Two new spermidine alkaloids from Chisocheton weinlandii

Article abstract of DOI:10.1002/hlca.200490129

The investigation of the MeOH extract of the leaves of Chisocheton weinlandii HARMS (Meliaceae) revealed two new open-chain spermidine alkaloids, chisitine 1 (1) and chisitine 2 (2). Their structures were elucidated by NMR spectroscopy, tandem-mass spectrometry, and independant syntheses (Scheme 3). Detailed MS/MS fragmentation pathways are discussed for both compounds based on H/D exchange and ¹⁸O-labeling experiments (Schemes 1 and 2).

Full text of DOI:10.1002/hlca.200490129

Helvetica Chimica Acta Vol. 87 (2004)
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Two New Spermidine Alkaloids from Chisocheton weinlandii
by Manuel Tzourosa), Laurent Biglera), Stefan Bienza), Manfred Hesse*a), Akira Inada*b), Hiroko Muratab),
c
Yuka Inatomib), Tsutomu Nakanishib), and DedyDarnaedi
)
a) Institute of Organic Chemistry, University of Z¸rich, Winterthurerstrasse 190, CH-8057 Z¸rich
b) Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101,
Japan
c) Herbarium Bogoriense, Jalan Ir, H. Juanda, Bogor 16122, Indonesia
The investigation of the MeOH extract of the leaves of Chisocheton weinlandii Harms (Meliaceae)
revealed two new open-chain spermidine alkaloids, chisitine 1 (1) and chisitine 2 (2). Their structures were
elucidated by NMR spectroscopy, tandem-mass spectrometry, and independant syntheses (Scheme 3). Detailed
MS/MS fragmentation pathways are discussed for both compounds based on H/D exchange and 18O-labeling
experiments (Schemes 1 and 2).
1. Introduction. Polyamine alkaloids are found in a large variety of natural sources
including plants and insects. These natural products are classified into two major
categories depending on whether the structure is cyclic or open-chained [1]. In general,
such polyamine alkaloids are composed of a putrescine (ˆ butane-1,4-diamine),
spermidine (ˆ N-(3-aminopropyl)butane-1,4-diamine), or spermine (ˆ N,N'-bis(3-
aminopropyl)butane-1,4-diamine) backbone that carries one or more substituents at
the N-atoms, mostly acyl groups. Their determination was usually performed by a
combination of several analytical techniques, the methods of choice being mass
spectrometry combined with NMR spectroscopy.
However, NMR spectroscopy often does not securely assess the exact framework of
the polyamine portion within these alkaloids because of signal overlap of the
methylene protons. Furthermore, acyl-substituted polyamines display an additional
range of complexity in their NMR spectra due to s-cis/s-trans isomerism of the amide
functions. The NMR spectra of such isomer mixtures, exhibiting multiple signals for the
NMR active nuclei, always leave doubt about whether the sample was pure or not.
Mass spectrometry in combination with tandem-mass spectrometry (MS/MS) is
gaining more and more importance in the identification and characterization of
polyamine alkaloids. Due to their high basicity and low volatility, polyamines are
excellent candidates for mass-spectrometric identification by means of electrospray
ionization (ESI-MS). This method allows to delimit the number of N-atoms present in
the molecule. In addition, MS/MS experiments can provide precious information on the
polyamine backbones and on the attachment sites of substituents. Furthermore, as
compared to NMR spectroscopy, data acquisition for MS is much faster and requires
considerably smaller amounts of material.
In connection with our ongoing search for new polyamine alkaloids from plants and
animal sources, we were interested in the MeOH extracts of the leaves of Chisocheton

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Helvetica Chimica Acta Vol. 87 (2004)
weinlandii Harms (Meliaceae). This tree is distributed throughout southern Irian Jaya
(Papua New Guinea, Indonesia). Around fifty putrescine alkaloids have already been
characterized from species belonging to the plant family Meliaceae [1]. Several
Chisocheton species have been screened for their natural-product content; among
them, Chisocheton paniculatus Hiern has been intensively investigated. Five apo-
tirucallol derivatives and four tetranortriterpenoids from the wood and seeds of C.
paniculatus were identified [2]. Four meliacin-type compounds were found in the fruits
[3], and five protolimonoids (called paniculatin B, C, D, G, and H) and one limonoid
(named arunachalin) were isolated and identified from the mature wood of the plant
[4]. For C. weinlandii, on the other hand, no study of the chemical constituents is found
in the literature. Only some insecticidal activity was reported for extracts of its leaves
and twigs [5]. These extracts showed inhibition to larval growth of the variegated
cutworm Peridroma saucia HÏbner (Noctuinidae). We have now studied the
constituents of the leaves of the plant and isolated the two new spermidine-type
alkaloids 1 and 2 (see Fig. 1). Their structures were established on the basis of NMR
spectroscopy, tandem-mass spectrometry, and comparison with their synthetic equiv-
alents.
Fig. 1. Structure and selected HMBC data of chisitine 1 (1) and 2 (2)
2. Results. 2.1. Isolation. Extraction of the crushed leaves of C. weinlandii with
MeOH and distribution of the extract between H2O and AcOEt yielded a crude
mixture that was separated by column chromatography (silica gel). With the eluent
CHCl3/MeOH 5 :1, a mixture was obtained that was further subjected to semi-prep.
reversed-phase HPLC, resulting in the isolation of two pure compounds named
1
)
Arbitrary numbering; for systematic name, see Exper. Part.

Helvetica Chimica Acta Vol. 87 (2004)
1413
chisitine 1 (1) and chisitine 2 (2) (Fig. 1). To the best of our knowledge, these are the
first spermidine alkaloids isolated from a Meliaceae species.
2.2. Structure Elucidation. Chisitine 1 (1) shows in its ESI-MS a series of three peaks
‡
‡
‡
at m/z 378 ([M ‡ H] ), 400 ([M ‡ Na] ), and 416 ([M ‡ K] ) that allow the
assignment of its relative molecular mass as 377. The value suggests the presence of
an odd number of N-atoms in the molecule. The H-and 13C-NMR spectra of 1,
1
showing a 1:1 ratio of two isomers (for data, see isomers 1a, and 1b in Tables 1 and 2),
indicate the presence of a cinnamoyl (ˆ(2E)-3-phenylprop-2-enoyl) substituent. After
detailed analysis of further NMR and MS data, the structure of chisitine 1 (1) was
elucidated as S-methyl [3-{[4-(formylamino)butyl][(2E)-3-phenylprop-2-enoyl]ami-
no}propyl]carbamothioate. This compound was synthesized, and the synthetic product
was found to be identical with the natural product with respect to all its spectroscopic
data (see Sect. 2.4).
Table 1. 1H-NMR Data (500.1 or 600.1 MHz, (D6)DMSO) of Chisitines 1 (1) and 2 (2)1). d in ppm, J in Hz.
1a)
2b)
Isomer 1a
Isomer 1b
8.11 (br. s)
Isomer 2a
8.49 8.47 (m)
Isomer 2b
HÀN(1)
CHO
8.23 (br. s)
8.57 8.55 (m)
ꢀ
8.00 (br. s)c)
8.00 (br. s)c)
HÀN(10)
HÀC(3'')
HÀC(5')
HÀC(5'')
HÀC(3')
HÀC(4'')
HÀC(6')
HÀC(7')
HÀC(2')
CH2(4)
CH2(6)
CH2(2)
CH2(9)
MeS
7.87 (d, J ˆ 7.4)
7.85 (d, J ˆ 7.4)
7.58 (d, J ˆ 7.1)
7.70 7.68 (m)c)
7.71 (d, J ˆ 7.4)
)
7.49 (br. d, J ꢀ 15.2)c)
7.54 7.45 (m)c)
ꢀ
7.44 7.36 (m)c)
7.44 7.27 (m)c)
7.10 (d, J ˆ 15.4)
3.49 3.45 (m)c)
3.32 3.29 (m)c)
3.21 3.15 (m)c)
7.05 (d, J ˆ 15.4)
3.32 3.29 (m)c)
3.49 3.45 (m)c)
3.13 3.08 (m)c)
7.13 (d, J ˆ 15.4)
7.05 (d, J ˆ 15.4)
3.44 3.42 (m)c)
3.55 3.50 (m)c)
3.29 3.26 (m)c)
3.55 3.50 (m)c)
3.38 3.34 (m)c)
3.38 3.34 (m)c)
3.13 3.08 (m)c)
2.21, 2.20 (2s)d)
1.68 1.65 (m)
3.13 3.08 (m)c)
CH2(3)
CH2(7)
CH2(8)
1.74 1.71 (m)
1.87 1.84 (m)
1.79 1.77 (m)
1.48 1.43 (m)
ꢀ
1.57 1.49 (m)c)
1.54 1.36 (m)c)
1.38 1.41 (m)
a) 500.1 MHz. b) 600.1 MHz. c) Signals of isomers a/b overlapped. d) Not distinguishable.
In the 1H-NMR spectrum of 1, the two d at d 7.10 and 7.05 and the broad d at d 7.49 (3J ˆ 15.4) are attributed
to the olefinic protons HÀC(2') and HÀC(3') of the two isomeric forms 1a and 1b1). The corresponding
13C-NMR signals for C(2') and C(3') are found at d 118.4 and 118.3, and 141.3 and 141.2 and secured by HSQC.
The signals at d(C) 161.0 and 160.9 (for 1a and 1b) and the broad s at d(H) 8.00 suggest the presence of a
formamide function [6]. A third substituent is identified by the signal at d(H) ca. 2.20 (3 H), which correlates to
the q (determined by DEPT) at d(C) 11.5 (two overlapping signals). These signals are attributed to the unusual
MeSC(O)N residue. The analytical data are in accordance with other carbamothioate-containing alkaloids
[7][8]. The presence of the MeSC(O)N group is further supported by the MS/MS results: the intense fragment
‡
ion l (m/z 330), corresponding to the loss of 48 Da from the quasi-molecular ion ([M ‡ H] ) at m/z 378,
indicates an easy loss of MeSH (see Fig. 3,a, below). 1H-NMR Signals registered between d 1.36 and 3.49
(integrating for 14 H) and the 13C-NMR signals found between d 26.3 and 46.8 (all t) suggest a spermidine

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Table 2. 13C-NMR Data (125.8 or 150.9 MHz, (D6)DMSO) of Chisitines 1 (1) and 2 (2)1). d in ppm, J in Hz.
1a)
2b)
Isomer 1a
Isomer 1b
Isomer 2a
Isomer 2b
C(1'')
C(1')
CHO
C(3')
C(4')
C(2'')
C(5'')
C(7')
C(6')
C(4'')
C(5')
C(3'')
C(2')
C(6)
C(4)
C(2)
C(9)
C(3)
C(8)
C(7)
MeS
166.2 (s)
165.2 (s)
161.0 (d)
141.3 (d)
135.1 (s)
166.0 (s)
164.9 (s)
160.9 (d)
141.2 (d)
135.0 (s)
166.4 (s)
165.3 (s)
161.0 (d)
141.3 (d)
135.1(s)
134.7 (s)
131.1 (d)
129.5 (d)
128.7 (d)
166.1 (s)
164.9 (s)
160.9 (d)
141.2 (d)
135.0 (s)
134.5 (s)
131.0 (d)
129.4 (d)
128.6 (d)
129.5 (d)c)
128.7 (d)c)
128.3 (d)c)
127.9 (d)c)
128.0 (d)
127.1 (d)
118.5 (d)
45.4 (t)
45.0 (t)
36.7 (t)
36.8 (t)
29.6 (t)
25.0 (t)
26.6 (t)
127.8 (d)
127.0 (d)
118.4 (d)
46.8 (t)
43.6 (t)
36.9 (t)
36.6 (t)
27.7 (t)
26.7 (t)
26.3 (t)
118.4 (d)
45.4 (t)
44.7 (t)
38.1 (t)
36.8 (t)
29.5 (t)
24.9 (t)
26.6 (t)
118.3 (d)
46.8 (t)
43.6 (t)
38.5 (t)
36.6 (t)
27.7 (t)
26.7 (t)
26.3 (t)
11.5 (q)c)
a) 125.8 MHz. b) 150.9 MHz. c) Signals of isomers a/b overlapped.
backbone for the compound. The sites of attachment of the three substituents at the polyamine core, as well as
the detailed assignments of the signals, were finally established on the basis of the MS/MS analysis (see Sect. 2.3)
as well as on the basis of the HMBC experiment (selected HMBC data in Fig. 1).
The second compound, chisitine 2 (2), shows three adduct ions in the ESI-MS at
‡
‡
‡
m/z 408 ([M ‡ H] ), 430 ([M ‡ Na] ), and 446 ([M ‡ K] ), consistent with a relative
molecular mass of 407. This indicates that chisitine 2 also contains an odd number of N-
atoms. The 1H-and 13C-NMR spectra of this alkaloid show the presence of the same 1:1
ratio of two isomers as found for 1 (for data, see isomers 2a and 2b in Tables 1 and 2).
Moreover, the data also indicate the presence of an acyl-substituted spermidine,
carrying the cinnamoyl and formyl moieties, but not the methyl carbamothioate unit.
Since the sites of attachment of the groups are identical to those found for chisitine 1,
the only difference between 2 and 1 concerns the nature of one substituent. Based on
further spectral data, the structure of 2 was determined to be N-{3-{[4-(formylami-
no)butyl][(2E)-3-phenylprop-2-enoyl]amino}propyl}benzamide and confirmed by
comparison with the analytical data of the synthetic chisitine 2 (Sect. 2.4).
The additional signals in the aromatic region of the 13C-NMR spectra of 2 at d 134.7 and 134.5 (2 s, C(2'')),
131.1 and 131.0 (2 d, C(5'')), 128.3 (2 overlapping d, C(4'')), and 127.1 and 127.0 (2 d, C(3'')) are assigned to a
benzoyl group [9]1). The replacement of the MeSCO in 1 by the PhCO group in 2 is consistent with the
difference of 30 Da in their relative molecular mass. The sites of attachment of the substituents at the
spermidine core for both isomers were established on the basis of a detailed MS/MS analysis (see Sect. 2.3) and
of the HMBC spectra (see data in Fig. 1).

Helvetica Chimica Acta Vol. 87 (2004)
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2.3. Tandem Mass Spectrometry of Chisitines 1 and 2. A special problem arises in the
correct placing of the two substituents (formyl and (methylthio)carbonyl in case of 1
and formyl and benzoyl in case of 2) at the two primary N-atoms of the spermidine
core. The question that arises is whether the amino groups are separated by three or
four CH2 residues from the third central N-atom. In addition to our earlier
investigations in this field of isomerism [10 12], we have studied very carefully this
problem. Indeed, it seems necessary to determine the general characteristic MS
fragmentation of this kind of compound, because similar natural products isolated in
much smaller amounts have to be investigated in the future.
Compounds 1 and 2 were examined by ESI-MS/MS on a quadrupole ion-trap mass
spectrometer. The two compounds produce two generations of daughter ions (MS2 and
MS3) on the basis of which fragmentation mechanisms are proposed. The aim of their
detailed elucidation is to detect characteristic fragmentation pathways that are
diagnostic for the structures proposed for 1 and 2. Characteristic ions could indicate the
location of the substituents as well as the sequence of the polyamine backbone (PA34
or PA43). The MS/MS data of compound 2 contain more informative ion responses
allowing the postulation of general fragmentation pathways, and are, thus, discussed
first.
‡
Upon MS2, quasi-molecular ion [2 ‡ H] at m/z 408 yields the four important
fragment ions a, d, and e/e' at m/z 390, 278, and 260 (Fig. 2,a). These ions are believed
‡
to arise from two major Pathways A and B (Scheme 1). Following Pathway A, [2 ‡ H]
could lose the cinnamoyl moiety (À130 Da) to form fragment ion d (m/z 278). This
suggested four-center cleavage has already been reported for N-coumaroylputrescine
[12]. Fragment ion d could lose H2O by reaction of the secondary amine (N(5)1)) with
either one of the two amide groups to form cyclic fragment ions e (1,6-cyclization) or e'
(1,7-cyclization), detected at m/z 260 as the base peaks in MS2. Analogous dehydrations
have been observed previously with peptides [13]. The cyclization d ! e and e' is
established by investigation of the labeled analog [18O]-2 (Fig. 2,c). The MS2 of [[18O]-
2 ‡ H] (m/z 410) reveals a doublet of peaks corresponding to e at m/z 260 and [18O]-e'
‡
(m/z 262), which attests that both proposed pathways are followed concurrently.
Isolation and further fragmentation (MS3) of e and e' at m/z 260 yield the four new
informative ions g, h, j, and k at m/z 162, 161, 105, and 99, respectively. Probably ion e'
leads to two cyclic fragment ions g (m/z 162) and k (m/z 99), and to the benzoyl ion j
(m/z 105). These three ions are established to originate from e' by investigation of
[18O]-2 (see Fig. 2,c): the corresponding signals for g and j are at m/z 164 (‡2 Da) and
107 (‡2 Da), respectively, whereas ion k is at the same m/z value 99 (see Scheme 1).
Fragment ion g (m/z 162) reveals that the benzoyl group is linked to the trimethylene
portion of the spermidine, while k (m/z 99) attests that the formyl moiety is located at
the end of the tetramethylene unit.
Fragmentation of the six-membered ion e (m/z 260) leads to the formation of ion h
at m/z 161, additionally supported by the MS3 (410 ! 260) of −pure× e in the spectrum of
[18O]-2 (Fig. 2,c). The diagnostic ions g, k, and h, therefore, justify the structure of
chisitine 2 (2) independently of the NMR spectra.
The second important fragmentation Pathway B starts with the direct dehydration
‡
‡
of quasi-molecular ion [2 ‡ H] giving rise to a signal at m/z 390 ([2 ‡ H À 18] ).
Again, the loss of H2O is explained by the formation of a cyclic structure, fragment ion a

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Helvetica Chimica Acta Vol. 87 (2004)
Fig. 2. MSn Plots of the quasi-molecular ions of chisitine 2 (2): a) MS2 (408) (black line) and MS3 (408 ! 260)
‡
‡
(grey line) of [2 ‡ H] , b) MS2 (411) (black line) and MS3 (411 ! 262) (grey line) of [(D2)-2 ‡ D] , and c) MS2
(410) (black line) and MS3 (410 ! 262)/(410 ! 260) (grey lines) of [[18O]-2 ‡ H] . Fragment ions are
‡
designated according to decreasing m/z values (a to k).
(see Scheme 1). The fragment ions b (m/z 380), c (m/z 286), f (m/z 189), and i (m/z
134) (Fig. 2,a) are not discussed because they are not characteristic for the structure
elucidation of 2.

Helvetica Chimica Acta Vol. 87 (2004)
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Scheme 1. MS Fragmentation of the Quasi-Molecular Ions of Chisitine 2 (2)2)
2
)
The * denotes the location of the 18O-atom in the case of the labeled analog [18O]-2. The boxes indicate the
selected m/z values submitted to MS2 and MS3. The structures of the fragment ions b, c, f, and i are not
depicted.

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To support the proposed mechanism, a H/D-exchange experiment was performed.
Thus, 2 was dissolved in an acidic solution of MeOD (0.1% of DCl), and the resulting
‡
deuterated compound [(D2)-2 ‡ D] (m/z 411) was investigated by ESI-MS and MS/
MS (Fig. 2,b). The mass shifts observed for the fragment ions, which depend on the
number of their exchangeable protons, support the proposed mechanism in Scheme 1.
All fragment ions are summarized in Table 3.
Table 3. Fragment Ions in the MSn of Chisitine 1 (1) and 2 (2)
Quasi-molecular ion
MSn Experiment Fragment ions, m/z (rel. intensity)
type
m/z
‡
1
[1 ‡ H]
378
MS2 (378)
l, 330(100); m, 302(1); n/n', 230(1); o, 200(1);
p, 182(14); q, 155(3)
MS3 (378 ! 330) m, 302(4); o, 200(6); p, 182(100); q, 155(4); r, 131(4)
‡
(D2)-1 [(D2)-1 ‡ D]
381
408
411
MS2 (381)
(D2)-l, 332(100); (D2)-m, 304(1); (D2)-n/(D2)-n', 232(2);
(D2)-o, 202(4); (D)-p, 183(14); (D)-q, 156(2)
MS3 (381 ! 332) (D2)-m, 304(6); (D2)-o, 202(7); (D)-p, 183(100);
(D)-q, 156(1); r, 131(4)
a, 390(2); b, 380(2); c, 286(9); d, 278(6); e/e', 260(100);
g, 162(9); h, 161(6)
‡
2
[2 ‡ H]
MS2 (408)
MS3 (408 ! 260) f, 189(9); g, 162(97); h, 161(100); i, 134(4); j, 105(14);
k, 99(84)
‡
‡
(D2)-2 [(D2)-2 ‡ D]
MS2 (411)
(D)-a, 391(2); (D3)-b, 383 (3); (D2)-c, 288(12);
(D3)-d, 281(8); (D2)-e/(D2)-e', 262(100);
(D)-g/(D2)-h, 163(14)
MS3 (411 ! 262) (D)-f, 190(2); (D)-g/(D2)-h, 163(100); (D)-i, 135(3);
j, 105(5); (D2)-k, 101(34)
MS2 (410)
[18O]-2 [[18O]-2 ‡ H]
410
[18O]-a, 392(3); [18O]-b, 382(6); c, 286(18); [18O]-d,
280(17); [18O]-e', 262(100); e, 260(84); [18O]-g,
164(13); h, 161(7)
MS3 (410 ! 262) [18O]-f, 191(10); [18O]-g, 164(100); [18O]-i,
136(1); [18O]-j, 107(11); k, 99(89)
MS3 (410 ! 260) h, 161(100)
In the case of chisitine 1 (1), the presence of the (methylthio)carbonyl substituent
instead of the benzoyl group of 2 has dramatic consequences on the MS behavior of 1
(Fig. 3). The S-containing group dominates the fragmentation paths (Scheme 2), which
results in less information to be extracted from the MS/MS data as compared to those
obtained with chisitine 2 (2). Pathway A, which begins with the cleavage of the
cinnamoyl group (À130 Da) and is followed by a dehydration reaction (À18 Da) to
form the two isomeric cyclic fragment ions n and n' at m/z 230 (in analogy to the ions e
and e' in the case of 1), is clearly unfavored (Fig. 3 and Scheme 2). Despite the low
intensity of the ions obtained, Pathway A is taking place and is attested by the
formation of fragment ions p (m/z 182) when n and n' are submitted to an MS3
experiment (spectra not shown).
‡
Quasi-molecular ion [1 ‡ H] (m/z 378) does not undergo dehydration (Pathway B)
but loses MeSH (À48 Da), following the favored Pathway C, to form the intense
fragment ion l at m/z 330 (base peak in MS2) (Scheme 2). The observation can be
rationalized by the fact that MeSH is a better leaving group than H2O. Isolation and
further fragmentation of ion l (m/z 330) leads to the loss of the cinnamoyl moiety

Helvetica Chimica Acta Vol. 87 (2004)
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Fig. 3. MSn plots of the quasi-molecular ions of chisitine 1 (1): a) MS2 (378) (black line) and MS3 (378 ! 330)
‡
‡
(grey line) of [1 ‡ H] , and b) MS2 (381) (black line) and MS3 (381 ! 332) (grey line) of [(D2)-1 ‡ D] .
Fragment ions are designated according to decreasing m/z value (l to r).
(À130 Da) under formation of fragment ion o (m/z 200). Subsequent dehydration
gives bicyclic ion p, recorded at m/z 182 (base peak in MS3). The latter ion can also
derive from ion n' (m/z 230) by expulsion of MeSH (À48 Da). In addition, fragment
ion l (m/z 330) produces the cinnamoyl cation r (m/z 131). Analogously to the above
mentioned H/D-exchange experiment, compound 1 was treated in the same way to give
‡
deuterated analog [(D2)-1 ‡ D] (m/z 381). The ion was investigated by ESI-MS and
MS/MS (Fig. 3,b) to support the mechanism proposed in Scheme 2.
This detailed MS analysis confirmed that the structures of both alkaloids 1 and 2
were correctly assigned. Nevertheless, it seemed important to ensure them by synthesis
and also to study their NMR spectra in detail.
2.4. Synthesis of Chisitine 1 (1) and 2 (2). The two target compounds 1 and 2 were
prepared starting from commercially available mono-Boc-protected butane-1,4-
diamine 3, which was benzylated by reductive amination of benzaldehyde to give
intermediate 4 (Scheme 3). Alkylation of 4 with N-(3-bromopropyl)phthalimide
afforded 5, which was debenzylated to the bis-protected spermidine derivative 6 by
hydrogenolysis. Subsequent derivatization of the secondary-amine function with
cinnamoyl chloride (ˆ(2E)-3-phenylprop-2-enoyl chloride) yielded compound 7. The

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Scheme 2. MS Fragmentation of the Quasi-Molecular Ions of Chisitine 1 (1)
Boc protecting group was removed by the action of CF3COOH, and compound 8 was
obtained as the CF3COOH salt. Formylation of 8 by treatment with 4-nitrophenyl
formate gave the spermidine derivative 9, carrying two of the desired three N-
substituents. Introduction of the last substituent was achieved after the phthalimide-
protecting group was removed by the addition of hydrazine. The resulting primary
amine 10 was treated with S-methyl carbonochloridothioate to provide the desired
alkaloid 1 in 16% overall yield. The reaction of 10 with benzoyl chloride gave

Helvetica Chimica Acta Vol. 87 (2004)
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Scheme 3. Synthetic Route to Chisitine 1 (1) and 2 (2) and Labeled Analog [18O]-2
a) 1. PhCHO, MeOH, r.t., 1 h; 2. NaBH4, 19 h; 95%. b) Br(CH2)3NPhth, K2CO3, 708, 23 h; 78%. c) H2 (4 bar),
Pd/C, EtOH, r.t., 19 h; quant. d) (E)-C6H5CHˆCHCOCl, Et3N, N,N-dimethylpyridin-4-amine (DMAP),
CH2Cl2, r.t., 6 h; 55%. e) CF3COOH, CH2Cl2, r.t., 3 h; quant. f) HCO2(4-NO2C6H4), Et3N, THF, r.t., 1 h; 82%.
g) N2H4 ¥ H2O, EtOH, reflux, 2 h; 60%. h) MeSCOCl or PhCOCl, pyridine, CH2Cl2, r.t., 1 h; 67% (1) or 64%
(2). i) 1. PhC[18O2]H, (COCl)2, DMF, CH2Cl2, r.t., 2 h; 2. 10, Et3N, CH2Cl2, r.t., 2 h; 78%.
1
compound 2 in 13% overall yield. All analytical data (HPLC-MS, MS/MS, H-and
13C-NMR, as well as UV spectra) measured for the two synthetic references were
identical with those of the corresponding natural alkaloids 1 and 2. To study the
fragmentation pathway during MS/MS analysis, labeled [18O]-2 was synthesized
analogously by coupling 10 with 18O-labeled benzoyl chloride, which was prepared
according to a reported procedure [14].
3. Conclusions. Two novel spermidine-derived triamide alkaloids were isolated
from the leaves of C. weinlandii, chisitine 1 (1) and chisitine 2 (2). The structures of the
two compounds were established on the basis of MS and NMR analysis and further
confirmed by comparison with their synthetic references. It was shown that 1 and 2 are
both present as a 1:1 mixture of two isomers when their NMR spectra are recorded at
300 K. The sites of attachment of the substituents were securely established by the
HMBC experiment.
During the mechanistic investigation of the MS fragmentation pathways of the two
compounds by ESI-MS/MS, it was observed that acyl-substituted spermidine deriva-

1422
Helvetica Chimica Acta Vol. 87 (2004)
tives undergo dehydration by a ring-closure reaction. Another distinction comes from
the dependence of the dissociation pathways on the lability of the substituents. The
replacement of PhCO by a MeSCO moiety, which possesses the good leaving group
MeSH, affects the MS/MS fingerprint. The MS/MS fragment ions recorded for the two
related compounds arise from alternative pathways. Attempts to elucidate their
structure solely on the basis of the MS/MS data are, therefore, risky.
We gratefully acknowledge the Swiss National Science Foundation for the generous support of this work,
Ms. N. Walch, and the NMR service of the Institute of Organic Chemistry University of Zurich for the
measurements. We also acknowledge the Ministry of Education, Sciences, Sports, and Culture, and the Ministry
of Health and Welfare, Japan, for financial help.
Experimental Part
1. General. All chemicals were of reagent grade and purchased from Fluka Chemie AG or Merck AG. All
solvents were of anal. grade and were used without further purification unless otherwise stated. THF was dried
over Na/benzophenone. Hydrogenation: Parr-Instruments Company Inc. Flash chromatography (FC): silica gel
Merck 60 (40 63 mm, 230 400 mesh). TLC: Merck precoated silica-gel-60-F254 plates; detection by UV at
254 nm, or by spraying Schlittler reagent (H2PtCl6/HCl/KI) or ninhydrine reagent and heating at 3008 for 1 min.
IR Spectra: Perkin-Elmer 297; in cmÀ1. NMR Spectra: Bruker ARX-300 (at 300.13 (1H) and 75.47 MHz (13C)) at
300 K for all 1D 1H, 13C, DEPT-135, and DEPT-90 experiments; DRX-600 (at 600.13 (1H) and 150.90 MHz
1
(13C)) or DRX-500 (at 500.13 (1H) and 125.76 MHz (13C)) at 300 K for the H, 13C,1H-HSQC, 13C,1H-HMBC,
13C,1H-HSQC-TOCSY experiments of 1 and 2; chemical shifts d in ppm, referenced against residual non-
deuterated solvent; J values in Hz. HPLC-MS: Hewlett-Packard 1100 HPLC system (Hewlett-Packard Co., Palo
Alto, CA, U.S.A.) equipped with a HTS-PAL autosampler (CTC Analytics, Zwingen, Switzerland), connected
to a Bruker ESQUIRE-LC quadrupole ion-trap instrument (Bruker Daltonik GmbH, Bremen, Germany)
equipped with a combined Hewlett-Packard atmospheric-pressure ion (API) source (Hewlett-Packard Co., Palo
Alto, CA, U.S.A.). Direct-infusion ESI-MS and ESI-MSn experiments were performed with a syringe infusion
pump (Cole-Parmer 74900-05; Cole-Parmer Instrument Company, Vernon Hills, IL, U.S.A.).
2. Isolation of Chisitine 1 (1) and 2 (2). The leaves of Chisocheton weinlandii Harms were harvested in 1993
in the garden of the Herbarium Bogoriense, Java, Indonesia, and voucher specimens have been deposited at the
Herbarium of the Faculty of Pharmaceutical Sciences, Setsunan University.
The crushed leaves (60.0 g) were extracted with MeOH (4 Â 1000 ml), and the solvent was distilled. The
MeOH extract (10.9 g) was then suspended in H2O (300 ml), and the aq. suspension was extracted with AcOEt
(4 Â 150 ml). After removal of the solvent, the extract (3.7 g) was chromatographed (silica gel, CHCl3/MeOH
gradient of increasing MeOH content). Fractions containing 1 and 2 (eluted with CHCl3/MeOH 5 :1) were
further purified by semi-prep. HPLC to afford pure chisitine 1 (1; 26.0 mg) and chisitine 2 (2; 15.3 mg). For the
anal. data of 1 and 2, which are identical in all respects to those of the synthetic references, see Sect. 4 below).
3. HPLC-ESI-MS and ESI-MS Investigation. Solvents and reagents: MeCN, MeOH (HPLC grade;
Scharlau, Barcelona, Spain); CF3COOH purum (Fluka, Buchs, Switzerland). H2O was purified with a Milli-QRG
apparatus (Millipore, Milford, MA, U.S.A). Samples preparation: 0.2 mg of solid material (natural or synthetic)
was dissolved in 800 ml of MeOH. For the HPLC-ESI-MS experiments, 10 ml of the stock solns. were injected for
the individual runs; for ESI-MS experiments, the solns were continuously introduced through the electrospray
interface at a flow rate of 6 ml minÀ1. For H/D-exchange experiments, 0.2 mg of solid material was dissolved in
500 ml of a 0.1% soln. of DCl in MeOD (Merck, Darmstadt, Germany) and stirred at 238 for 15 min prior to use.
Column and chromatographic conditions: Interchrom-Uptisphere-C18-HDO column (UP3 HDO#20QS, 3 mm,
2.1 Â 200 mm; Interchim, MontluÁon, France); flow rate 0.2 ml minÀ1; mobile phase: gradient within 30 min
from 20 to 50% of solvent B and finally within 5 min from 50 to 100% of B (solvent A ˆ 0.1% CF3COOH soln. in
H2O, solvent B ˆ 0.1% CF3COOH soln. in MeCN). MS Conditions: nebulizer gas (N2) 40 psi, dry gas (N2) 8.5 l
minÀ1, dry temp. 3008, capillary voltage 4200 V, end plate À 500 V, capillary exit 86 V, and skimmer 1 18 V; data
acquisitions at normal resolution (0.6 Da at half peak height), under ion charge control (ICC) conditions
(10000) in the mass range from m/z 50 to 600 and an m/z 39.3 trap drive value. To get representative mass
spectra, 8 scans were averaged for each spectrum.

Helvetica Chimica Acta Vol. 87 (2004)
1423
4. Synthesis of Chisitine 1 (1) and 2 (2). tert-Butyl [4-(Benzylamino)butyl]carbamate (4). To a soln. of tert-
butyl (4-aminobutyl)carbamate (3; 1.40 g, 7.42 mmol) in MeOH (40 ml), benzaldehyde (0.79 g, 7.43 mmol) was
added and the mixture stirred at 238 for 45 min. It was cooled to 08 (ice/water), NaBH4 (0.32 g, 8.46 mmol) was
added portionwise within 1 h, and the soln. allowed to warm to 238 and stirred for an additional 17 h. After
evaporation, the residue was dissolved in H2O (50 ml) and extracted with CH2Cl2 (3 Â 50 ml) and the org. phase
dried (MgSO4) and evaporated: 4 (1.96 g, 95%). Colorless oil. Rf (CH2Cl2/MeOH/25% NH4OH 9 :9 :1): 0.64. IR
(film): 3340s, 2960s, 2920s, 2850s, 2810s, 1950w, 1875w, 1810w, 1700s, 1610m, 1520s, 1455s, 1390s, 1370s, 1275s,
1250s, 1170s, 1030s, 920m, 870m, 780m, 740s, 700s. 1H-NMR (CDCl3): 7.31 7.17 (m, 5 H); 4.83 (br. s, 1 H); 3.73
(s, 2 H); 3.08 3.06 (m, 2 H); 2.62 2.57 (m, 2 H); 1.50 1.46 (m, 4 H); 1.39 (s, 9 H). 13C-NMR (CDCl3): 156.1
(s); 140.6 (s); 128.5 (d); 128.3 (d); 127.1 (d); 79.1 (s); 54.2 (t); 49.1 (t); 40.6 (t); 28.6 (q); 28.0 (t); 27.6 (t). ESI-MS
‡
(MeOH): 279.2 ([M ‡ H] ).
tert-Butyl {4-{Benzyl[3-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)propyl]amino}butyl}carbamate (5).
K2CO3 (0.52 g, 3.78 mmol) was added to a soln. of 4 (0.96 g, 3.43 mmol) in MeCN (15 ml) followed by N-(3-
bromopropyl)phthalimide (1.01 g, 3.78 mmol), and the heterogeneous mixture was stirred at 708 for 23 h. The
soln. was poured on H2O (50 ml) and extracted with CH2Cl2 (3 Â 50 ml), the combined org. phase dried
(MgSO4) and evaporated, and the crude mixture purified by FC (hexane/AcOEt 3 :2 ! 1:1): pure 5 (1.80 g,
78%). Slightly yellow oil. Rf (CH2Cl2/MeOH 1:1): 0.58. IR (film): 3360s, 2990s, 2900s, 2830s, 2780s, 1940w, 1755s,
1690s, 1600s, 1500s, 1435s, 1380s, 1230s, 1155s, 1020s, 900s, 870s, 855s, 705s, 685s, 655s. 1H-NMR (CDCl3): 7.84
7.81 (m, 2 H); 7.71 7.68 (m, 2 H); 7.31 7.17 (m, 5 H); 4.74 (br. s, 1 H); 3.69 (t, J ˆ 7.6, 2 H); 3.52 (s, 2 H); 3.07
3.06 (m, 2 H); 2.50 2.40 (m, 4 H); 1.83 (quint., J ˆ 7.3, 2 H); 1.50 1.45 (m, 4 H); 1.43 (s, 9 H). 13C-NMR
(CDCl3): 168.5 (s); 156.2 (s); 139.8 (s); 134.0 (d); 132.4 (s); 129.1 (d); 128.3 (d); 127.0 (d); 123.3 (d); 79.1 (s); 58.8
(t); 53.6 (t); 51.4 (t); 40.7 (t); 36.6 (t); 28.6 (q); 28.0 (t); 26.3 (t); 24.7 (t). ESI-MS (MeOH ‡ NaI): 488.3 ([M ‡
‡
Na] ).
tert-Butyl {4-{[3-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)propyl]amino}butyl}carbamate (6). A mixture
of 5 (1.18 g, 2.54 mmol) and Pd/C (200 mg) in EtOH (100 ml) was stirred for 19 h at 238 under a H2 pressure of
¾
4 bar [15]. The catalyst was filtered off via Celite and washed with MeOH (150 ml). The combined filtrate was
evaporated: 6 (1.26 g, quant.). Colorless solid. Rf (CH2Cl2/MeOH 1:1): 0.61. IR (film): 3370m, 2940m, 2790m,
2440w, 2780s, 1770w, 1705s, 1690s, 1600w, 1530m, 1465m, 1440m, 1400m, 1365m, 1270m, 1250m, 1175m, 715m.
1H-NMR (CDCl3): 7.84 7.81 (m, 2 H); 7.72 7.69 (m, 2 H); 5.14 (br. s, 1 H); 3.85 (t, J ˆ 6.5, 2 H); 3.17 3.11
(m, 2 H); 3.04 2.95 (m, 4 H); 2.32 2.27 (m, 2 H); 1.95 1.85 (m, 2 H); 1.66 1.47 (m, 2 H); 1.41 (s, 9 H).
13C-NMR (CDCl3): 168.6 (s); 156.5 (s); 134.3 (d); 132.1 (s); 123.6 (d); 79.5 (s); 47.8 (t); 45.8 (t); 39.9 (t); 35.2 (t);
‡
‡
28.6 (q); 27.3 (t); 25.8 (t); 23.3 (t). ESI-MS (MeOH): 320.2 (5), 376.3 (100, [M ‡ H] ), 398.2 (6, [M ‡ Na] ).
tert-Butyl {4-{[3-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)propyl][(2E)-3-phenylprop-2-enoyl]amino}bu-
tyl}carbamate (7). According to [16], Et3N (3.67 g, 36.29 mmol) and DMAP (42 mg, 0.34 mmol) were added
to a soln. of 6 (948 mg, 2.54 mmol) in CH2Cl2 (25 ml). After stirring at 238 for 30 min, (2E)-3-phenylprop-2-
enoyl chloride (847 mg, 5.08 mmol) was added, and the mixture was stirred at 238 for an additional 6 h. The soln.
was washed with sat. aq. NH4Cl soln. (3 Â 30 ml) followed by sat. aq. Na2CO3 soln. (3 Â 30 ml). The org. layer
was dried (MgSO4) and evaporated. The crude mixture was purified by FC (hexane/AcOEt 1:1 ! 2 :3): pure 7
(707 mg, 55%). Yellow oil. Rf (hexane/AcOEt 1:1): 0.11. IR (film): 3350m, 3070m, 2980s, 2940s, 2870m, 1775s,
1710s, 1655s, 1610s, 1525s, 1475s, 1460s, 1445s, 1405s, 1375s, 1340s, 1280s, 1260s, 1180s, 1150m, 1100m, 1080m,
1045m, 985m, 900m, 865m, 775s, 730s, 695m, 675m. 1H-NMR (CDCl3): 7.88 7.82 (m, 2 H); 7.74 7.63 (m, 3 H);
7.52 7.43 (2 m, 2 H); 7.36 7.31 (m, 3 H); 6.83, 6.76 (2 d, J ˆ 15.4 each, ratio 1:1, 1 H); 4.65 (br. s, 1 H); 3.77
3.72 (m, 2 H); 3.55 3.44 (m, 4 H); 3.18 3.12 (m, 2 H); 2.10 1.95 (m, 2 H); 1.75 1.62 (m, 2 H); 1.57 1.47
(m, 2 H); 1.41 (s, 9 H). 13C-NMR (CDCl3): 168.4 (s); 166.5, 166.4 (2 s); 156.2 (s); 143.0 (d); 135.5 (s); 134.3,
134.1 (2 d); 132.3, 132.1 (2 s); 129.7, 128.9, 128.0 (3 d); 123.6, 123.4 (2 d); 117.5, 117.3 (2 d); 79.4 (s); 48.0, 46.6,
46.1, 44.6 (4 t); 40.2 (t); 36.2, 35.7 (2 t); 29.0 (t); 28.6 (q); 27.8, 27.6, 27.4, 27.2, 25.4 (5 t). ESI-MS (MeOH ‡ NaI):
‡
528.3 ([M ‡ Na] ).
4-{[3-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)propyl][(2E)-3-phenylprop-2-enoyl]amino}butan-1-ammo-
nium Trifluoroacetate (8). CF3COOH (1.58 g, 13.85 mmol) was added dropwise to a soln. of 7 (700 mg,
1.39 mmol) in CH2Cl2 (10 ml), and the mixture was stirred at 238 for 3 h. Evaporation gave 8 (929 mg, quant.).
Brownish oil. Rf (CH2Cl2/MeOH 1:1): 0.50. IR (film): 3310m, 1805s, 1745s, 1680s, 1615m, 1535m, 1505m, 1475m,
1440s, 1420m, 1370m, 1340m, 1240s, 1205s, 1110m, 1065m, 1010m, 850m, 830m, 805m, 760s, 745m. 1H-NMR
(CDCl3): 7.74 7.62 (m, 4 H); 7.47 (d, J ˆ 15.4, 1 H); 7.35 7.18 (m, 5 H); 6.64 (d, J ˆ 15.4, 1 H); 3.68 3.58
(m, 2 H); 3.47 3.41 (m, 4 H); 3.12 3.10 (m, 2 H); 2.03 1.94, 1.92 1.87 (2 m, ratio 3 :1, 2 H); 1.72 1.65
(m, 4 H). 13C-NMR (CDCl3): 169.5, 169.3 (2 s); 168.9 (s); 160.7 (q, J(CO,F) ˆ 41.2); 146.0 (d); 134.8, 134.7
(2 d); 134.2 (s); 131.7 (2 s); 130.9, 129.2, 128.3 (3 d); 123.8, 123.7 (2 d); 115.2 (q, J(C,F) ˆ 286.9); 115.3, 115.2

1424
Helvetica Chimica Acta Vol. 87 (2004)
(2 d); 48.6, 47.0, 46.5, 45.8 (4 t); 40.5, 40.2 (2 t); 35.9, 35.5 (2 t); 28.3, 27.4, 26.4, 24.7, 24.4, 24.0 (6 t). ESI-MS
‡
‡
(MeOH): 406.3 (100, [M ‡ H] ), 428.2 (6, [M ‡ Na] ).
(2E)-N-[3-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)propyl]-N-[4-(formylamino)butyl]-3-phenylprop-2-
enamide (9). According to [17], 4-nitrophenyl formate (231 mg, 1.38 mmol) in THF (4 ml) was added dropwise
to a soln. of 8 (700 mg, 1.35 mmol) and Et3N (684 mg, 6.76 mmol) in THF (10 ml). The mixture was stirred at
238 for 1 h. Evaporation and purification of the crude product by FC (CH2Cl2/MeOH 93 :7 ! 9 :1) gave pure 9
(481 mg, 82%). Yellowish oil. Rf (CH2Cl2/MeOH 9 :1): 0.39. IR (film): 3370m, 3140m, 3020m, 2950m, 1810s,
1750s, 1715s, 1690s, 1640s, 1575m, 1540m, 1475s, 1440s, 1420s, 1370m, 1345m, 1230m, 1175m, 1115m, 1070m,
1020m, 930m, 805s, 760s. 1H-NMR (CDCl3): 8.16 (br. s, 1 H); 7.86 7.81 (m, 2 H); 7.74 7.62 (m, 3 H); 7.51 7.31
(m, 5 H); 6.81, 6.76 (2 d, J ˆ 15.4 each, ratio 1 :1, 1 H); 6.40, 6.01 (2 br. s, 1 H); 3.78 3.71 (m, 2 H); 3.55 3.43
(m, 4 H); 3.38 3.23 (m, 2 H); 2.10 1.92 (m, 2 H); 1.68 1.52 (m, 4 H). 13C-NMR (CDCl3): 168.4 (s); 166.6 (s);
164.7, 161.5 (2 d); 143.3, 143.1 (2 d); 135.4, 135.3 (2 s); 134.4, 134.3 (2 d); 132.2, 132.1 (2 s); 129.8, 129.0, 128.0,
123.6, 123.4 (5 d); 117.4, 117.1 (2 d); 48.1, 46.4, 46.3, 44.8 (4 t); 37.7 (t); 36.2, 35.7 (2 t); 29.0, 27.3, 27.2, 26.3, 25.5
‡
(5 t). ESI-MS (MeOH ‡ NaI): 456.3 ([M ‡ Na] ).
(2E)-N-(3-Aminopropyl)-N-[4-(formylamino)butyl]-3-phenylprop-2-enamide (10). According to [16],
N2H4 ¥ H2O (412 mg, 8.23 mmol) was added to a soln. of 9 (360 mg, 0.83 mmol) in EtOH (10 ml), and the
mixture was stirred under reflux for 2 h. After cooling, the solvent was evaporated, the residue treated with 2n
aq. HCl (10 ml), the mixture filtered, and the filtrate basified with sat. aq. Na2CO3 soln. (15 ml). The aq. layer
was extracted with CH2Cl2 (3 Â 20 ml) and the combined org. layer dried (MgSO4) and evaporated: 10 (152 mg,
60%). Yellowish oil. Rf (CH2Cl2/MeOH 1:1): 0.05. IR (film): 3360m, 3130m, 3010m, 2940m, 1710s, 1685s, 1635s,
1535m, 1495s, 1465s, 1420m, 1365m, 1340m, 1320m, 1270m, 1240m, 1220m, 1020m, 805m, 750m, 725m. 1H-NMR
(CDCl3): 8.14 (s, 1 H); 7.67 7.66, 7.62 7.60, 7.57 7.56 (3 m, ratio 43 :50 :7, 1 H); 7.51 7.42, 7.34 7.28
(2 m, 5 H); 6.96, 6.79, 6.41 (3 d, J ꢀ 15.4 each, 1 H); 6.77, 6.54 (2 br. s, 1 H); 3.51 3.39 (m, 4 H); 3.34 3.21
(m, 2 H); 2.76 2.58 (m, 2 H); 1.74 1.50 (m, 8 H). 13C-NMR (CDCl3): 166.8, 166.7 (2 s); 164.8, 161.6 (2 d);
142.9, 142.7 (2 d); 135.4, 135.3 (2 s); 129.7, 128.9, 128.0 (3 d); 117.9, 117.4 (2 d); 47.6, 46.2, 45.8, 43.9, 39.3, 39.1,
37.7, 37.5 (8 t); 33.2, 31.4 (2 t); 27.1, 26.9, 26.4, 25.4 (4 t). ESI-MS (MeOH ‡ HCOOH): 286.2 (6), 304.2 (100,
‡
‡
[M ‡ H] ), 326.2 (6, [M ‡ Na] ).
S-Methyl{3-{[4-(Formylamino)butyl][(2E)-3-phenylprop-2-enoyl]amino}propyl}carbamothioate
(ˆChisitine 1; 1). In analogy to [18], S-methyl carbonochloridothioate (43 mg, 0.39 mmol) was added dropwise
to a soln. of 10 (107 mg, 0.35 mmol) and pyridine (31 mg, 0.39 mmol) in CH2Cl2 (3 ml) and the mixture was
stirred at 238 for 1 h. Evaporation and purification of the crude mixture by FC (CH2Cl2/MeOH 95 :5 ! 9 :1)
gave pure 1 (90 mg, 67%). Yellowish oil that solidified upon standing at 48. M.p. 89 908. Rf (CH2Cl2/MeOH
9 :1): 0.31. IR (film): 3350s, 3120m, 3000m, 2940m, 1705s, 1685s, 1635s, 1570s, 1535s, 1520s, 1495s, 1470s, 1420s,
1365m, 1340m, 1255s, 1160m, 1015m, 800m, 745m, 725m. NMR: Tables 1 and 2. ESI-MS (MeOH): 378.2 (26,
‡
‡
‡
[M ‡ H] ), 400.3 (88, [M ‡ Na] ), 416.1 (100, [M ‡ K] ). MSn: Table 3. HPLC-UV-MS: tR 23.8 min; lmax 204,
218, 282 nm.
N-{3-{[4-(Formylamino)butyl][(2E)-3-phenylprop-2-enoyl]amino}propyl}benzamide (ˆChisitine 2; 2).
According to [19], benzoyl chloride (18 mg, 0.13 mmol) was added dropwise to a soln. of 10 (36 mg,
0.12 mmol) and pyridine (10 mg, 0.13 mmol) in CH2Cl2 (2 ml) and the mixture was stirred at 238 for 2 h.
Evaporation and purification of the crude mixture by FC (CH2Cl2/MeOH 95 :5 ! 9 :1) gave pure 2 (31 mg,
64%). Yellow oil that solidified upon standing at 48. M.p. 102 1038. Rf (CH2Cl2/MeOH 9 :1): 0.25. IR (film):
3290s, 3050m, 2920s, 2860m, 2230w, 1645s, 1595s, 1535s, 1485s, 1455s, 1430s, 1380s, 1325s, 1300s, 1240m, 1190s,
1120m, 1090m, 1070m, 1025m, 975m, 910m, 855m, 800m, 765s, 700s. NMR: Tables 1 and 2. ESI-MS (MeOH):
‡
‡
‡
408.3 (63, [M ‡ H] ), 430.2 (100, [M ‡ Na] ), 446.1 (70, [M ‡ K] ). MSn: Table 3. HPLC-UV-MS: tR 25.0 min;
lmax 220, 280 nm.
N-{3-{[4-(Formylamino)butyl][(2E)-3-phenylprop-2-enoyl]amino}propyl}benz[18O]amide ([18O]-2).
(COCl)2 (9 mg, 0.07 mmol) was added to a cold (ice/water) soln. of [18O2]benzoic acid (8 mg, 0.07 mmol;
prepared according to [14]) and DMF (ca. 0.5 ml, catalyst) in CH2Cl2 (1.5 ml), and the resultant mixture was
allowed to warm to 238 and stirred for an additional 2 h. After evaporation of the oil, the residue was dissolved in
CH2Cl2 (1 ml) and the mixture cooled to 08 (ice/water) and treated with a soln. of 10 (10 mg, 0.03 mmol) and
Et3N (7 mg, 0.07 mmol) in CH2Cl2 (1.5 ml). After stirring at 238 for 2 h, the solvent was evaporated and the
crude mixture purified by prep. TLC (CH2Cl2/MeOH 9 :1): [18O]-2 (11 mg, 78%). Rf (CH2Cl2/MeOH 9 :1): 0.25.
‡
ESI-MS (MeOH ‡ NaI): 432.3 ([M ‡ Na] ). MSn: Table 3.

Helvetica Chimica Acta Vol. 87 (2004)
1425
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Received December 17, 2003