out at 0 °C to prevent a facile
cyclization13 to form 12 (eq 1).
14
The substrate 13 for the key inverse electron demand
Diels-Alder reactions was prepared by clean, selective
displacement of one of the methylthio groups from 3,6-
9
,10,15
bis(methylthio)-1,2,4,5-tetrazine (6)
-butyne16 (CH
OH, Et N, 51%). The first intramolecular
4 + 2] cycloaddition was accomplished at room temper-
ature in superb conversion upon N-acylation of 13 with
with 1-amino-
3
[
3
3
9
2
BOC O and catalytic DMAP (THF, 23 h, 96%), and the
intermediate N-BOC derivative prior to cyclization was
not detected. The resulting 1,2-diazine 14 was N-BOC-
deprotected (4 M HCl/EtOAc, 2 h, 25 °C, 100%) and used
directly in the coupling reaction with 11.
Sch em e 2
The ease with which 11 cyclized to 12, even in the
absence of an acid catalyst, presented an impediment to
the preparation of 16, which could be overcome by the
addition of excess i-Pr
equiv of HOBt, 10 equiv of i-Pr
C, 68%). This set the stage for the second key intra-
molecular [4 + 2] cycloaddition and subsequent aroma-
2
NEt and HOBt (2 equiv of EDCI,
2
2
NEt, DMF, 24 h, 25
°
(
12) Corey, E. J .; Gilman, N. W.; Ganem, B. E. J . Am. Chem. Soc.
968, 90, 5616.
13) This acid-catalyzed cyclization has been observed for the benzoic
1
(
acid analogue of 11. See: Kirby, A. J .; Williams, N. H. J . Chem. Soc.,
Perkin Trans. 2 1994, 643. For 7-methoxy-7,8-dihydro[1,3]dioxolo[4,5-
1
g]isochromen-5-one (12): H NMR (CDCl
6
3
, 400 MHz) δ 7.49 (s, 1H),
.66 (s, 1H), 6.03 (d, J ) 1.2 Hz, 1 H), 6.02 (d, J ) 1.2 Hz, 1H), 5.39 (t,
J ) 3.8 Hz, 1H), 3.55 (s, 3H), 3.21 (dd, J ) 3.5 Hz, J ) 18.2 Hz, 1H),
2
2
.98 (dd, J ) 3.8 Hz, J ) 16.4 Hz, 1H); MALDIFTMS (DHB) m/z
+
23.0598 (C11
14) Similar cyclizations prevented the preparation of the o-ethynyl
and o-dibromovinyl acids by producing i and ii, respectively. For
10 5
H O + H requires 223.0601).
(
1
6
-acetylbenzo[1,3]dioxole-5-carboxylic acid (i): H NMR (CD
3
OD, 250
MHz) δ 7.10 (s, 1H), 7.03 (s, 1H), 6.14 (s, 2H), 1.78 (s, 3H); FABHRMS
+
(
NBA/NaI) m/z 231.0267 (C10
8 5
H O + Na requires 231.0269). For
7
-bromomethylene-7H-furo[3′,4′:4,5]benzo[1,2-d][1,3]dioxol-5-one (ii):
1
H NMR (CDCl
3
, 500 MHz) δ 7.17 (s, 1H), 6.93 (s, 1H), 6.15 (s, 2H),
.14 (s, 1H); MALDIFTMS (DHB) m/z 268.9451 (C10 BrO requires
68.9449).
6
2
H
5
4
Et
2
O, -30 °C, 15 min) and subsequent DMF trap (-78
°
C to 25 °C, 18 h) (Scheme 2). Aldehyde 7 was trans-
(
15) Reviews: Boger, D. L. Chemtracts: Org. Chem. 1996, 9, 149.
formed to the corresponding methyl ester 8 by oxidation
according to a modification of the Corey-Ganem proce-
Boger, D. L.; Weinreb, S. M. Hetero Diels-Alder Methodology in
Organic Synthesis; Academic: San Diego, 1987. Boger, D. L. Chem.
Rev. 1986, 86, 781. Boger, D. L. Tetrahedron 1983, 39, 2869.
dure12 (NaCN, MnO
, CH
lowing mild deprotection of the ketal 8 with p-TsOH
2
3
OH, 25 °C, 18 h, 61%). Fol-
(16) Koziara, A.; Osowska-Pacewicka, K.; Zawadzki, S.; Zwierzak,
A. Synthesis 1985, 202.

9
122 J . Org. Chem., Vol. 65, No. 26, 2000
Boger and Wolkenberg
Sch em e 3
20 and 22 to the corresponding sulfones 23 and 24
(
[
m-CPBA, -78 to 25 °C, 79-94%) did not improve their
4 + 2] cycloaddition reactivity. This lack of reactivity is
due to the intrinsically poor reactivity of aromatic 1,2-
diazines toward Diels-Alder reactions, especially in
comparison to the successful, closely analogous pyrone
system of Castedo et al.7
Con clu sion s
A convergent total synthesis of anhydrolycorinone,
anhydrolycorinium chloride, and hippadine was described
enlisting a tetrazine f diazine f benzene strategy
featuring two sequential intramolecular [4 + 2] cyclo-
addition reactions of an unsymmetrical 6-(acylamino)-
1
,2,4,5-tetrazine.17
Exp er im en ta l Section
Met h yl 6-(Diet h oxym et h yl)b en zo[1,3]d ioxole-5-ca r -
boxyla te (8). A solution of 711 (5.2 g, 20.6 mmol) in 200 mL
of anhydrous CH OH was treated with NaCN (5.05 g, 103
3
tization via elimination of methanol, which was ac-
complished in superb yield to provide the anhydroly-
corinone precursor 17 (N,N-diethylaniline, 265 °C, 20 h,
mmol) and the mixture was stirred at 25 °C until all solid had
dissolved (15 min). The reaction mixture was then treated with
activated MnO (27 g, 309 mmol) and stirred at 25 °C for 24 h
2
before being filtered through Celite and concentrated under
reduced pressure. The resulting residue was dissolved in
1
00%). Reductive desulfurization with Raney nickel
provided anhydrolycorinone (5, EtOH, 25 °C, 18 h, 97%),
CH
2
Cl
2
(100 mL) and washed with H
2
O (100 mL). The organic
5
which was subsequently converted to hippadine (1, DDQ,
extract was dried (Na
2
SO ), concentrated under reduced
4
6
8
0 °C, 24 h, 65%) and anhydrolycorinium chloride (4,
pressure, and subjected to flash chromatography (SiO , 4.5 ×
2
Red-Al, 25 °C, 2 h; O
2
, HCl/EtOH, 25 °C, 24 h, 82%). The
18 cm, 15% EtOAc/hexanes) to afford 8 (4.03 g, 61%) as a
1
colorless oil: H NMR (CDCl
3
, 400 MHz) δ 7.30 (s, 1H), 7.27 (s,
synthetic samples of 1, 4, and 5 were identical in all
1
13
1H), 6.17 (s, 1H), 6.00 (s, 2H), 3.85 (s, 3H), 3.74-3.48 (m, 4H),
respects ( H and C NMR, IR, mp) with properties
13
reported for authentic materials.1,4,5
1.21 (t, J ) 7.0 Hz, 6H); C NMR (CDCl
3
, 100 MHz) δ 167.0,
1
(
50.6, 147.1, 137.1, 123.1, 110.0, 107.3, 101.8, 99.0, 62.9, 52.1
Sequ en ce of Aza d ien e Diels-Ald er Rea ction s.
Complementary to these studies in which the five-
membered ring of the pyrrolophenanthridine system was
formed first, a series of approaches in which the six-
membered ring was formed first were also examined. In
these approaches, the aminotetrazines 13, 18, and 19
were coupled directly to 11 (Scheme 3). The aminotetra-
zines 13 and 18 were produced upon direct displacement
of one methylthio group of 3,6-bis(methylthio)-1,2,4,5-
-1
2C), 15.1 (2C); IR (film) νmax 2975, 1720, 1487, 1281 cm .
Anal. Calcd for C14
H, 6.71.
18 6
H O : C, 59.57; H, 6.43. Found: C, 59.71;
Meth yl 6-F or m ylben zo[1,3]d ioxole-5-ca r boxyla te (9).
A solution of 8 (4.03 g, 14.3 mmol) in 100 mL of CH Cl was
treated with p-TsOH (280 mg, 1.4 mmol) and H O (10 mL),
and the mixture was stirred at 25 °C for 18 h. The reaction
mixture was diluted with H O (90 mL) and extracted with
CH Cl
(3 × 100 mL). The organic extracts were dried (Na
SO ) and concentrated under reduced pressure. Flash chro-
matography (SiO Cl ) provided 9 (2.9 g, 98%)
, 6.5 × 15 cm, CH
as a white solid: mp 112-113 °C; H NMR (CDCl , 500 MHz)
δ 10.53 (s, 1H), 7.42 (s, 1H), 7.38 (s, 1H), 6.12 (s, 2H), 3.94 (s,
2
2
2
2
2
2
2
-
4
tetrazine (6) (CH
produced in 51% overall yield by methylthio group
displacement with 3-amino-1-propanol (CH OH, 25 °C),
oxidation to the corresponding aldehyde (PCC, CH Cl
5 °C, 5 h), and Wittig olefination (ClCHdPPh , NHMDS,
78 to 25 °C, 3 h). As expected, acylation with 11 was
3
OH, 25 °C). The tetrazine 19 was
2
2
1
2
3
3
3
1
1
H); 13C NMR (CDCl
34.1, 128.8, 110.6, 107.9, 103.1, 53.1; IR (film) νmax 2919, 1714,
3
, 125 MHz) δ 190.8, 166.3, 151.8, 151.5,
2
2
,
2
3
-1
493, 1283 cm ; MALDIFTMS (DHB) m/z 209.0447 (C10
8 5
H O
-
+
+
H
requires 209.0450).
accompanied by room temperature [4 + 2] cycloadditions
and with the enol ether serving as the exclusive dieno-
phile. Thus, [4 + 2] cycloaddition reaction with closure
of the fused six-membered ring was observed in prefer-
ence to the fused five-membered ring presumably dic-
tated by the electron-rich character of the competing
dienophiles. The modest conversions proved to be a
consequence of a poor N-acylation reaction of 11 with 13
and 18-19 (75-95% recovered tetrazine), with the
subsequent in situ cycloaddition proceeding without
detection of the intermediate N-acylaminotetrazine. The
resulting 1,2-diazines 20-22 were subjected to thermal
conditions to induce a second intramolecular Diels-Alder
reaction. Despite the use of a range of solvents (1,3,5-
triisopropylbenzene, 2-chloronaphthalene, o-dichloroben-
zene, N,N-diethylaniline), high temperatures (150-280
Meth yl 6-(2-Meth oxyvin yl)ben zo[1,3]d ioxole-5-ca r box-
yla te (10). A solution of methoxymethyltriphenylphosphonium
chloride (362 mg, 1.06 mmol) in 2.0 mL of anhydrous THF at
-78 °C was treated with t-BuLi (0.76 mL of a 1.26 M solution
in pentane, 0.96 mmol) dropwise and stirred for 20 min. The
resulting deep red solution was warmed to -40 °C for 30 min
and then cooled to -78 °C before 9 (100 mg as a solution in
2
.0 mL THF, 0.48 mmol) was added dropwise. The reaction
mixture was warmed to 25 °C and stirred for 15 h. The reaction
mixture was quenched with the addition of H O (20 mL) and
extracted with EtOAc (3 × 25 mL). The combined organic
extracts were dried (Na SO ), concentrated under reduced
pressure, and subjected to flash chromatography (SiO
, 4 ×
4 cm, 10% EtOAc/hexanes) to provide 10 as a 1:1 mixture of
cis and trans isomers (71 mg, 63%) as an off-white solid: mp
2
2
4
2
1
1
6
1-63 °C; H NMR (CDCl , 500 MHz) cis, δ 7.66 (s, 1H), 7.41
3
(
(
s, 1H), 6.24 (d, J ) 7.3 Hz, 1H), 6.22 (d, J ) 7.3 Hz, 1H), 6.05
s, 2H), 3.91 (s, 3H), 3.82 (s, 3H); trans, δ 7.46 (s, 1H), 6.97 (d,
°
C), and prolonged reaction times (2-30 h), only recov-
ered starting materials and small amounts of the corre-
sponding sulfoxides were obtained. Oxidation of diazines
(
17) L1210 IC50 values: 1, >100 µM; 4, 0.9 µM; 5, 23 µM; 17, 9 µM;
16, 16 µM.

Total Synthesis of Amaryllidaceae Alkaloids
J . Org. Chem., Vol. 65, No. 26, 2000 9123
J ) 13.2 Hz, 1H), 6.91 (s, 1H), 6.89 (d, J ) 12.9 Hz, 1H), 6.05
4.19 (t, J ) 8.1 Hz, 2H), 3.51 (s, 3H), 3.07 (t, J ) 8.5 Hz, 2H),
2.60 (s, 3H); cis, δ 7.56 (s, 1H), 6.78 (s, 1H), 6.75 (s, 1H), 5.96
(s, 2H), 5.95 (d, J ) 7.4 Hz, 1H), 5.21 (d, J ) 7.4 Hz, 1H), 4.17
(t, J ) 8.1 Hz, 2H), 3.67 (s, 3H), 3.07 (t, J ) 8.5 Hz, 2H), 2.60
1
3
(
3
s, 2H), 3.92 (s, 3H), 3.78 (s, 3H); C NMR (CDCl , 125 MHz)
δ 167.7 and 167.5, 151.4 and 150.6, 150.3 and 148.5, 146.1
and 145.6, 135.4 and 133.5, 121.6 and 120.7, 110.8 and 110.5,
1
5
10.4 and 106.1, 104.7 and 103.2, 102.0 and 101.9, 61.0 and
(s, 3H); 13C NMR (CDCl
3
, 125 MHz) δ 168.9 (2C), 157.7 and
6.7, 52.2 and 52.1; IR (film) νmax 2950, 1710, 1633, 1484, 1293
157.6, 157.5 and 157.4, 150.2 and 149.8, 149.1 and 148.3, 146.4
and 145.8, 133.3 and 133.1, 129.5 and 128.6, 128.4 and 127.7,
123.2 (2C), 110.1 and 108.4, 107.7 and 106.4, 102.9 and 102.3,
101.8 and 101.7, 61.0 and 57.1, 47.0 (2C), 24.9 (2C), 14.0 (2C);
-
1
+
cm ; MALDIFTMS (DHB) m/z 237.0759 (C12
requires 237.0763).
12 5
H O + H
6
-(2-Meth oxyvin yl)ben zo[1,3]dioxole-5-car boxylic Acid
11). A solution of 10 (63 mg, 0.27 mmol) in 1.0 mL of 3:1:1
THF/CH OH/H O was treated with LiOH (57 mg, 1.35 mmol)
and stirred at 55 °C for 8 h. The reaction mixture was
concentrated under reduced pressure, diluted with H O (1.0
mL), and washed with Et O (1.0 mL). The aqueous phase was
cooled to 0 °C, acidified with 3 M aqueous HCl, and quickly
extracted with Et
O (3 × 1.5 mL). The combined organic
extracts were dried (Na SO ) and concentrated under reduced
-1
(
IR (film) νmax 2923, 1636, 1412, 1250, 1037 cm ; MALDIFTMS
+
3
2
17 3 4
(DHB) m/z 394.0844 (C18H N O S + Na requires 394.0832).
2-Meth ylth io-4,5-dih ydr o[1,3]dioxolo[4,5-j]pyr r olo[3,2,1-
d e]p h en a n th r id in -7-on e (17). A solution of 16 (11 mg, 0.03
mmol) in degassed N,N-diethylaniline was warmed at 265 °C
2
2
in a sealed tube under N
cooled to 25 °C and loaded directly onto SiO
equilibrated in CH Cl . N,N-Diethylaniline was eluted with
, and the column was then eluted with 1% CH OH/
to afford 17 (9.4 mg, 100%) as a white solid: mp 218-
2
for 20 h. The reaction mixture was
2
2
(2.5 × 10 cm)
2
4
2
2
pressure to provide pure 11 (60 mg, 100%) as an unstable
white solid which was used immediately in subsequent reac-
CH
CH
2
2
Cl
Cl
2
2
3
1
1
tions: H NMR (CDCl
7
)
3
, 400 MHz) δ 7.61 (s, 1H), 7.49 (s, 1H),
219 °C; H NMR (CDCl
7.53 (s, 1H), 7.29 (s, 1H), 6.13 (s, 2H), 4.48 (t, J ) 7.9 Hz, 2H),
, 125
3
, 400 MHz) δ 7.92 (s, 1H), 7.71 (s, 1H),
.47 (s, 1H), 6.91 (d, J ) 12.9 Hz, 1H), 6.85 (s, 1H), 6.84 (d, J
12.9 Hz, 1H), 6.25 (d, J ) 7.3 Hz, 1H), 6.19 (d, J ) 7.6 Hz,
3.41 (t, J ) 7.9 Hz, 2H), 2.55 (s, 3H); 13C NMR (CDCl
3
1
2
H), 3.76 (s, 3H), 3.71 (s, 3H); MALDIFTMS (DHB) m/z
MHz) δ 159.6, 152.3, 149.1, 138.4, 132.9, 132.2, 130.3, 125.0,
+
23.0598 (C11
H
10
O
5
+ H requires 223.0601).
123.7, 120.0, 117.4, 107.3, 102.5, 101.2, 47.1, 27.7, 18.6; IR
-
1
Bu t-3-yn yl-(6-m eth ylth io-1,2,4,5-tetr azin -3-yl)am in e (13).
(film) νmax 2913, 1641, 1610, 1492, 1390 cm ; MALDIFTMS
(DHB) m/z 312.0691 (C17H13NO S + H requires 312.0689).
3
1
5
+
A solution of 3,6-bis(thiomethyl)-1,2,4,5-tetrazine (6, 622 mg,
.6 mmol) in 15 mL of anhydrous CH OH at 25 °C was treated
with Et N (1.0 mL, 7.2 mmol) and 1-amino-3-butyne (460 mg,
.4 mmol). After the reaction mixture was stirred for 18 h,
the solvent was removed under reduced pressure and the
resulting oil was dissolved in CH Cl (30 mL), washed with
saturated aqueous NaHCO (30 mL), dried (Na SO ), and
concentrated under reduced pressure. Flash chromatography
SiO
, 4.5 × 15 cm, 10-15% EtOAc/hexanes gradient elution)
provided 13 (360 mg, 51%) as a bright red solid: mp 75-76
3
3
An h yd r olycor in -7-on e (5). A slurry of Raney nickel (110
mg wet, 50 wt equiv) and 17 (2.3 mg, 0.007 mmol) in 0.3 mL
of absolute EtOH was stirred vigorously at 25 °C for 18 h. The
Raney nickel was removed by filtration (EtOAc wash) through
Celite. The removal of solvents under reduced pressure and
1
6
3
4
2
2
3
2
4
PTLC (SiO
white solid: mp 257-259 °C (lit. mp 262-264 °C); H NMR
(CDCl , 500 MHz) δ 7.93 (s, 1H), 7.75 (d, J ) 8.1 Hz, 1H),
2 2 2
, 3% MeOH/CH Cl ) afforded 5 (1.8 mg, 97%) as a
5
1
(
2
3
7.56 (s, 1H), 7.29 (d, J ) 7.3 Hz, 1H), 7.19 (t, J ) 7.7 Hz, 1H),
6.12 (s, 2H), 4.48 (t, J ) 8.1 Hz, 2H), 3.43 (t, J ) 8.5 Hz, 2H);
1
°
C; H NMR (CDCl
.4, 6.4 Hz, 2H), 2.66 (s, 3H), 2.59 (dt, J ) 2.6, 6.4 Hz, 2H),
.05 (t, J ) 2.5 Hz, 1H); 13C NMR (CDCl
, 100 MHz) δ 167.5,
60.8, 80.9, 70.7, 39.9, 18.9, 13.6; IR (film) νmax 3298, 2928,
3
, 250 MHz) δ 5.83 (br s, 1H), 3.73 (dt, J )
1
3
6
2
1
2
1
3
C NMR (CDCl , 125 MHz) δ 160.0, 152.3, 148.9, 139.9, 131.3,
3
131.1, 124.3, 123.7, 123.6, 119.9, 117.2, 107.3, 102.5, 101.3,
-
1
47.0, 27.9; IR (film) νmax 2923, 1608, 1557, 1254 cm
;
-
1
+
855, 1699, 1574, 1435 cm ; FABHRMS (NBA/NaI) m/z
3
MALDIFTMS (DHB) m/z 266.0821 (C16H11NO + H requires
+
96.0656 (C
-ter t-Bu tyloxyca r bon yl-3-m eth ylth io-5,6-d ih yd r op y-
r r olo[2,3-c]p yr id a zin e (14). A solution of 13 (179 mg, 0.92
mmol) in 10 mL of anhydrous THF at 25 °C under N was
treated with BOC O (800 mg, 3.67 mmol) and DMAP (17 mg,
.14 mmol) and stirred for 24 h. After solvent was removed
under reduced pressure, the residue was subjected to flash
7
H
9
N
5
S + H requires 196.0657).
266.0812).
7
Hip p a d in e (1). According to the method of Ghosal et al.,5
a solution of anhydrolycorin-7-one (5, 2.1 mg, 0.007 mmol) and
DDQ (12 mg, 0.048 mmol) in 2.0 mL of anhydrous benzene
was stirred at 80 °C. After 24 h, the reaction mixture was
cooled to 25 °C, solvents were removed under reduced pressure,
2
2
0
and the residue was subjected to PTLC (SiO
CH Cl ) to afford 1 (1.3 mg, 65%) as a white solid: mp 218-
219 °C (lit. mp 217-218 °C); H NMR (CDCl , 500 MHz) δ
2 3
, 2.5% CH OH/
chromatography (SiO
gradient elution) to provide 14 (256 mg, 96%) as a tan oil:
2
, 2.5 × 11 cm, 20-40% EtOAc/hexanes
2
2
1
5
1
H
3
NMR (CDCl
2
NMR (CDCl
3
, 500 MHz) δ 7.00 (s, 1H), 3.98 (t, J ) 8.0 Hz,
8.04 (d, J ) 3.7 Hz, 1H), 7.99 (s, 1H), 7.92 (d, J ) 7.7 Hz, 1H),
7.75 (d, J ) 7.7 Hz, 1H), 7.66 (s, 1H), 7.47 (t, J ) 7.7 Hz, 1H),
H), 3.01 (t, J ) 8.0 Hz, 2H), 2.64 (s, 3H), 1.56 (s, 9H); 13
C
, 125 MHz) δ 157.7, 155.9, 150.8, 132.3, 122.4,
6.89 (d, J ) 3.7 Hz, 1H), 6.16 (s, 2H); 13C NMR (CDCl
3
, 125
3
8
1
2.2, 46.0, 28.2, 24.4, 13.5; IR (film) νmax 2977, 2927, 1728,
622, 1538, 1484 cm ; FABHRMS (NBA/NaI) m/z 290.0948
MHz) δ 158.4, 152.8, 148.8, 131.9, 131.2, 128.7, 124.2, 123.8,
-
1
122.8, 118.6 (2C), 117.0, 111.0, 108.3, 102.5, 102.0; IR (film)
+
-1
(
C
12
H
17
N
3
O
2
S + Na requires 290.0939).
-(2-Meth oxyvin yl)ben zo[1,3]dioxol-5-yl-(3-m eth ylth io-
,6-d ih yd r op yr r olo[2,3-c]p yr id a zin -7-yl)m eth a n on e (16).
νmax 2921, 1707, 1591, 1447 cm ; MALDIFTMS (DHB) m/z
+
6
9 3
264.0662 (C16H NO + H requires 264.0655).
5
An h yd r olycor in iu m Ch lor id e (4). According to a modi-
fication of the method of Humber et al.,6 a solution of
anhydrolycorin-7-one (5, 0.7 mg, 0.003 mmol) in 0.3 mL of
anhydrous toluene was treated with Red-Al (7 µL of a 65 wt
% solution in toluene, 0.02 mmol) and stirred at 25 °C under
A sample of 14 (11 mg, 0.048 mmol) was dissolved in 4.0 M
HCl/EtOAc (2.5 mL), and the solution was stirred for 2 h at
2
5 °C. The solvents were removed under reduced pressure, and
the residual salt 15 was dried thoroughly under high vacuum.
The salt was treated with a solution of EDCI (18 mg, 0.09
Ar. After 1.5 h, saturated aqueous NH
and the mixture was extracted with EtOAc (5 × 0.5 mL). The
combined organic extracts were dried (Na SO ) and the
solvents were removed under reduced pressure. The residue
was immediately dissolved in 0.5 mL EtOH and treated with
0.1 mL of concentrated HCl, and air was bubbled through the
solution for 18 h. The solvents were removed under reduced
pressure to afford 4 (0.7 mg, 82%) as a light yellow solid: dec
4
Cl (0.5 mL) was added
mmol), HOBt (12 mg, 0.09 mmol), i-Pr
and 11 in anhydrous DMF (1.0 mL), and the reaction was
stirred under N at 25 °C for 24 h. The reaction mixture was
diluted with CH OH (3.0 mL), and solvents were removed
under reduced pressure. The resulting residue was diluted
with H O (1.5 mL) and extracted with CH Cl
(3 × 1.5 mL).
The combined organic extracts were dried (Na SO ), concen-
trated under reduced pressure, and subjected to flash chro-
matography (SiO OH/CH Cl
, 1.5 × 12 cm, 0.5-2% CH
gradient elution) to provide 16 as a 2:1 mixture (trans:cis) of
2
NEt (79 µL, 0.45 mmol),
2
4
2
3
2
2
2
2
4
6
1
2
279-281 °C (lit. dec 280-285 °C); H NMR (D O, 500 MHz)
2
3
2
2
δ 9.33 (s, 1H), 8.22 (d, J ) 8.4 Hz, 1H), 7.95 (s, 1H), 7.84 (t, J
) 7.3 Hz, 1H), 7.77 (d, J ) 7.4 Hz, 1H), 7.58 (s, 1H), 6.33 (s,
1
13
isomers (11.3 mg, 68%) as a colorless oil: H NMR (CDCl
00 MHz) trans, δ 7.09 (s, 1H), 6.78 (s, 1H), 6.76 (s, 1H), 6.68
d, J ) 12.9 Hz, 1H), 5.96 (s, 2H), 5.81 (d, J ) 12.5 Hz, 1H),
3
,
2H), 5.13 (t, J ) 6.6 Hz, 2H), 3.72 (t, J ) 6.6 Hz, 2H);
C
5
(
NMR (D
2
O, 125 MHz) δ 156.7, 150.5, 144.4, 136.7, 136.6, 133.4,
131.5, 126.2, 123.7, 122.7, 120.2, 107.6, 104.4, 101.2, 60.0, 27.6;

9
124 J . Org. Chem., Vol. 65, No. 26, 2000
Boger and Wolkenberg
IR (film) νmax 3439, 1502, 1478, 1267, 1031, 925 cm-1;
(S.E.W.). We thank Michael P. Hedrick for conducting
cytotoxicity assays.
-
MALDIFTMS (DHB) m/z 250.0836 (C16
H
12ClNO
2
- Cl re-
quires 250.0836).
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
spectra of bromopiperonal diethyl ketal, 7-14, 16-24, 1, 4,
and 5 and experimental details for the preparation of 18-24
are provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
Ack n ow led gm en t. We gratefully acknowledge the
financial support of the National Institutes of Health
CA42056) and the Skaggs Institute for Chemical Biol-
ogy, as well as the Achievement Rewards for College
Scientists Foundation for a predoctoral fellowship
(
J O0012546