Synthetic studies of carzinophilin. Part 4: Chemical and biological properties of carzinophilin analogues

Article abstract of DOI:10.1016/S0040-4020(03)00380-6

Chemical and biological properties of carzinophilin congeners obtained in the course of our synthetic studies were investigated. These studies revealed feasibility for the use of some analogues as a double alkylating agent. Further, analogues carrying the

Full text of DOI:10.1016/S0040-4020(03)00380-6

Tetrahedron 59 (2003) 3089–3097
Synthetic studies of carzinophilin. Part 4: Chemical and biological
properties of carzinophilin analoguesq
†
Masaru Hashimoto, Miyoko Matsumoto, Kaoru Yamada and Shiro Terashima
,
*
*
Sagami Chemical Research Center, 2743-1, Hayakawa Ayase, Kanagawa 252-1193, Japan
Received 30 January 2003; accepted 6 March 2003
Abstract—Chemical and biological properties of carzinophilin congeners obtained in the course of our synthetic studies were investigated.
These studies revealed feasibility for the use of some analogues as a double alkylating agent. Further, analogues carrying the naphthalene
and the epoxide parts were found to show remarkable in vitro cytotoxicity and in vivo antitumor activity. q 2003 Elsevier Science Ltd. All
rights reserved.
1. Introduction
we have also made synthetic studies on 1,1 and reported
preparation of the protected form of 11e as well as its various
congeners.1a–d Our studies revealed that the 1-azabicyclo-
[3.1.0]hexane system, the central framework of 1, is
susceptible to nucleophilic attacks resulting in aziridine
ring-opening. We have further disclosed that some carzino-
philin-related compounds exhibit potent in vitro cyto-
toxicity against P388 murine leukemia in communication
forms.1a–c,e Now, we would like to describe full details of our
studies on chemical and biological properties of the
compounds prepared in this series of synthetic studies.
Antitumor antibiotic carzinophilin (¼azinomycin B2,3, 1)
isolated in 19544 has been known as one of the oldest DNA
intercalative bis-alkylating agents, before elucidation of its
structure.5 Interaction between 1 and DNA have been
reported by Armstrong,6,7 Saito,8 Coleman,9–11 Gates,12
Shipman13 and Imanishi.14 They mainly explored the
selectivity in DNA cleavage about the DNA sequence and
its denaturing patterns. Interstrand cross-linkage is believed
to occur by both aziridine and epoxide ring-openings
induced by attacks of bases in DNA. However, the reaction
mode in which 1 reacts with nucleophiles has been little
discussed.7,8 On the other hand, the unique structure and
strong antitumor activity of 1 attracted organic chemists
toward its total synthesis.15 Recently, Coleman achieved an
elegant total synthesis of azinomycin A,16 the natural
analogue of 1 lacking the C4 enol system. In the last decade,
2. Results and discussion
2.1. Chemical stability and reaction mode of the 1-aza-
bicyclo[3.1.0]hexane ring system against nucleophiles
It was found that diethyl (1-azabicyclo[3.1.0]hex-2-ylidene)-
malonate (ent-2) and ethyl (N-pivaloyl-1-azabicyclo[3.1.0]-
hex-2-ylidene)glycinate (ent-3) were quite susceptible to
nucleophilic attacks. Silica gel column chromatography
employed for the purification process decomposed ent-2
completely. Florisilw column chromatography performed
quickly only provided a pure sample of ent-2 in low yield
(34%) although the crude yield of ent-2 was estimated to
be around 80% in its preparation.1a The glycine derivative
ent-3 seemed to be slightly more stable than ent-2, because
Florisilw column chromatography afforded a pure sample in
a little higher yield (54%). The aziridine rings of both ent-2
and ent-3 were found to be cleaved easily at the C10
position (secondary carbon, carzinophilin numbering) by
treating with thiophenol in the presence of one equivalent of
triethylamine, giving phenylthiomethyl, derivatives, ent-4a
and ent-5a, respectively. Similar aziridine ring-opening
took place with an acetate anion under weakly acidic
q See Ref. 1a–e.
Keywords: antitumor activity; carzinophilin; anticancer activity; DNA
alkylation.
*
Corresponding authors. Tel./fax: þ81-172-35-0869;
e-mail: hmasaru@cc.hirosaki-u.ac.jp; terashima@sagami.or.jp
Present address: Department of Agriculture and Life Science, Hirosaki
University, 3 Bunkyo-Cho, Hirosaki 036-8561, Japan.
†
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00380-6

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M. Hashimoto et al. / Tetrahedron 59 (2003) 3089–3097
We have also succeeded in synthesizing 8, the model
compound further carrying an epoxide function in the
molecule as well as 13-O-desacetyl-12,13-di-O-benzyl-4-
O-methylcarzinophilin 9. These were found to be more
stable than the simple models 2 and 3. For example, 8 and 9
were readily purified by silica gel column or thin-layer
chromatography, which conditions had decomposed almost
all of 2 and 3. However, treatment of 8 with thiophenol took
place the aziridine ring opening to give the mono-adduct 10
smoothly. Interestingly, the epoxide moiety of 10 was also
cleaved gradually by thiophenol, providing the bis-adduct
11 in 76% yield after 11 hours. Under those conditions,
amides 12 and 13 were obtained as minor products.
Formation of 12 and 13 is tentatively considered to be
induced by removal of N16H and following hydrolysis as
shown for I. However, direct hydrolysis of the C7N16
enamine in 8 might be also possible. Similarly, the
O-protected carzinophilin derivative 9 also underwent the
same reaction to give the bis-adduct 14 in 63% yield.
Michael addition of thiophenol to the C4 position of 14 was
also conceivable, and subsequent elimination of the
methoxy group may give rise to the tris-adduct 15.
However, 15 was not detected so far as we examined.
These observations may mimic a putative double-linking
property of DNA with 1 although thiophenol was employed
as the nucleophile in place of nucleotides (Scheme 2).
Scheme 1. Reactions of the 1-azabicyclo[3.1.0]hexane derivatives 2 and 3
with nucleophiles. Reagents and conditions: (a) PhSH, Et3N, THF, rt, 62%
(4a), 67% (5a). (b) AcOH, THF, rt, 74% (4b), 86% (5b), MeOH, rt, 80%
(4c), 68% (5c).
conditions. In those reactions, nucleophilic attack to the C11
position (tertiary carbon, azinomycin numbering), giving
the piperidine derivatives 6 or 7 was not observed at all.
These bicyclic compounds 2 and 3 were not stable enough
even under neutral conditions. Pure samples of both 2 and 3
gradually decomposed in MeOH giving 5-methoxymethyl-
pyrrolidine derivatives, ent-4c and ent-5c in good yields
after standing for several days. The structures of ent-4a–c
were confirmed by observing the fragment ion peaks
assigned as [M2RCH2]þ (R¼PhS, AcO, or MeO) at
m/z¼226 with relatively strong intensity (ent-4a:11%, ent-
2.2. In vitro cytotoxicity of carzinophilin analogues
synthesized in our synthetic studies
We also aimed to develop a novel anticancer drug from the
compounds obtained in the series of synthetic studies on 1.
Thus, 51 related compounds were subjected to cytotoxicity
assay employing P388 murine leukemia in vitro. The values
of IC50 are summarized in Table 1. Adriamycin was
1
4b:4.1%, ent-4c:66%). The H NMR spectra of 4a–c also
supported the existence of the pyrrolidine rings. Similar
observations on ent-5a–c proved their structures as depicted
(Scheme 1).
Scheme 2. Reactions of thiophenol with highly functional analogues 8 and 9.

M. Hashimoto et al. / Tetrahedron 59 (2003) 3089–3097
3091
Table 1. IC50 Values of carzinophilin related analogues against P388 murine leukemia in vitro (mg/mL)
employed as a reference compound showing the IC50 to be
1–3£1023 mg/mL. Most of the monocyclic malonyl-
idenepyrrolidines 16–19 and their enantiomers ent-16–
ent-19 showed no cytotoxicity, however, bromide or iodide
with dibenzyl ester 18b, ent-18b, 19b, and ent-19b
exhibited weak activity (18b:0.84. ent-18b:2.7, 19b:2.4,
ent-19b:5.4). Enantiomers carrying 11R configuration
showed a tendency to exhibit a little stronger activity than
those of the 11S-isomers. Both enantiomers of bicyclic
malonylidene diethyl esters 2 and ent-2 showed weak
cytotoxicity, but neither enantiomer of dibenzyl ester 20 nor
ent-20 had cytotoxicity. Although the bicyclic models with
simple glycinate ester groups (3, ent-3, 21a, and ent-21a,b)
exhibited no cytotoxicity, replacement of the N-acyl group
with the C17–C21 unit significantly intensified the activity.
Especially, mesylates 23b and iso-23b inhibited increasing
of cell numbers as strongly as adriamycin17 which is now in
clinical usage. Bicyclic molecule iso-8, which carries the

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M. Hashimoto et al. / Tetrahedron 59 (2003) 3089–3097
Table 2. Antitumor activity against P388 leukemia in vivo
inactive constituent from the same culture broth as 1.2 The
amide 12 possesses the C20C21 epoxide but not the
aziridine moiety. Thus, 12 may act as a single alkylating
agent. Interestingly, sulfide 13, the analogue of 12 without
epoxide, also showed relatively strong cytotoxicity. The
naphthoate part seemed to be the most important for the
activity. But simple naphthoic acid 32 was inactive. Quite
recently, Coleman et al. also described the role of the left-
hand moiety of 1 in cross-link formation with DNA.11
Finally, the C1–C6 fragment 33 was found to show no
activity. As mentioned above, we have succeeded in finding
some congeners such as 12, 23b, and ent-23b to be
promising as an antitumor agent in the first screening.
However, we found those compounds except for 8, iso-8 and
30 bearing both the epoxide and aziridine moieties, can not
make double linked complex with DNA. Those cytotoxi-
cities might be caused by a different mechanism from that of
the double alkylation of DNA. Thus, it is difficult at this
stage to summarize the structure activity relationship from
those results.
Compound
Dose (mg/kg)
T/C (%)
12
40
20
10
109
109
146
23b
60
30
15
141
139
146
ent-23b
40
20
10
127
127
114
Table 3. Anticellular activity against human uterine cervix carcinoma Hela
S3 cells in vitro
Compounds
IC50 (mM)
1 h
72 h
12
23b
ADM
MMC
0.12
1.8
0.53
2.0
0.035
0.049
0.074
0.13
Congeners 12, 23b, and ent-23b, showing promising
cytotoxic activity were further subjected to in vivo
antitumor assay against P388 murine leukemia (Table 2).
These experiments revealed that 12 and 23b showed weak
antitumor activity. As for 12 and 23b, anticellular activity
was further determined employing Hela S3 cells in vitro. It
was found that that both of them showed strong activity
which is almost the same level as that of adriamycin (ADM)
and mitomycin C (MMC)18 as shown in Table 3. These
congeners 12 and 23b were further subjected to in vivo
antitumor activity against murine Sarcoma 180 employing
MMC as a reference (Table 4). Although amide 12 did not
show remarkable activity in this assay, carzinophilin
analogue 23b was found to shrink Sarcoma 180 more
significantly by administrating 60 mg/kg of 23b intrave-
nously on days 1 and 4 (run 11). This antitumor activity is
stronger than that of MMC (see run 12). However, these
experiments suggested that a large amount of 23b was
required to be administered for effective tumor degener-
ation. The amount is excessive from the viewpoint of use as
a medicinal drug. In other words, more than two grams of
23b must be taken once in four days based on the simple
calculations. On the other hand, body weight of the mice
Hela S3 cells (8£102/well) were cultured on day 0, and treated with the
compounds for 1 and 72 h from day 1. On day 4, the anticellular activity
was determined by the neutral red dye uptake method.
left-hand functionalities, also inhibited the cell number
growth. The naphthalene and the epoxide moieties seem
to be required for the enhanced cytotoxicity, because the
activity of benzoates 22a,b, iso-22a,b 24, and iso-24 was
found to be very weak. Functionalization at the C12 and
C13 positions did not enhance the cytotoxicity (see 25–27).
The analogues with C1–C21 carrying the naphthalene
moiety exhibited acceptable cytotoxicity although these
compounds did not have the aziridine ring in their molecules
(see 28–30). Based on our experiments, these congeners
may react with nucleophiles only at the C21 epoxide carbon.
Deleting the epoxide function decreased the cytotoxicity
remarkably as shown for 13 and 14. Our studies also
revealed that amide 12 exhibits cytotoxicity in a similar
level to that of adriamycin though it has been isolated as an
Table 4. Antitumor activity against murine Sarcoma 180 in vitro
Run
Compounds
Dose (mg/kg/day)
Schedule
Tumor size (mm3)
T/C
Body weighta (g)
Mortality
1
2
3
4
5
6
Control
12
0
1958^270
2074^275
1666^308
1751^460
1682^309
1524^197
1.00
1.06
0.85
0.89
0.86
0.78
þ7.6
þ7.3
þ5.1
þ7.2
þ6.1
þ4.7
0/5
0/5
0/5
0/5
0/5
0/5
1.3
2.5
5.0
10
Days 1, 4
Days 1, 4
Days 1, 4
Days 1, 4
Days 1, 4
20
7
8
9
10
11
23b
3.8
7.5
15
30
60
Days 1, 4
Days 1, 4
Days 1, 4
Days 1, 4
Days 1, 4
1788^645
1800^667
1818^409
1465^432
372^208
0.91
0.92
0.93
0.75
0.19
þ7.4
þ5.2
þ6.6
þ3.9
þ0.3
0/5
0/5
0/5
0/5
0/5
12
MMC
6.0
Day 1
648^179
0.33
þ2.8
0/5
Sarcoma 180 cells (5£106/mouse) were inoculated into male mice (body weight¼18–20 g) on day 0. Compounds are administrated intravenously following
the indicated schedule.
Body weight change between day 1 and day 7 in Sarcoma 180 bearing mice.
a

M. Hashimoto et al. / Tetrahedron 59 (2003) 3089–3097
3093
must increase during the experiments. Actually, mice
without treatment gained 7.6 g after a week (run 1 as
control). However, mice treated with 60 mg/kg of 23b
gained only 0.3 g of their weight. These observations
suggest that administering 23b resulted in health declines of
mice for some reasons including toxicity. Although we
could not succeed in developing an antitumor drug from
carzinophilin-related compounds, we found some molecules
which show strong cytotoxicity in vitro and antitumor
activity in vivo. We believe these studies provide founda-
tional knowledge for developing a novel antitumor drug
from carzinophilin congeners.
was identical to that of the authentic sample independently
prepared described below.
4.2.2. Authentic ent-4a. A solution of NaSPh in DMF
[prepared with NaH (3.0 mg, 125 mmol), and PhSH (20 mg,
181 mmol) in DMF (500 mL)] was added to a solution of
(S)-2-bis(ethoxycarbonyl)-methylidene-5-methanesulfoxy-
methylpyrrolidine1a (35.0 mg, 85.0 mmol) in DMF
(1.0 mL) at room temperature. After stirring at 1308C for
3 h, the mixture was poured into water and extracted with
Et2O. The combined ethereal extracts were washed with
brine, dried over MgSO4, then concentrated in vacuo.
Purification of the residue by silica gel column chroma-
tography (benzene/AcOEt¼90:10) gave ent-4a (25.9 mg,
87%) as a colourless caramel.
3. Conclusion
4.2.3. Reaction with AcOH giving (S)-2-bis(ethoxycarbo-
nyl)methylidene-5-acetoxymethylpyrrolidine (ent-4b). A
mixture of ent-2 (14.3 mg, 59.8 mmol) and AcOH (100 mL)
in CH2Cl2 (5.0 mL) was stirred at room temperature. After
stirring for 3 h, the mixture was concentrated in vacuo.
Purification of the residue by silica gel column chroma-
tography (CH2Cl2/acetone¼93:7) gave 4b (13.3 mg, 74%)
as a colourless oil. [a]D20¼þ43.88 (c 1.06, CHCl3). IR (film):
3300, 2970, 1740, 1690, 1645, 1570, 1440, 1370, 1250,
1080, 1040, 800 cm21. 1H NMR (400 MHz, CDCl3): d 1.22,
1.24 (each 3H, t, J¼7.1 Hz, CH3CH2O£2), 1.67 (1H, dddd,
J¼6.2, 6.8, 9.3, 13.1 Hz, C4HH), 2.03 (3H, s, CH3CO), 2.10
(1H, dddd, J¼5.8, 8.0, 9.4, 13.1 Hz, C4HH), 3.02 (1H, ddd,
J¼6.9, 9.4, 18.5 Hz, C3HH), 3.13 (1H, ddd, J¼5.8, 9.5,
18.5 Hz, C3HH), 3.93 (1H, dd, J¼7.6, 11.2 Hz, C5CHHO),
4.04 (1H, m, C5H), 4.11, 4.13 (each 2H, q, J¼7.1 Hz,
CH3CH2O£2), 4.16 (1H, dd, J¼3.9, 11.2 Hz, C5CHHO),
9.53 (1H, br s, NH). EI-MS (rel. int. %) m/z¼299 (2.7, Mþ),
254 (4.4, [M2EtO]þ), 239 (0.94, [M2AcOH]þ), 226 (4.1,
[M2AcOCH2]þ), 180 (100, [M2EtOH–AcOCH2]þ).
EI-HRMS calcd for C14H21NO6 (Mþ): m/z¼299.1369;
found m/z¼299.1338. The 1H NMR spectrum of this sample
was identical to that of the authentic sample independently
prepared described below.
We succeeded in finding several potent cytotoxic carzino-
philin congeners, which inhibit P388 cell increasing in
vitro among compounds obtained in our synthetic studies on
1. Especially, 12 and 23b also exhibited promising
antitumor activity against P388 murine leukemia in vivo.
Both 12 and 23b were also found to induce decline of
uterine cervix carcinoma Hela S3 in vitro. Further
investigation revealed 23b induces decline of Sarcoma
180 remarkably although it brings about some health
deterioration in mice.
4. Experimental
4.1. General
See General in the experimental part for Part 1 of this series
of papers. Compounds with large molecular weight
(MW.750) could not be subjected to high-resolution
mass spectroscopy.
4.2. Reactions of diethyl [(S)-azabicyclo[3.1.0]hex-2-
ylidene]malonate (ent-2)
4.2.4. Authentic ent-4b. A mixture of (S)-2-bis(ethoxy-
carbonyl)methylidene-5-hydroxymethylpyrrolidine1a (28 mg,
109 mmol), and Ac2O (100 mL, excess) in a mixture of
CH2Cl2 (1.0 mL) and pyridine (200 mL) was stirred at room
temperature for 12 h. After concentration in vacuo,
purification of the residue by silica gel column chroma-
tography (acetone/CH2Cl2¼7:93) gave ent-4b (30.5 mg,
93%) as a colourless oil. [a]2D0¼þ44.78 (c 1.07, CHCl3).
4.2.1. Reaction with PhSH giving (S)-2-bis(ethoxycarb-
onyl)methylidene-5-phenylthiomethylpyrrolidine (ent-
4a). A solution of ent-2 (2.1 mg, 8.8 mmol), PhSH
(10 mL), and Et3N (30 mL) in THF (200 mL) was stirred
at room temperature for 1 h, and the mixture was
concentrated in vacuo. Purification of the residue by
preparative silica gel TLC (benzene/AcOEt¼80:20) gave
thiophenol adduct ent-4a (1.9 mg, 62%) as a colourless
caramel. IR (film): 3300, 2970, 1645, 1565, 1435, 1280,
1250, 1080, 895, 840, 790 cm21 1H NMR (400 MHz,
.
4.2.5. Reaction with MeOH giving (S)-2-bis(ethoxy-
carbonyl)methylidene-5-methoxymethylpyrrolidine
(ent-4c). A solution of ent-2 (16.0 mg, 66.9 mmol) in
MeOH (1.0 mL) was stirred at room temperature for 7 days.
After concentration in vacuo, purification of the residue by
silica gel column chromatography (benzene/AcOEt¼90:10)
gave 4c (14.5 mg, 80%) as an oil. IR (film): 3300, 2970,
CDCl3): d 1.29, 1.30 (each 3H, t, J¼7.1 Hz, CH3CH2O£2),
1.78 (1H, dddd, J¼6.0, 6.8, 9.4, 12.9 Hz, C4HH), 2.21 (1H,
dddd, J¼5.7, 7.7, 9.1, 12.9 Hz, C4HH), 2.96 (1H, dd, J¼7.3,
13.4 Hz, C5CHHO), 3.05 (1H, ddd, J¼6.8, 9.5, 18.5 Hz,
C3HH), 3.07 (1H, dd, J¼5.8, 13.4 Hz, C5CHHO), 3.23 (1H,
ddd, J¼5.7, 9.4, 18.5 Hz, C3HH), 4.98 (1H, m, C5H), 4.18,
4.20 (each 2H, q, J¼7.1 Hz, CH3CH2O£2), 7.2–7.4 (5H, m,
aromatic protons), 9.67 (1H, br s, NH). EI-MS (rel. int. %)
m/z¼349 (2.1, Mþ), 304 (2.1, [M2EtO]þ), 226 (11,
[M2PhSCH2]þ), 180 (100, [M2EtOH–PhSOCH2]þ).
EI-HRMS calcd for C18H23NO4S (Mþ): m/z¼349.1349;
found m/z¼349.1343. The 1H NMR spectrum of this sample
1690, 1640, 1570, 1280, 1245, 1080 cm21 1H NMR
.
(200 MHz, CDCl3): d 1.28, 1.29 (each 3H, t, J¼7.1 Hz,
CH3CH2O£2), 1.68 (1H, dddd, J¼6.2, 7.0, 9.3, 12.8 Hz,
C4HH), 2.10 (1H, dddd, J¼5.7, 8.2, 8.7, 12.8 Hz, C4HH),
3.03 (1H, ddd, J¼7.3, 9.1, 18.4 Hz, C3HH), 3.20 (1H, ddd,
J¼5.7, 9.5, 18.4 Hz, C3HH), 3.25 (1H, dd, J¼7.6, 9.4 Hz,

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M. Hashimoto et al. / Tetrahedron 59 (2003) 3089–3097
C5CHHO), 3.35 (3H, s, CH3O), 3.42 (1H, dd, J¼4.2,
9.4 Hz, C5CHHO), 4.03 (1H, m, C5H), 4.15, 4.17 (each
2H, q, J¼7.1 Hz, CH3CH2O£2), 9.60 (1H, br s, NH).
EI-MS (rel. int. %) m/z¼271 (15, Mþ), 226 (66,
[M2MeOCH2]þ), 180 (100, M2EtOH–MeOCH2)þ).
EI-HIMS calcd for C13H21NO5: m/z¼271.1420; found
m/z¼271.1426.
CH3CH2O (Z-isomer)], 4.15 [1H£b, dd, J¼3.3, 11.0 Hz,
C11CHHO (Z-isomer)], 4.22 [1H£a, dd, J¼4.0, 11.0 Hz,
C11CHHO (E-isomer)], 5.95 [1H£b, br, NH (Z-isomer)],
6.45 [1H£a, br, NH (E-isomer)], 7.13 [1H£b, br, NH
(Z-isomer)], 8.03 [1H£a, br, NH (E-isomer)]. EI-MS (rel.
int. %) m/z¼326 (2.9, Mþ), 269 (11, [M2t-Bu]þ), 253 (12,
[M2AcOCH2]þ), 241 (61, [M2t-BuCO]þ), 57 (100, tBuþ).
The 1H NMR spectrum of this sample was identical to that
of the authentic sample independently prepared described
below.
4.3. Reaction of ethyl N-(pivaloyl-[(S)-1-azabicyclo-
[3.1.0]hex-2-ylidene]glycinate (ent-3)
4.3.1. Reaction with PhSH giving ethyl N-(pivaloyl-[(S)-
5-phenylthiomethylpyrrolidin-2-ylidene]glycinate (ent-
5a). The same treatments of ent-3 (8.7 mg, 32.8 mmol) as
described in Section 4.2.1 gave ent-5a (8.3 mg, 67%) as an
4.3.3. Authentic ent-5b. Treatment of ethyl N-pivaloyl [(S)-
5-hydroxymethylpyrrolidine-2-ylidene]glycinate1a (8.0 mg,
28.1 mmol) in the same manner as described in Section
4.2.4. ent-5b (8.0 mg, 87%) as an oil after silica gel column
chromatography (CH2Cl2/acetone¼88:12).
1
oil after silica gel column chromatography. The H NMR
spectrum of this sample showed that it consists of the two
tautomers arising from its enamine moiety (E/Z¼90:10).
Assignments of signals for the main isomer and some for the
4.3.4. Reaction with MeOH giving ethyl N-(pivaloyl-[(S)-
5-methoxymethylpyrrolidin-2-ylidene]glycinate (ent-5c).
Treatments of ent-3 (10.0 mg, 37 mmol) in the same manner
to that disclosed in Section 4.2.4 gave ent-5c (7.1 mg, 68%)
1
minor isomer are described. H NMR (400 MHz, CDCl3,
a¼0.90, b¼0.10, carzinophilin numbering): d 1.25 [3H£a,
t, J¼7.1 Hz, CH3CH2O (E-isomer)], 1.25 [9H£a, s,
(CH3)3CSi (E-isomer)], 1.28 [9H£b, s, (CH3)3CSi
(Z-isomer)], 1.72 [1H£a, dddd, J¼5.8, 7.2, 9.2, 13.0 Hz,
C12HH (major)], 1.80 [1H£b, m, C12HH (minor)], 2.14
[1H£a, dddd, J¼5.3, 7.3, 9.2, 13.0 Hz, C12HH (major)],
2.20 [1H£b, m, C12HH (minor)], 2.62 [1H£a, ddd, J¼7.3,
9.2, 17.2 Hz, C13HH (E-isomer)], 2.70 [1H£a, ddd, J¼5.8,
9.3, 17.2 Hz, C13HH (E-isomer)], 3.00 [2Hþ1H£b, m,
C11H2, C13HH (Z-isomer)], 3.19 [1H£b, ddd, J¼5.3, 9.0,
18.0 Hz, C13HH (Z-isomer)], 3.88 [1H£b, m, C11H
(Z-isomer)], 3.95 [1H£b, m, C11H (E-isomer)], 4.11
[2H£a, br q, J¼7.1 Hz, CH3CH2O (E-isomer)], 4.13
[2H£b, q, J¼7.1 Hz, CH3CH2O (Z-isomer)], 5.95 [1H£b,
br, NH (Z-isomer)], 6.46 [1H£a, br, NH (E-isomer)], 6.98
[1H£b, br, NH (Z-isomer)], 7.2–7.4 (5H, m, aromatic
protons), 8.16 [1H£a, br, NH (E-isomer)]. EI-MS (rel. int.
%) m/z¼376 (4.7, Mþ), 291 (61, [M2tBuCO]þ), 253 (45,
[M2PhSCH2]þ), 207 (27, [M2PhSCH2–EtOH]þ), 57
(100, tBuþ).
1
as an oil after silica gel column chromatography. The H
NMR spectrum of this sample showed that it consists of
the two tautomers arising from its enamine moiety (E/Z¼
90:10). Assignments of signals for the main isomer and
some for the minor isomer are described. 1H NMR
(400 MHz, CDCl3, a¼0.90, b¼0.10, carzinophilin number-
ing): d 1.23 [3H£a, t, J¼7.2 Hz, CH3CH2O (E-isomer)],
1.25 [9H£a, s, (CH3)3CSi (E-isomer)], 1.26 [3H£b, t, J¼
7.1 Hz, CH3CH2O (E-isomer)], 1.28 [9H£b, s, (CH3)3CSi
(Z-isomer)], 1.64 [1H£a, dddd, J¼7.7, 7.6, 9.2, 13.3 Hz,
C12HH (E-isomer)], 1.76 [1H£b, m, C12HH (Z-isomer)],
2.05 [1H£a, dddd, J¼5.9, 7.7, 8.9, 13.3 Hz, C12HH
(E-isomer)], 2.13 [1H£b, m, C12HH (Z-isomer)], 2.63
[1H£a, ddd, J¼7.6, 8.9, 17.2 Hz, C13HH (E-isomer)], 2.69
[1H£a, ddd, J¼5.9, 9.2, 17.2 Hz, C13HH (E-isomer)], 3.05,
3.13 [each 1H£b, m, C13H2 (Z-isomer)], 3.28 [1H£a, dd,
J¼7.6, 9.3 Hz, C11CHHO (E-isomer)], 3.35 (3H, s, CH3O),
3.40 [1H£a, dd, J¼4.5, 9.3 Hz, C11CHHO (E-isomer)],
3.90 [1H£b, m, C11H (Z-isomer)], 4.02 [1H£a, m, C11H
(E-isomer)], 4.10 [2H£a, br q, J¼7.2 Hz, CH3CH2O
(E-isomer)], 4.13 [2H£b, q, J¼7.2 Hz, CH3CH2O
(Z-isomer)], 5.75 [1H£b, br, NH (Z-isomer)], 6.47 [1H£a,
br, NH (E-isomer)], 7.08 [1H£b, br, NH(Z-isomer)], 8.07
[1H£a, br, NH(E-isomer)]. EI-MS (rel. int. %) m/z¼298
(25, Mþ), 253 (1.0, [M2MeOCH2]þ), 241 (8.2,
[M2tBu]þ), 213 (5.4, [M2tBuCO]þ), 57 (100, t-Buþ).
4.3.2. Reaction with AcOH giving ethyl (N-pivaloyl-[(S)-
5-acetoxymethylpyrrolidin-2-ylidene]glycinate (ent-5b).
Similar treatments of ent-3 (11.5 mg, 43.2 mmol) to those
described in Section 4.2.3 gave ent-5b (12.1 mg, 86%) as an
1
oil after silica gel column chromatography. The H NMR
spectrum of this sample showed that it consists of the two
tautomers arising from its enamine moiety (E/Z¼90:10).
Assignments of signals for the main isomer and some for the
4.4. Reaction of ethyl 2-[(R)-1-azabicyclo[3.1.0]hex-2-
ylidene]-2-[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-
naphthoyl)bytyryl]aminoacetate (8) with PhSH
1
minor isomer are described. H NMR (400 MHz, CDCl3,
a¼0.90, b¼0.10, carzinophilin numbering): d 1.24 [3H£a,
t, J¼7.1 Hz, CH3CH2O (E-isomer)], 1.25 [9H£a, s,
(CH3)3CSi (E-isomer)], 1.29 [9H£b, s, (CH3)3CSi
(Z-isomer)], 1.67 [1H£a, dddd, J¼6.3, 7.6, 9.0, 12.9 Hz,
C12HH (major)], 2.09 [3H£a, CH3CO (E-isomer)], 2.12
[1H£a, dddd, J¼6.5, 7.5, 8.2, 12.9 Hz, C12HH (major)],
2.17 [3H£b, s, CH3CO (Z-isomer)], 2.67 [1H£a, dt, J¼17.3,
7.7 Hz, C13HH (E-isomer)], 2.70 [1H£a, ddd, J¼6.2, 9.0,
17.3 Hz, C13HH (E-isomer)], 3.14 [2H£b, m, C13H2
(Z-isomer)], 3.88 [1H£a, dd, J¼7.8, 11.0 Hz, C11CHHO
(E-isomer)], 3.90 (1H£b, dd, J¼7.1, 11.0 Hz, C11CHHO
(Z-isomer)), 4.09 [1H, m, C11H, 4.11 [2H£a, br q, J¼
7.1 Hz, CH3CH2O (E-isomer)], 4.15 [2H£b, q, J¼7.1 Hz,
4.4.1. 4 h’s reaction. A solution of 815 (5.0 mg, 10.1 mmol),
PhSH (10 mL, 91 mmol), and Et3N (10 mg, 99 mmol) in
THF (1.0 mL) was stirred at room temperature. After 4 h,
the mixture was directly adsorbed on silica gel column.
After elution of excess PhSH with benzene, further elution
with AcOEt gave the crude products. Purification by silica
gel preparative TLC (benzene/acetone¼85:15) gave 10
(3.8 mg, 62%) and 11 (2.0 mg, 27%), amide 12 (trace), and
13 (trace).
4.4.2. 10 h’s reaction. A solution of 8 (5.4 mg, 11.0 mmol),

M. Hashimoto et al. / Tetrahedron 59 (2003) 3089–3097
3095
PhSH (10 mL, 91 mmol), and Et3N (10 mg, 99 mmol) in
THF (1.0 mL) was stirred at room temperature. After 10 h,
TLC analysis of the mixture indicated disappearance of
starting 8. Work-up similar to that described in Section 4.4.1
gave 11 (6.0 mg, 76%), 12 (0.3 mg, 11%), and 13 (0.2 mg,
1680, 1610, 1600, 1410, 1270, 1230, 1200, 1180,
1
1080 cm21. H NMR (400 MHz, CDCl3): d 1.48 (3H, s,
C4H3), 2.69 (3H, s, C50CH3), 3.26, 3.38 (each 1H, d,
J¼13.8 Hz, C3CH3SPh), 3.76 (1H, br, OH), 3.98 (3H, s,
C30OCH3), 5.54 (1H, s, C2H), 5.55, 6.36 (each 1H, br,
NH2), 7.13–7.40 (7H, aromatic protons), 7.49 (1H£a, d,
J¼2.6 Hz, C40H), 7.83 (1H£b, d, J¼2.6 Hz, C20H), 8.62
(1H£0.9, m, C80H). EI-MS (rel. int. %) m/z¼439 (0.8, Mþ),
421 (1.3, [M2H2O]þ), 404 (1.0, [M2H2O–NH3]þ), 315
(2.0, M2PhSCH2)þ), 216 (36, ArCO2Hþ), 199 (100.
ArCOþ). EI-HRMS calcd for C24H25NO5S (Mþ): m/z¼
439.1455; found m/z¼439.1433. The 1H NMR spectrum of
this sample was identical to that of the authentic sample
independently prepared described below.
1
4%). The H NMR and IR spectra of 12 were identical to
those of the natural product.2
4.4.3. Physical data of mono-adduct 10. Rf¼0.40 (silica
gel, acetone/CH2Cl2¼15:85). IR (film): 3350, 2930, 1720,
1
1700, 1670, 1600, 1280, 1240, 1080 cm21. The H NMR
spectrum of this sample showed that it consists of the two
tautomers arising from its enamine moiety (E/Z¼85:15).
Assignments of signals for the main isomer and some for the
1
minor isomer are described. H NMR (400 MHz, CDCl3,
a¼0.85, b¼0.15 carzinophilin numbering): d 1.17 [3H£b, t,
J¼7.1 Hz, CH3CH2O (Z-isomer)], 1.21 [3H£a, t, J¼7.1 Hz,
CH3CH2O (E-isomer)], 1.59 [3H£a, s, C20H3 (E-isomer)],
1.60 [3H£b, s, C20H3 (Z-isomer)], 1.71 [1H£a, m, C12HH
(E-iso0mer)], 2.15 [1H£a, m, C12HH (E-isomer)], 2.69 (3H,
s, C5 CH3), 2.70 [2H£a, m, C13H2 (E-isomer)], 2.79
[1H£a, d, J¼4.6 Hz, C21HH (E-isomer)], 2.92 [1H£a, dd,
J¼7.2, 13.3 Hz, C11CHH (E-isomer)], 3.04 [1H£a, dd, J¼
5.7, 13.3 Hz, C11CHH (E-isomer)], 3.05 [1H£a, d, J¼
4.6 Hz, C21HH (E-isomer)], 0 3.95[1H£a, m, C11H
(E-isomer)], 3.98 [3H£a, s, C3 OCH3 (E-isomer)], 3.99
[3H£b, s, C3’OCH3 (Z-isomer)], 4.12 (2H, m, CH3CH2O),
5.27 [1H£a, s, C18H (E-isomer)], 5.33 [1H£b, s, C18H (Z-
isomer)], 5.75 [1H£b, br s, NH (Z-isomer)], 6.93 [1H£a, br
s, NH (E-isomer)], 7.2–7.4 (7H, aromatic protons), 7.49
[1H£a, d, J¼2.6 Hz, C40H (E-isomer)], 7.93 [1H£b, d,
J¼2.6 Hz, C20H (E-isomer)], 7.95 [1H£a, d, J¼2.6 Hz,
C20H (Z-isomer)], 8.25 [1H£a, br, NH (E-isomer)], 8.66
[1H£a, m, C8’H]. EI-MS (rel. int. %) m/z¼604 (18, Mþ),
481 (8.9, [M2PhSCH2]þ), 199 (100, ArCOþ). EI-HRMS
calcd for C33H36N2O7S: m/z¼604.2245; found m/z¼604.2239.
4.4.6. Authentic 13. A solution of 12 (4.0 mg, 11.2 mmol)
PhSH (10 mg, 91 mmol) and Et3N (10 mg, 99 mmol) in
THF (1.0 mL) was stirred at room temperature for 12 h.
After the mixture was concentrated in vacuo, the residue
was purified by silica gel column chromatography (acetone/
CH2Cl2¼10:90) gave 13 (4.0 mg, 9.1 mmol, 81%) as an oil.
4.5. Reaction of 13-O-dibenzyl-4-O-methyl carzinophilin
(9) with thiophenol giving (3R,4R,5S)-3,4-dibenzyloxy-2-
[(E)-1-((2S,3S)-3-hydroxy-3-methyl-2-(3-methoxy-5-
methyl-1-naphthoxy)-4-phenyl-thiobutyrylamino)-1-(N-
((Z)-1-methoxymethylidene-2-oxopropyl)carbamoyl)]-
methylidene-5-phenylthiomethyl-pyrrolidine (14)
A solution of 9 (2.5 mg, 3.2 mmol), PhSH (35 mg,
31.8 mmol), and Et3N (2.5 mL, 1.8 mg, 18 mmol) was
stirred in THF (1.0 mL) at room temperature for 3 h. After
concentration, purification of the residue by preparative
silica gel TLC developed with AcOEt/hexane (50:50) gave
14 (2.0 mg, 63%) as a caramel. Rf¼0.40 silica gel, AcOEt/
hexane¼50:50). IR (film): 3370, 2940, 1720, 1700, 1650,
1510, 1500, 1480, 1280, 1240, 1210, 1190, 1090, 740,
1
4.4.4. Physical data of bis-adduct 11. Rf¼0.46 (silica gel,
acetone/CH2Cl2¼15:85). IR (film): 3350, 2970, 2920, 1720,
690 cm21. H NMR (200 MHz, CDCl3): d 1.51, 2.18, 2.67
(each 3H, s, C20H3, C1H3, CH3Ar, respectively), 3.08 (1H,
dd, J¼7.4, 13.6 Hz, C11CHHSPh), 3.24 (1H, dd, J¼6.8,
13.6 Hz, C11CHHSPh), 3.33, 3.37 (each 1H, d, J¼13.9 Hz,
C21H2SPh), 3.43 (3H, s, CH3O), 3.87 (1H, d, J¼3.1 Hz,
C12H), 3.96 (3H, s, CH3O), 4.22, 4.47 (each 1H, d, J¼
11.6 Hz, PhCH2O), 4.68 (1H, d, J¼11.1 Hz, PhCHHO),
4.78 (1H, br s, alcoholic proton), 4.86 (1H, d, J¼11.1 Hz,
PhCHHO), 5.00 (1H, s, C18H), 5.13 (1H, d, J¼3.1 Hz,
C13H), 6.40 (1H, br s, NH), 7.04 (1H, d, J¼0.8 Hz, C4H),
7.05–7.50 (25H, NH, aromatic protons), 7.81 (1H, d, J¼
2.5 Hz, C20H), 8.66 (1H, br d, J¼7.8 Hz, C80H). SIMS
(3-nitrobenzylalcohol) m/z¼996 (MHþ). Neither EI- nor
CI-MS of this sample gave informative signals.
1
1690, 1670, 1595, 1275, 1230, 1080, 730 cm21. H NMR
spectrum of this sample showed that it consists of the two
tautomers arising from its enamine moiety (E/Z¼85:15).
The 1H NMR (400 MHz, CDCl3, a¼0.85, b¼0.15, carzino-
philin numbering): d 1.21 [3H, br t, J¼7.2 Hz, CH3CH2O
(E-isomer)], 1.52 [3H£a, s, C20H3 (E-isomer)], 1.68
[1H£a, m, C12HH (E-isomer)], 1.81 [1H£b, m, C12HH
(Z-isomer)], 2.13 [1H£a, m, C12HH (E-isomer)], 2.20
[1H£b, m, C12HH (Z-isomer)], 2.68 (3H, s, C50CH3), 2.70
[2H£a, m, C13H2 (E-isomer)], 2.90, 3.04 (each 1H, br,
C11CH2), 3.12, 3.35 (each 1H, d, J¼13.6 Hz, C21H2),
3.89[1H£a, m, C11H (E-isomer)], 3.97 [3H£b, s, C30OCH3
(Z-isomer)], 3.98 [3H£a, s, C30OCH3 (E-isomer)], 4.12 (2H,
m, CH3CH2O), 5.52 [1H£a, br s, C18H (E-isomer)], 5.81
[1H£b, br s, NH (Z-isomer)], 7.03 [1H£a, br s, NH
(E-isomer)], 7.05–7.40 0 (12H, aromatic protons), 7.49
[1H£a, d, J¼2.6 Hz, C4 H (E-isomer)], 7.79 [1H£b, C20H
(E-isomer)], 8.26 [1H£0.9, br, NH (E-isomer)], 8.61
(1H£0.9, m, C80H). EI-MS (rel. int. %) m/z¼714 (7.3, Mþ),
704 (8.4, [M2PhSH]þ), 548 (3.4, [M2PhSCH2 CMeþH]þ),
498 (7.6, [M2ArCO2H]þ). EI-HRMS calcd for
C39H42N2O7S2 (Mþ): m/z¼714.2436; found m/z¼714.2420.
4.6. Physical data of novel compounds in Table 1
The compounds 22a,b, 24, and their diastereomers iso-
22a,b, and iso-24 were prepared only for cytotoxicity assay
by employing almost the same procedure as described for
the preparation of 2 and 3a–c in Part 1 of this series of
papers. So, their physical data are only reported here.
4.6.1. Ethyl 2-[(R)-5-hydroxymethylpyrrolidin-2-yl-
idene]-2-[(S)-2-benzoxy-3-methylbutyrylamino]acetate
(22a). IR (film): 3360, 2960, 2920, 1720, 1665, 1590, 1260,
4.4.5. Physical data of 13. IR (film): 3400, 2920, 1720,

3096
M. Hashimoto et al. / Tetrahedron 59 (2003) 3089–3097
1
1090, 710 cm21. The H NMR spectrum of this sample
showed that it consists of the two tautomers arising from its
enamine moiety (E/Z¼80:20). Assignments of signals for
the main isomer and some for the minor isomer are
(each 3H, d, J¼6.5 Hz, C19 (CH3)2), 1.25 (3H, t, J¼7.2 Hz,
CH3CH2O), 1.74 (1H, m, C12HH), 2.00 (1H, m, C12HH),
2.48 (1H, m, C19H), 2.57 (1H, ddd, J¼6.1, 9.8, 17.4 Hz,
C12HH), 2.74 (1H, ddd, J¼6.4, 9.8, 17.4 Hz, C12HH), 3.40
(1H, br, OH), 3.52 (1H, dd, J¼6.0, 11.2 Hz, C11CHHOH),
3.57 (1H, br dd, J¼2.6, 11.7 Hz, C13CHH), 3.69 (1H, dd,
J¼3.8, 11.2 Hz, C13CHHO), 3.90 (1H, m, C11H), 4.05 (2H,
m, CH3CH2O), 5.29 (1H, d, J¼4.4 Hz, C18H), 6.84 (1H, br,
NH), 7.50 (2H, m, aromatic protons), 7.60 (1H, m, aromatic
proton), 8.10 (3H, NH, aromatic protons). EI-MS (rel. int.
%) m/z¼404 (13, Mþ), 373 (1.7, [M2CH2OH]þ), 227
(7.2, [M2PhCOOCHCH(Me)2]þ), 199 (18, [M2
PhCOOCHCH(Me)2CO]þ), 105 (100, PhCOþ). EI-HIMS
calcd for C21H22N2O6 (Mþ): m/z¼404.1945; found m/z¼
404.1960.
1
described. H NMR (400 MHz, CDCl3, a¼0.80, b¼0.20,
carzinophilin numbering): d 1.10 [3H£a, d, J¼6.9 Hz,
C20CH3 (major)], 1.15 [3H£a, d, J¼6.8 Hz, C19CH3
(major)], 1.16 [3H£a, t, J¼7.2 Hz, C19CH3 (major)], 1.90
[1H£b, dq, J¼4.3, 9.0 Hz, C12HH (minor)], 2.05 [1H£b,
C12HH (minor)] 2.48 [1Hþ1H£a, m, C19H, C12H
(minor)], 2.52 (1H£a, br, OH), 2.65 [1H£a, ddd, J¼6.5,
8.9, 17.6 Hz, C13H (major)], 2.68 [1H£a, ddd, J¼7.1, 9.2,
17.6 Hz, C13H (major)], 3.06 [1H£b, ddd, J¼5.1, 10.3,
18.4 Hz, C13HH (minor)], 3.15 [1H£b, ddd, J¼7.2, 9.4,
18.4 Hz, C13HH (minor)], 3.30 [1H£b, ddd, J¼br, OH
(minor)], 3.48 [1H£a, dd, J¼5.9, 11.8 Hz, C11CHHO
(major)], 3.51 [1H£b, C11HH (minor)], 3.68 [1H£a, dd,
J¼3.6, 11.3 Hz, C11CHH (major)], 3.73 [1H£b, br d, J¼
11.8 Hz, C11CHH (minor)], 3.87 [1H£b, m, C11H
(minor)], 3.99 [1H£a, C11H (major)], 5.06 [1H£b, d,
J¼4.9 Hz, C18H (minor)], 5.37 [1H£a, d, J¼4.4 Hz, C18H
(major)], 5.50 [1H£b, br, NH (minor)], 6.83 [1H£a, br, NH
(major)], 7.19 [1H£b, br, NH (minor)], 7.50 (2H, br t,
J¼7.5 Hz, aromatic protons), 7.63 (1H, m, aromatic proton),
8.11 [2Hþ1H£a, NH aromatic protons, (major)]. EI-MS
(rel. int. %) m/z¼404 (10, Mþ), 373 (7.2, [M2CH2OH]þ),
227 (5.8, [M2PhCOOCHCH(Me)2]þ), 199 (18, [M2
PhCOOCHCH(Me)2CO]þ), 105 (100, PhCOþ). EI-HIMS
calcd for C21H28N2O6 (Mþ): m/z¼404.1945; found m/z¼
404.1961.
4.6.4. Ethyl 2-[(S)-5-methanesulfoxymethylpyrrolidin-2-
ylidene]-2-[(S)-2-benzoxy-3-methylbutyrylamino]ace-
tate (iso-22b). IR (film): 3350, 2960, 1720, 1670, 1600,
1350, 1260, 1170, 1100, 950, 710 cm21. The 1H NMR
spectrum of this sample showed that it consists of the two
tautomers arising from its enamine moiety (E/Z¼85:15).
Assignments of signals for the main isomer are only
described. 1H NMR (400 MHz, CDCl3, carzinophilin
numbering): d 1.10, 1.16 (each 3H, d, J¼6.5 Hz, C19
(CH3)2), 1.19 (3H, t, J¼7.2 Hz, CH3CH2O), 1.80 (1H, m,
C12HH), 2.14 (1H, m, C12HH), 2.47 (1H, m, C19H), 2.62
(1H, ddd, J¼5.9, 9.6, 17.5 Hz, C13HH), 2.77 (1H, ddd,
J¼6.6, 8.5, 17.5 Hz, C13HH), 3.07 (3H, s, CH3SO3), 4.02–
4.19 (4H, CH3CH2O, C11H, C19H), 4.28 (1H, dd, J¼3.8,
10.1 Hz, C11CHHOMs), 5.26, (1H, d, J¼4.4 Hz, C18H),
6.81 (1H, br, NH), 7.51 (2H, br t, J¼7.9 Hz, aromatic
protons), 7.64 (1H, br tt, J¼1.4, 7.9 Hz, aromatic protons),
8.12 (2H, br dd, J¼1.4, 7.9 Hz, aromatic protons). EI-MS
(rel. int. %): m/z¼482 (10, Mþ), 386 (2.6, [M2MsOH]þ),
305 (5.9), 105 (100, PhCO). EI-HIMS calcd for
C22H30N2O8S (Mþ): m/z¼482.1724; found m/z¼1702.
4.6.2. Ethyl 2-[(R)-5-methanesulfoxymethylpyrrolidin-2-
ylidene]-2-[(S)-2-benzoxy-3-methylbutyrylamino]ace-
tate (22b). IR (film): 3350, 2950, 1710, 1670, 1595, 1510,
1350, 1250, 1170, 1100, 950, 710, 520 cm21. The 1H NMR
spectrum of this sample showed that it consists of the two
tautomers derived from its enamine moiety (E/Z¼85:15).
Assignments of signals for the main isomer are only
described. 1H NMR (400 MHz, CDCl3, carzinophilin
numbering): d 1.11, 1.16 (each 3H, d, J¼6.5 Hz, C19
(CH3)2), 1.18 (3H, t, J¼7.2 Hz, CH3CH2O), 1.74 (1H, dddd,
J¼6.0, 6.6, 9.7, 19.9 Hz, C12HH), 2.18 (1H, ddd, J¼6.5,
7.9, 19.9 Hz, C12HH), 2.47 (1H, m, C19H), 2.70 (1H, ddd,
J¼6.7, 9.5, 17.6 Hz, C13HH), 2.71 (1H, ddd, J¼6.7, 9.5,
17.6 Hz, C13HH), 3.04 (3H, s, CH3SO3), 4.05 (1H, dd, J¼
6.7, 10.1 Hz, C11CHHOMs), 4.07 (2H, q, J¼7.2 Hz,
CH3CH2O), 4.17 (1H, m, C11H), 4.26 (1H, dd, J¼3.8,
10.1 Hz, C11CHHOMs), 5.26 (1H, d, J¼4.4 Hz, C18H),
6.80 (1H, br, NH), 7.51 (2H, br t, J¼7.9 Hz, aromatic
protons), 7.64 (1H, br tt, J¼1.4, 7.9 Hz, aromatic protons),
8.12 (2H, br dd, J¼1.4, 7.9 Hz, aromatic protons). EI-MS
(rel. int. %) m/z¼482 (12, Mþ), 305 (6.2), 105 (100, PhCO).
EI-HIMS calcd for C22H30N2O8S (Mþ): m/z¼482.1724;
found m/z¼482.1740.
4.6.5. Ethyl 2-[(R)-1-azabicyclo[3.1.0]hexan-2-ylidene]-
2-[(S)-2-benzoxy-3-methylbutyrylamino]acetate (24). IR
(film): 3350, 2960, 1720, 1680, 1490, 1360, 1100,
.
710 cm21 1H NMR (400 MHz, CDCl3, carzinophilin
numbering): d 1.08 (3H, d, J¼7.0 Hz, C19CH3), 1.14 (3H,
d, J¼6.8 Hz, C19CH3), 1.27 (3H, t, J¼7.2 Hz, CH3CH2O),
1.68 (1H, dd, J¼0.8, 4.3 Hz, C10H), 2.16 (2H, m, C12H2),
2.31 (1H, d, J¼5.1 Hz, C10H), 2.50 (1H, m, C19H), 2.70
(1H, dt, J¼19.5, 9.3 Hz), 2.76 (1H, m, C11H), 3.15 (1H,
ddd, J¼4.0, 10.1, 19.5 Hz, C13H), 4.19, 4.22 (each 1H, dq,
J¼10.0, 7.2 Hz, CH3CH2O), 5.44 (1H, d, J¼4.2 Hz, C18H),
7.48 (2H, tt, J¼1.5, 7.9 Hz, aromatic protons), 7.61 (1H, tt,
J¼1.5, 7.9 Hz, aromatic proton), 7.68 (1H, br, NH), 8.11
(2H, m, aromatic protons). EI-MS (rel. int. %) m/z¼386
(1.5, Mþ), 105 (100, PhCOþ).
4.6.6. Ethyl 2-[(R)-1-azabicyclo[3.1.0]hexan-2-ylidene]-
2-[(S)-2-benzoxy-3-methylbutanoyl-amino]acetate (iso-
4.6.3. Ethyl 2-[(S)-5-hydroxymethylpyrrolidin-2-yl-
idene]-2-[(S)-2-benzoxy-3-methylbutyrylamino]acetate
(iso-22a). IR (film): 3360, 2960, 1720, 1670, 1590, 1380,
1250, 1100, 710 cm21. The 1H NMR spectrum of this
sample showed that it consists of the two tautomers arising
from the enamine moiety (E/Z¼85:15). Assignments of
24). IR (film): 3350, 2960, 1720, 1680, 1380, 1100 cm21
.
1H NMR (400 MHz, CDCl3, carzinophilin numbering): d
1.10, 1.15 (each 3H, d, J¼6.9 Hz, C19CH3), 1.17 (3H, t,
J¼7.1 Hz, CH3CH2O), 1.70 (1H, d, J¼5.1 Hz, C10H), 2.20
(2H, m, C12H2), 2.35 (1H, d, J¼5.1 Hz, C10H), 2.46 (1H,
m, C19H), 2.71 (1H, ddd, J¼9.1, 9.1, 19.6 Hz, C13H), 2.81
(1H, m, C11H), 3.16 (1H, ddd, J¼4.1, 10.6, 19.6 Hz,
1
signals for the main isomer are only described. H NMR
(400 MHz, CDCl3, carzinophilin numbering): d 1.11, 1.15

M. Hashimoto et al. / Tetrahedron 59 (2003) 3089–3097
3097
C13H), 4.20 (2H, m, CH3CH2O), 5.38 (1H, d, J¼4.6 Hz,
Acknowledgements
C18H), 7.48 (2H, tt, J¼1.5, 7.9 Hz, aromatic protons), 7.61
(1H, tt, J¼1.5, 7.9 Hz, aromatic proton), 7.68 (1H, br, NH),
8.11 (2H, m, aromatic protons). EI-MS (rel. int. %) m/z¼
386 (1.8, Mþ), 105 (100, PhCO).
We are grateful to Professor Tadzuko Tashiro (Cancer
Chemotherapy Center, Japanese Foundation for Cancer
Research) for assaying antitumor activity against P388, and
to Drs Tadashi Wahiawa, Kazuyo Sugiyama, and Katsuya
Gomi (Kyowa Hakko Kogyo Co. Ltd.) for determining
antitumor activity against Sarcoma 180.
4.7. Cytotoxicity assay against P388 murine leukemia in
vitro
Growing cells of murine P388 lymphocytic leukemia were
suspended at 2£104 cells/mL in PRMI-1640 medium
containing 10% fetal bovine, 10 mM 2-hydroxyethyldisul-
fate and kanamycin (100 mg/mL), and the samples dis-
solved in MeOH were added. The mixtures were incubated
at 378C for 4 days in a CO2 incubator with an atmosphere
containing 5% CO2. MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] solution (1.0 mg/mL
PBS) was added and incubation was continued for 4 h. To
the supernatant solution was added DMSO and it was mixed
thoroughly to dissolve the formazan. The absorbance was
measured at 550 nm (Ref 630 nm), indicating the mass of
viable cells. A 50% cell growth inhibitory concentration
(IC50) was determined from the calculated cell growth
inhibitory rates at several concentrations.
References
1. Parts of this series of papers have been the subject of five
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Matsumoto, M.; Yamada, K.; Terashima, S. Tetrahedron Lett.
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3. Moran, E. J.; Armstrong, R. W. Tetrahedron Lett. 1991, 32,
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4.7.1. Antitumor assay against P388 murine leukemia in
vivo. P388 murine leukemia cells (106 cells/mouse) were
inoculated i.p. into CDF1 mice (6 mice). After 1 and 5 days,
solutions of the sample were administered. The effectively
(T/C value) was calculated by the following method:
4. Hata, T.; Koga, F.; Sano, Y.; Kanamori, K.; Matsumae, A.;
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T=C ¼ T=C £ 100
T: median survival days for group incubated the samples, C:
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T/C values are .120, were regarded to be effective.
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Chem. Soc. 2002, 124, 13008.
4.8. Cytotoxicity assay against Sarcoma 180
Human uterine cervix Hela S3 cells (8£102/well) were
cultured in MEM medium containing 10% bovine serum at
378C for 12 h in a CO2 incubator with an atmosphere
containing 5% CO2. Samples dissolved in DMSO were
added to the culture medium. After incubation for 72 h, the
cytotoxicity was estimated by neutral red dye uptake
method.
12. Zang, H.; Gates, K. S. Biochemistry 2000, 39, 14968.
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4.9. Antitumor assay against Sarcoma 180
Sarcoma 180 cells (5£106/mouse) were inoculated s.c. into
male ddY mice (body weight¼18–20 g). After 1 and 4
days, methanol solutions of the sample were administered.
The size of the tumor was measured by the following
method;
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2
ðmajor axisÞ £ ðminor axisÞ
tumor volume ¼
2