Biomimetic formation of macrocyclic spermine alkaloids

Article abstract of DOI:10.1002/1522-2675(20010711)84:7<2108::AID-HLCA2108>3.0.CO;2-G

Dihydroxyverbacine (10), a precursor for oxidative phenol coupling, was obtained via (±)-buchnerine (14), whose synthesis is described. The oxidizing system hemin (ferriprotoporphyrin IX chloride)/H₂O₂ promoted intramolecular coupling of 10 to give the alkaloids aphelandrine (1), orantine (2), and chaenorpine (7). The alkaloids were identified by on-line coupled HPLC and atmospheric-pressure chemical-ionization (APCI) mass spectrometry.

Full text of DOI:10.1002/1522-2675(20010711)84:7<2108::AID-HLCA2108>3.0.CO;2-G

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Helvetica Chimica Acta ± Vol. 84 (2001)
Biomimetic Formation of Macrocyclic Spermine Alkaloids
by Vladimir Dimitrov, Herve Geneste, Armin Guggisberg, and Manfred Hesse*
Organisch-chemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich
Dedicated to Professor Heinz Heimgartner on the occasion of his 60th birthday
Dihydroxyverbacine (10), a precursor for oxidative phenol coupling, was obtained via (Æ)-buchnerine (14),
whose synthesis is described. The oxidizing system hemin (ferriprotoporphyrin IX chloride)/H2O2 promoted
intramolecular coupling of 10 to give the alkaloids aphelandrine (1), orantine (2), and chaenorpine (7). The
alkaloids were identified by on-line coupled HPLC and atmospheric-pressure chemical-ionization (APCI) mass
spectrometry.
Introduction. ± The polyamines spermine and spermidine, which naturally occur in
a variety of living organisms, have been shown to be crucial, e.g., for cell growth,
carcinogenesis, and neurotransmission [1][2]. The polyamine derivatives have been
observed as alkaloids in linear conjugates or as macrocyclic lactams [1]. In the case of
putrescine alkaloids, the base is preferably incorporated in a chain, whereas, in
spermidine and spermine alkaloids, the cyclic form is predominant mostly in 13- or 17-
membered lactam rings [1].
Aphelandrine (1), its diastereoisomer orantine (2), and chaenorpine (7) are
examples of spermine alkaloids (Scheme 1). Both alkaloids 1 and 2 have been found in
several Aphelandra species [3 ± 5]. Aphelandrine was identified also in Encephalos-
phaera lasiandra (Acanthaceae) [4]. Orantine was further observed in Chaenorhinum
minus [6], Schweinfurthia papilionacea (Scrophulariaceae) [7], and in Ephedra sp.
(Ephedraceae) [8]. Finally, the alkaloid chaenorpine was identified in the basic extract
of Chaenorhinum minus [6]. The biosynthesis of these alkaloids, in particular that of
aphelandrine (1), has long been in the center of our interest [9]. The biogenetic
precursor should be a macrocyclic compound of type 9, which provides, after oxidation
and oxidative phenol coupling, the alkaloids presented in Scheme 1. The hypothesis of
Barton and Cohen was that the biosynthesis of a variety of natural products includes
oxidative coupling of phenols [10]. This hypothesis has been confirmed by the
identification of the enzyme, membrane-bound cytochrome P-450, that catalyzes the
transformation of the benzyltetrahydroisoquinoline alkaloid (R)-reticuline into
salutaridine; the latter is a key intermediate in morphine synthesis [11].
For the biogenesis of the alkaloids shown in Scheme 1, (S)-dihydroxyverbacine
((S)-10) seems to be very suitable as a precursor. Several pathways are possible for the
formation of (S)-10 (Scheme 2). The open-chained species 11a and 11b can lead, after
aza-Michael addition, to protoverbine (12) and prelandrine (13), respectively. In an
additional step, prelandrine has to be transformed into (S)-dihydroxyverbacine ((S)-
10). Another possibility is the formation of buchnerine (14) from 11c, after aza-Michael

Helvetica Chimica Acta ± Vol. 84 (2001)
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Scheme 1
addition, its further acylation to [(E)-4-methoxycinnamoyl]buchnerine (15), followed
by transformation to 10. It is also possible that 10 is formed by an aza-Michael addition
of the N,N'-dicoumaroylspermine (16) [9]. Actually, some of the compounds presented
in Scheme 2 are alkaloids that have been identified in different plants. Protoverbine
(12) was isolated from Verbascum pseudonobile Soj. et Stef. (Scrophulariaceae) [12],
and buchnerine (14) was found in Clerodendrum buchneri (Verbenaceae) [13]. An
analogue of dihydroxyverbacine (10), the [(Z)-4-methoxycinnamoyl]buchnerine, was
identified in C. buchneri [13].
Three recent important findings considerably support the biosynthetic pathway of
aphelandrine (1) proceeding via compounds 12, 13, and (S)-10. Protoverbine (12) could
be transformed to prelandrine (13) by incubation with cytochrome P-450-containing
microsomes from the roots of A. squarrosa in the presence of NADPH [14].
Prelandrine (13) was detected in the roots of A. squarrosa [14]. The formation of
aphelandrine (1) was realized after microsome-assisted oxidation of (S)-dihydroxy-
verbacine ((S)-10) and its D8-labeled analogue [15][16].
The task of the present work was to find a biomimetic way for oxidative phenol
coupling of 10 leading to the formation of any of the above discussed alkaloids by
means of the simple ꢀinstrumentsꢁ of synthetic organic chemistry. In this paper, we
describe the results of the nonenzymatic oxidative phenol coupling of dihydroxyver-
bacine (10) leading to aphelandrine (1), orantine (2), and chaenorpine (7).
Results and Discussion. ± Dihydroxyverbacine (10) as Precursor for Oxidative
Phenol Coupling. There are two possible synthetic pathways to 10 proceeding via
buchnerine (14): a) the route to the racemic product 10, and b) those providing the

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Helvetica Chimica Acta ± Vol. 84 (2001)
Scheme 2
possibility to prepare (S)-dihydroxyverbacine ((S)-10) (Scheme 3). Racemic buchner-
ine (14) was synthesized in two steps via 19 from spermine (17) by the reaction with b-
keto ester 18 [17], followed by Sb(OEt)3-promoted macrolactamization, as described
by Yamamoto and co-workers [18]. (S)-Buchnerine ((S)-14) was prepared starting
from (S)-b-amino-b-(4-methoxyphenyl) ester 20 via 21 ± 24, which was further
transformed in four steps to (S)-dihydroxyverbacine ((S)-10) according to the recently
published procedures [16][19].
In the synthetic step from 23 to 24, the formation of an interesting by-product was observed, which is worth
mentioning. The transformation of 23 was performed on a 1 ± 2 g scale, and, therefore, it was possible to isolate
the by-product 25 in sufficient quantity. We suggest for 25 the structure of a carbamic acid (see Scheme 4), based
on the arguments presented below. Two feats were critical for the structure elucidation of 25, namely to
establish the presence of the (N)COOH group as a substituent and to determine the place of the substitution.
The occurrence of the (N)COOH group at the macrocyclic framework of 25 was deduced by means of chemical
transformations and MS investigations. The MS experiments with 25 showed the presence of an additional 44-
amu moiety, which we presume to be CO2 in view of the reagents used in the reaction 23 ! 24. Indeed, the
formation of 25 may be understood to be the result of CO2 insertion into the N H bond leading to the
carbamic-acid derivative. Similar insertion reactions with CO2 have recently been reported [20]. The molecular
‡
‡
mass of 25 was determined by ESI-MS. The quasi-molecular ions at m/z 849 ([M ‡ H] ), 871 ([M ‡ Na] ), and
‡
887 ([M ‡ K] ) showed the characteristic isotope pattern resulting from the presence of one Br-atom, which was
confirmed by microanalysis. The ESI-MS/MS experiments with 25 gave the fragments shown in Scheme 4, that,
with m/z 805, indicate the loss of CO2.
Compound 25 was treated with morpholine to afford 26, which was detosylated to 27. The quasi-molecular
‡
‡
ions at m/z 856 ([M ‡ H] ) and 548 ([M ‡ H] ) in the ESI-MS of 26 and 27, respectively, were in accordance

Helvetica Chimica Acta ± Vol. 84 (2001)
2111
Scheme 3
with the ꢀnitrogen ruleꢁ (odd number of N-atoms). The loss of CO2 (m/z 504), was also observed in the CI-MS of
27, and the CI-MS was in accordance with the ESI investigations.
The NMR data (1H, 13C, HSQC, HMBC, NOESY, and TOCSY of 25 ± 27 in different solvents (CDCl3,
CD3OD, (D6)DMSO, (D8)toluene, and (D5)pyridine) and at different temperatures (from 508 up to 808) were
difficult to analyze. The spectra were temperature-dependent and exhibited broad signals in all cases. Obviously,
several possible conformations of 25 ± 27 are present, due to the flexibility of the macrocyclic ring, which
prevented access to detailed structure information. However, the NMR data established the presence of two
different carbonyl C-atoms, which is an additional argument for the suggested COOH group. However, no
conclusions concerning the position of the COOH and C4H8X (XˆBr or morpholino) substituents could be
drawn. Moreover, 25 ± 27 could not be crystallized, and, thus, the question of the exact structure of 25 ± 27
remains open.
Oxidative Phenol Coupling and On-Line HPLC/APCI-MS and HPLC/APCI-MS/
MS Investigations. We checked several reagents known for their potential to induce
oxidative phenol coupling [21 ± 24]. Thus, the complex CuCl(OH) ´ TMEDA
(TMEDA ˆ N,N,N',N'-tetramethylethylenediamine) is an excellent catalyst for the
oxidative binaphthyl coupling [21]. For the oxidation of dihydroxyverbacine (10), its
CH2Cl2 solution at 08 was saturated with O2 in the presence of 10 mol-% of
CuCl(OH) ´ TMEDA, according to the conditions that were recently used for the
preparation of steroidal binaphthyl derivatives [22]. However, after two days stirring at
room temperature, HPLC coupled on-line with atmospheric-pressure chemical-

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Helvetica Chimica Acta ± Vol. 84 (2001)
Scheme 4
‡
ionization mass spectrometry (APCI-MS) showed that 10 ([M ‡ H] at m/z 495) had
not reacted. Thus, we prepared an analogous Cu complex with 10 itself as ligand instead
of TMEDA, anticipating a possible template effect, the generation of radicals within
the macrocyclic framework, and the participation of the metal center in close proximity
to the phenol moiety of the molecule. For this purpose 10 was treated with CuCl in 95%
aqueous MeOH under the same conditions as for the preparation of CuCl(OH) ´
TMEDA [21]. The formation of a complex CuCl ´ 10 was strongly suggested by the
observed color change from initially slightly yellow to blue-green and finally blue-
violet, in accordance with the observations made on formation of CuCl(OH) ´ TME-
DA. After solvent evaporation, CH2Cl2 was added to CuCl ´ 10 and the mixture
saturated with O2 at 08 and stirred for two days at room temperature. But, also in this
case, the precursor 10 remained unchanged. The use of other metal salts, CuCl2 and
FeCl3 in CHCl3/MeOH or 0.1m aqueous K2CO3/MeCN did not produce any coupling
products.
It is known that oxoammonium salts, such as 4-(acetylamino)-2,2,6,6-tetramethyl-1-
oxopiperidinium tetrafluoroborate, are reagents for oxidative phenol coupling in
MeCN/H2O in the presence of KHCO3 [23]. However, when the oxoammonium
oxidizing reagent was applied to 10, no coupling products could be detected, and 10

Helvetica Chimica Acta ± Vol. 84 (2001)
2113
remained unchanged after 3 days. Thus, dihydroxyverbacine (10) seems to be
surprisingly stable under oxidative conditions.
In further experiments, we used hemin (ferriprotoporphyrin IX chloride) in
combination with H2O2 as oxidizing agent. The following arguments led to this choice.
There are several types of heme proteins that contain the iron protoporphyrin IX group
± hemoglobins and myoglobins that are responsible for the storage and the transport of
O2, and cytochromes that are involved in the transfer of electrons. The ability of
cytochrome P-450 to promote the oxidative phenol coupling of 10 to aphelandrine (1)
has recently been demonstrated [15]. Although, to the best of our knowledge, neither
hemin (see Scheme 5) nor any analogous heme protein has been used for oxidative
phenol coupling, it seemed appropriate to apply it as reagent in H2O2 oxidation
experiments. Metalloporphyrin-catalyzed oxidations, also those with H2O2, have been
the object of a variety of investigations [24]. Thus, a dark red-brown solution of
dihydroxyverbacine (10) and 30 mol-% of hemin in 0.1m aqueous K2CO3/MeCN 1:1
Scheme 5

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Helvetica Chimica Acta ± Vol. 84 (2001)
was treated with 10% aqueous H2O2 solution at room temperature. On-line coupled
HPLC/APCI-MS and HPLC/APCI-MS/MS (reversed-phase HPLC column and
conditions) of the homogeneous reaction mixture, revealed the formation of the
‡
desired coupling products at m/z 493 ([M ‡ H] ), already 15 min after the addition of
H2O2 (Mr 492 for the isomeric alkaloids aphelandrine (1), orantine (2), and
chaenorpine (7)). The main component of the reaction mixture during the first 1.5 h
‡
was unreacted 10 (m/z 495 ([M ‡ H] ), tR 8.60 min). Besides the desired products at
m/z 493, some decomposition products with tR between 13 and 21 min originating from
10, but possibly also from hemin, were observed. The by-product at m/z 347
(13.03 min), formed from 10 by loss of the cinnamoyl fragment, became the main
component after 3 h reaction time. It must be pointed out that, when 10 was stirred with
H2O2 alone for several hours, the main component in the mixture was unchanged 10,
and only an insufficient quantity of an oxidation product at m/z 527 (tR 4.57 min; [(10 ‡
‡
32) ‡ H] ) was observed.
The aim of the HPLC/MS investigations was to detect coupling products with Mr
492. The best way to observe the products of oxidative phenol coupling was to detect
them selectively in the selected-ion-monitoring mode (SIM). However, the additional
use of MS/MS techniques (tandem mass spectrometry) provided more structural
information. The triple-stage quadrupole mass spectrometer allowed the isolation of
the quasi-molecular ions with the first quadrupole, the collision-induced dissociation
(CID) with the second quadrupole used as collision cell, and the recording of the
corresponding daughter-ion spectra with the third quadrupole [25]. Several products
possessing Mr 492 and MS/MS closely similar to each other were thus observed (Fig.)
and the alkaloids aphelandrine (1), orantine (2), and chaenorpine (7) could
unambiguously be identified by the addition of authentic 1, 2, or 7, respectively, to
the reaction mixture. The on-line MS/MS of the coupling products obtained and those
of 1, 2, and 7 were almost identical (Table). The fragments at m/z 251, 265, and 476
(Fig.) were the most intense signals in the MS/MS of the coupling products and are
typical for aphelandrine (1), orantine (2), and chaenorpine (7). The fragment ions B2
Table. MS/MS Data of the Coupling Products of Dihydroxyverbacine (10) and of Aphelandrine (1), Orantine
(2) and Chaenorpine (7)
Peaks in the HPLC/APCI-MS/MS
A1 A2 A3 A4 A5
(Orantine)
Pure alkaloids
A6
1
2
7
(Aphelandrine) (Chaenorpine) (Aphelandrine) (Orantine) (Chaenorpine)
m/z m/z
m/z m/z m/z
m/z
m/z
m/z
m/z
476 476
±
±
±
±
464
±
476
±
±
476
±
419
348
±
265
251
±
476
464
419
348
322
265
251
198
155
129
112
476
464
419
348
322
265
251
198
155
129
112
476
464
419
348
322
265
251
±
464
±
419 419
348 348
322 322
265 265
251 251
348 348 348
±
265 265 265
251 251 251
±
155
129
±
±
±
±
±
198
155
±
±
±
198
±
129
±
129
112
155
129
112
129 129
112 112
112 112

Helvetica Chimica Acta ± Vol. 84 (2001)
2115
Figure. HPLC/APCI-MS Chromatogram of the coupling products of dihydroxyverbacine (10): a) reconstructed
ion chromatogram (RIC) after CID of the quasi-molecular ions m/z 493 and b) ± d) extracted-ion chromatograms
of daughter fragment ions m/z 198, 251, 265, and 476

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Helvetica Chimica Acta ± Vol. 84 (2001)
and B5 at m/z 198 were characteristic only for 1 and 2 and can, therefore, be used as a
finger print for these alkaloids. The structure of the coupling products A1 (3.59 min),
A3 (8.30 min), and A4 (9.22 min) could not definitively be elucidated (see also below).
Some conclusions and propositions according to the coupling possibilities seem
appropriate. The hemin forms, after reaction with H2O2, an oxoiron(IV) radical cation
that can act as a two-electron oxidizing reagent [24][26]. The heme proteins perform
their oxidations within a catalytic cycle, which is possible also for the hemin
(Scheme 5). In the case of the precursor 10, radical cations can be formed in both
phenol moieties of the molecule. Concerning the coupling reaction, Barton and Cohen
[10] favored the ꢀradical pairing hypothesisꢁ, although they envisaged as well the
ꢀoxidation substitution oxidationꢁ mechanism. However, it is possible that in the case of
compound 10, both mechanisms play a role, e.g., the ꢀoxidation substitution oxidationꢁ
mechanism would provide 1 and 2, whereas the radical pairing would be more
appropriate for the formation of chaenorpine or chaenorhine analogue (Scheme 5). It
is reasonable to assume that the coupling occurs predominantly at positions with higher
electron density. Computer calculations suggested the location of the electron density
at the positions shown in Scheme 5. Additionally, a high electron density is located at
the para position to the OH group, which is, however, highly hindered. These
calculations are in accordance with the observed coupling products. For the formation
of aphelandrine (1) and orantine (2), the configuration of the double bond (E or Z) in
the precursor 10 should not affect the cyclization. In this case, the orientation (Re or Si
face of the CˆC bond) should play a significant role. It must be pointed out that in the
observed chaenorpine (7), the CˆC bond possesses the (Z)-configuration. That, from
the (E)-configurated precursor 10 chaenorpine (7) was obtained, indicates an
isomerization after the formation of the radical-cation species (Scheme 5). The
coupling products A1, A3, and A4 (see above and Fig.) are probably chaenorhine
analogues ((E) and (Z) isomers are possible) and (E)-chaenorpine. It should be
further pointed out that the oxidative phenol coupling presented was relatively fast, but
obviously not selective, according to the reagents and conditions applied.
In conclusion, it was demonstrated that the oxidative phenol coupling of
dihydroxyverbacine (10), according to the biogenetic pathway, leading to the naturally
occurring aphelandrine (1), orantine (2), and chaenorpine (7) is possible by means of
the simple ꢀinstrumentsꢁ of synthetic organic chemistry.
We thank the Swiss National Science Foundation and the Dr. Helmut Legerlotz Stiftung of the Organisch-
chemisches Institut der Universität Zürich for generous financial support. V. D. thanks Dr. K. Drandarov for
providing his experience for the synthesis of dihydroxyverbacine and Dr. L. Bigler for the introduction in the
secrets of mass spectrometry. We thank Dr. R. Kunz for the computer calculations.
Experimental Part
General. Materials and solvents: ( )-(8S)-1-[(E)-3-(4-Hydroxyphenyl)prop-2-enoyl]-8-(4-hydroxyphen-
yl)-1,5,9,13-tetraazacycloheptadecan-6-one ((S)-10) was prepared according to [16][19]; MeCN (HPLC-grade,
Scharlau, E-Barcelona); AcOH (Fluka, purum, Switzerland); the water was purified with an Milli-QRG
apparatus (Millipore, Milford, MA, USA). TLC: precoated silica gel 60 F254 plates (Merck); visualization by
irradiation with UV light, by Schlittlerꢁs reagent, and Ce(SO4)2/H2SO4 soln. Column chromatography: silica gel
Merck 60 (230 ± 400 mesh). IR Spectra: Perkin-Elmer 781; nÄ in cm 1. 1H- and 13C-NMR Spectra: Bruker ARX-
300; Bruker DRX-500; d in ppm rel. to Me4Si as internal standard. HPLC: Waters 626-LC system, with 996-

Helvetica Chimica Acta ± Vol. 84 (2001)
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photodiode-array detector, 600S controller, and Millenium Chromatography Manager 2010 v. 2.15 (Waters
Corp., Milford, MA, USA); Rheodyne 77251 rotary valve fitted with a 5-ml loop (Rheodyne, Cotati, CA, USA);
1
Waters SymmetryTM-C8 column with integrated guard column (5 mm, 3.9 Â 150 mm); flow rate 0.5 ml min
;
mobile phase: A, H2O; B, MeCN; C 10% AcOH in H2O; linear gradient A/B/C 87:3 :10 ! 10 :80 :10 within
30 min. APCI-MS and APCI-MS/MS: Finnigan TSQ-700 triple-stage quadrupole instrument equipped with an
Â
atmospheric pressure chemical ionization (APCI) ion source (Finnigan, San Jose, CA, USA); APCI operating
conditions in the positive mode: vaporizer temp. 4508, corona voltage 5 kV, heated capillary temp. 2208, cheat
gas N2 with an inlet pressure of 40 psi, conversion dynode 15 kV; MS/MS experiments: collision gas Ar with a
relative pressure 2.5 ± 3.3 mTorr, collision-induced dissociation offset (Coff) 35 eV.
Ethyl (Æ)-3-{{3-{{4-[(3-Aminopropyl)amino]butyl}amino}propyl}amino}-3-(4-methoxyphenyl)propanoate
(19). To a soln. of spermine (17; 20.23 g, 99.98 mmol) in EtOH (100 ml) were added ethyl 3-(4-methoxyphenyl)-
3-oxopropanoate (18; 7.41 g, 33.34 mmol) in EtOH (35 ml) and AcOH (24.60 g, 409.66 mmol). The mixture was
refluxed for 2 h, cooled to r.t., and then MeOH (100 ml), AcOH (16.70 g, 278.10 mmol), and a soln. of
NaCNBH3 (2.40 g, 38.19 mmol) in MeOH (30 ml) were added. The mixture was stirred overnight at r.t. After
evaporation H2O (100 ml) was added, and the mixture was extracted with CHCl3 (3 Â 100 ml). The aq. phase
was alkalinized with K2CO3 and extracted with CHCl3 (1Â) and CHCl3/i-PrOH 4 :1 (5Â). After evaporation of
the extract, the residue was dissolved in EtOH and the soln. acidified with 32% aq. HCl soln. The precipitated
tetrahydrochloride of 19 was filtered off, washed with EtOH and Et2O, and recrystallized from AcOH (40 ml):
9.35 g (51% rel. to 18) of 19 ´ 2 HCl. TLC (silica gel, CHCl3/MeOH/25% aq. NH3 soln. 7:3 :1): Rf 0.40. IR
(KBr): 3435, 2986, 1735, 1613, 1515, 1463, 1300, 1255, 1188, 1180, 1112, 1030, 992, 845, 825, 750. 1H-NMR
(CD3OD): 7.48 (d, J ˆ 8.8, 2 arom. H); 7.02 (d, J ˆ 8.8, 2 arom. H); 4.66 (m, H C(3)); 4.06 (q, J ˆ 7.1, MeCH2);
3.82 (s, MeO); 3.18 ± 2.84 (m, 14 H); 2.24 ± 2.04 (m, 4 H); 1.83 (br. m, 4 H); 1.14 (t, J ˆ 7.1, MeCH2. 13C-NMR:
172.5 (s, CˆO); 162.5 (s, Cp); 131.2 (d, arom. C); 126.4 (s, Cipso); 115.8 (d, arom. C); 62.4 (t, C(2)); 60.2
(d, C(3)); 56.0 (q, MeO); 48.3, 48.2, 46.0, 44.1, 38.8, 38.0, 25.4, 24.2, 24.1 (9t, 9 CH2); 14.3 (q, Me). CI-MS: 409
‡
‡
(38, [M ‡ 1] ), 337 (12), 224 (26), 207 (86), 203 (100, [spermine ‡ H] ).
(Æ)-8-(4-Methoxyphenyl)-1,5,9,13-tetraazacycloheptadecan-6-one (ˆ(Æ)-Buchnerine; 14). For the prepa-
ration of the free base, 19 ´ 4 HCl (4.18 g, 7.54 mmol) was dissolved in H2O (85 ml), the soln. alkalinized with
solid K2CO3 and extracted with CHCl3/i-PrOH 4 :1 (5 Â 50 ml), the extract evaporated, the residue dissolved in
CHCl3, and the soln. filtered and evaporated. The free base 19 was refluxed with benzene (200 ml) for 2 h in a
flask equipped with a H2O remover. The soln. was cooled to 08, 1m Sb(OEt)3 in toluene (9.65 mmol) was added,
and the mixture was refluxed for 14 h. After cooling to 08, the mixture was quenched with MeOH (50 ml) and
chromatographed (silica gel, CHCl3/MeOH/25% aq. NH3 soln. 78 :19 :3 ! 7:3 :1): 1.46 g (53%) of 14. Colorless
oil. TLC (silica gel, CHCl3/MeOH/25% aq. NH3 soln. 7 :3 :1): Rf 0.45. IR (CHCl3): 2995, 2930, 2840, 2675, 1650,
1612, 1510, 1465, 1305, 1250, 1178, 1120, 1035, 909, 833, 658. 1H-NMR (CDCl3): 8.48 (t, J ˆ 4.8, CONH); 7.26 ±
7.12 (m, 2 arom. H); 6.90 ± 6.79 (m, 2 arom. H); 4.03 ± 2.27 (m, 21 H); 1.89 ± 1.36 (m, 8 H). 13C-NMR (CDCl3):
171.7 (s, CˆO); 158.6 (s, Cp); 135.4 (s, Cipso); 127.8, 113.9 (2d, arom. C); 59.7 (d, C(8)); 55.2 (q, MeO); 48.8, 48.6,
‡
48.2, 47.7, 46.7, 45.7, 38.6, 28.2, 27.9, 27.3, 27.0 (11t, 11 CH2). ESI-MS: 363 ([M ‡ H] ).
8-(4-Methoxyphenyl)-1,13-(4-tolylsulfonyl)-1,5,9,13-tetraazacycloheptadecan-6-one (24) and 9-(4-Bromo-
butyl)-8-(4-methoxyphenyl)-6-oxo-1,13-(4-tolylsulfonyl)-1,5,9,13-tetraazacycloheptadecane-5-carboxylic Acid
(25). To a soln. of 23 (2.74 g, 4.44 mmol) in dry DMF (445 ml) Cs2CO3 (3.25 g, 9.97 mmol) was added. The
mixture was heated at 608 for 40 min and stirred for a further 40 min at r.t. Then 1,4-dibromobutane (1.07 g,
4.96 mmol) in DMF (94 ml) was added dropwise. The mixture was stirred for 30 h at r.t. and evaporated. The
residue was chromatographed (AcOH, then AcOH/MeOH 12 :1, 10 :1, and 9 :1): 2.59 g (87%) of 24 and 0.22 g
(6%) of 25.
‡
Data of 24: TLC (silica gel, AcOH/MeOH 6 :1): Rf 0.38. ESI-MS: 671 ([M ‡ H] ). Spectroscopic data: see
[16][19].
Data of 25: Colorless glass-like solid. TLC (silica gel, AcOH/MeOH 6 :1): Rf 0.57. 1H-NMR: br. signals, no
interpretation possible. 13C-NMR (CDCl3): 180.00 ± 131.18 (br. s, several C); 129.65, 128.36, 126.98, 113.93
(d, arom. CH); 62.82 (br. d, C(8)); 55.19 (q, MeO); 50.00 ± 24.15 (br. t, several CH2); 28.38 (q, Me). ESI-MS:
‡
‡
‡
849 ([M ‡ H] ), 871 ([M ‡ Na] ), 887 ([M ‡ K] ). Anal. calc. for C39H53BrN4O8S2 (849.90): C 55.11, H 6.29,
Br 9.40, N 6.59, S 7.55; found: C 56.43, H 6.29, Br 8.84, N 6.87, S 7.56.
Hemin/H2O2 Oxidation of Dihydroxyverbacine (10). Dihydroxyverbacine (10) (3 mg, 0.0060 mmol) and
hemin (1.3 mg, 0.0019 mmol) were dissolved in MeCN (0.15 ml) and 0.1m eq. K2CO3 (0.15 ml). Then 10% H2O2
soln. (20 ml, ca. 0.0588 mmol) was added, and the mixture was shaken for 10 min. For analysis, the mixture was
filtered through a 0.45-mm centrifugal filter tube (Eppendorf-Netheler-Hinz GmbH, Hamburg). The mixture
was analyzed by HPLC/MS and HPLC/MS/MS (aliquots of 5 ml were injected; see Fig.).

2118
Helvetica Chimica Acta ± Vol. 84 (2001)
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Received March 22, 2001