Synthesis of a doubly labelled concanamycin derivative for ATPase binding studies

Article abstract of DOI:10.1002/1099-0690(200112)2001:23<4525::AID-EJOC4525>3.0.CO;2-S

The synthesis of a doubly labelled concanamycin derivative for binding studies with V- and P-type ATPases is described. The starting point was 21-deoxyconcanolide A (6), which was generated from concanamycin A (1) in three steps and which exhibited the full ATPase inhibitor activity, with the advantage of a stability better than that of 1. Through use of a suitable protecting group for 6, the carbene-generating diazirine residue and ¹²⁵I were introduced regio- and stereo-selectively. The inhibitory efficacy of the resulting 23-iodo(¹²⁵I)-9-O-[p-(trifluoroethyldiazirinyl)benzoyl]-21,23- dideoxyconcanolide A (11b) turned out to be high enough for labelling studies. Photoaffinity labelling experiments clearly showed that 11b is a suitable derivative with which to determine the binding site of concanamycin-like compounds in different ATPases.

Full text of DOI:10.1002/1099-0690(200112)2001:23<4525::AID-EJOC4525>3.0.CO;2-S

FULL PAPER
Synthesis of a Doubly Labelled Concanamycin Derivative for ATPase Binding
Studies[‡]
Gudrun Ingenhorst,[a] Kai Uwe Bindseil,[a] Claudia Boddien,[a] Stefan Dröse,[b]
Michael Gaßel,[b] Karlheinz Altendorf,[b] and Axel Zeeck*[a]
Keywords: Enzymes / Iodine / Natural products / Photoaffinity labelling
The synthesis of a doubly labelled concanamycin derivative
for binding studies with V- and P-type ATPases is described.
The starting point was 21-deoxyconcanolide A (6), which
selectively. The inhibitory efficacy of the resulting 23-
iodo(125I)-9-O-[p-(trifluoroethyldiazirinyl)benzoyl]-21,23-
dideoxyconcanolide A (11b) turned out to be high enough for
was generated from concanamycin A (1) in three steps and labelling studies. Photoaffinity labelling experiments clearly
which exhibited the full ATPase inhibitor activity, with the
advantage of a stability better than that of 1. Through use
of a suitable protecting group for 6, the carbene-generating
diazirine residue and 125I were introduced regio- and stereo-
showed that 11b is a suitable derivative with which to deter-
mine the binding site of concanamycin-like compounds in
different ATPases.
Introduction
Asymmetric plecomacrolides[2] with 18- and 16-mem-
bered macrolactone skeletons, such as concanamycins A (1;
Figure 1) and C (2) and the bafilomycins, respectively, have
been shown to be the most specific inhibitors (nanomolar
concentrations) of V-type (vacuolar) ATPases.[3,4] Some P-
type ATPases are also inhibited by these plecomacrolides
(micromolar concentrations), whereas the F-type ATPases
are not affected. The fact that the V-type ATPases are in-
volved in the process of osteoclast bone resorption[5] made
the plecomacrolides interesting compounds for pharmaco-
logical studies.[6] However, concanamycin A (1), the most
potent inhibitor, exhibits an undesirably high toxicity,[7,8]
which reduces its prospects for pharmaceutical use. Never-
theless, the concanamycins are important tools for the study
of the physiological and biochemical properties of V-type
and P-type ATPases.[9]
Despite the intensive use of plecomacrolides in biochem-
ical research, the exact molecular mechanism of inhibition
and the binding site within the enzyme complex is still un-
known.[10,11] We therefore set out to synthesise a concana-
mycin derivative containing both a photoreactive group and
an additional moiety acting as a tracer group, to enable the
inhibitor binding site to be detected after successful label-
ling. The first encouraging results from structure/activity
studies[4] indicated that the incorporation of functional
groups without serious effects on the inhibitory potential of
Figure 1. Structure of concanamycins
the plecomacrolide should be possible. In order to prepare
suitable semisynthetic concanamycin derivatives, we studied
the reactivities of different functional groups in the antibi-
otics by means of O-acylation, O-alkylation and nucleo-
philic substitution.[1,12Ϫ14] The derivatives were tested for
their ATPase inhibitory activity. As a result, the pharmaco-
phore of the plecomacrolide was identified[12,15] and specific
labelling of the V-type ATPase of Manduca Sexta was
achieved.
On the basis of these results we introduced tracer groups
(fluorescent or radioactive) and nitrene-generating azido or
carbene-generating diazirine groups to achieve the covalent
binding of concanamycin derivatives to their target.[13,15]
[‡]
Chemistry of Unusual Macrolides, 4. Ϫ Part 3: Ref.[1]
Institut für Organische Chemie, Universität Göttingen,
[a]
Tammanstrasse 2, 37077 Göttingen, Germany
Fax: (internat.) ϩ 49-(0)551/12593
E-mail: azeeck@gwdg.de
[b]
Abteilung Mikrobiologie, Fachbereich Biologie/Chemie,
Universität Osnabrück,
Barbarastrasse 11, 49076 Osnabrück, Germany
Eur. J. Org. Chem. 2001, 4525Ϫ4532
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/1223Ϫ4525 $ 17.50ϩ.50/0
4525

A. Zeeck et al.
FULL PAPER
The first concanamycin derivative for photoaffinity label- ate’s leaving group properties would simultaneously be ad-
ling was 9-O-([3,5-3H]-4-azidobenzoyl)-21-deoxyconcana- vantageous for substitution with Na125I in the last step. Es-
mycin A (3), with a specific radioactivity of 10 mCi/ terification with p-(trifluoroethyldiazirinyl)benzoic acid at
mmol.[13] With this derivative, labelling of the Kdp-ATPase 9-OH before introduction of the radioactive tracer ap-
(P-type ATPase) was possible, but experiments with V-type peared feasible.
ATPases were unsuccessful, probably due to the relatively
low specific radioactivity and the low traceability of tri-
tium.[16]
Results
In this paper we present the synthesis of a photoreactive
21-Deoxyconcanolide A (6)
concanamycin derivative that exhibits an inhibitory activity
against V- and P-type ATPases and possesses a high specific
radioactivity due to the presence of 125I as a tracer element.
The covalent binding to the enzymes was achieved through
the introduction of a diazirine into the plecomacrolide.
The transformation of concanamycin A (1) into the more
stable 21-deoxyconcanolide A (6) was performed in three
steps (Scheme 1). The reversible 21-O-methylation with
10Ϫ3  HCl in MeOH has already been used for the separa-
tion of concanamycins A (1) and B.[17] When we followed
the reported reaction conditions, we observed partial de-
gradation of 1. The quantitative transformation of 1 into
its 21-O-methyl derivative 4 could, however, be achieved in
methanol with catalytic amounts of Lewis acids, such as 0.1
Synthetic Strategy
The concanamycins were originally isolated from Strepto-
myces diastatochromogenes.[17] As described previously,[2,14]
we reisolated them from the mycelium extract of a new soil
isolate Streptomyces sp. (Gö 22/15) in the course of our
chemical screening program. Concanamycin A (1) was the
main component synthesised by this strain,[18] and so we
used it as the starting point for derivatisation.
Because of their hemiacetal groups at C(21), concanamy-
cin A (1) and, especially, its aglycone concanolide A are ra-
ther sensitive to acids and bases. The conditions for any
derivatisation are thus limited, as is the half-time during
biological tests and photoaffinity labelling experiments. We
decided to enhance the stability of the hemiacetal not by
acetalisation, which is easily possible, but by production of
a deoxy derivative, turning the hemiacetal into a cyclic
ether. The method for this reduction was first reported by
Horne and Jordan.[20] Furthermore, we decided to liberate
the aglycone of the plecomacrolide by hydrolysis, because
we had seen previously that the loss of the sugar moiety
does not affect the inhibitory activity.[4] We thus focused
our interest on 21-deoxyconcanolide A (6), which exhibited
an inhibitory potential on ATPases almost identical to that
of 1.[15]
1
equivalent of FeCl3, within a few minutes. The H NMR
spectroscopic data of the product showed the introduction
of a methoxy group (δH ϭ 3.02). As the NMR chemical
shifts and coupling constants of 1 and 4 were almost ident-
ical, it could be deduced that the acetalisation had pro-
ceeded regio- and stereoselectively with retention of the
configuration at C(21).
As photolabel, the very effective carbene-generating di-
azirine group was chosen as part of a p-(trifluoroethyldiazi-
rinyl)benzoyl residue,[19] which could easily be introduced
by esterification. Because of its high traceability, 125I was
selected as a radioactive tracer and introduced by means of
Scheme 1. Synthesis of 21-deoxyconcanolide A (6) (Reagents: i,
NaBH3CN, HCl, EtOH)
For the reduction of the acetal into a cyclic ether we
a simple nucleophilic substitution of a suitable leaving chose the mild approach described by Horne and Jordan
group with 125IϪ. For reasons of expense and safety, this with NaBH3CN/HCl(g)/MeOH.[20] Modified conditions,
reaction was planned as the last step of the whole synthesis. with aqueous HCl and EtOH as solvent, enabled 21-O-
As a consequence, the esterification with p-(trifluoroethyldi- methylconcanamycin A (4) to be transformed into 21-de-
azirinyl)benzoic acid had to be performed one step earlier.
oxyconcanamycin A (5) by a quick route with a yield of
For 21-deoxyconcanolide A (6), it had previously been 46%. This reaction, which probably involves the formation
shown that esterification of the hydroxy groups can be per- of an oxonium ion as an intermediate, proceeds highly ste-
formed regioselectively, starting with 23-OH and followed reospecifically. Direct assignment of the absolute stereo-
1
by 9-OH.[12,13] As 7-OH and 19-OH remained unaffected, chemistry at C(21) of 5 from its H NMR coupling con-
it appeared that they were hindered by intramolecular con- stants was impossible because of the overlapping proton
formational constraints and by a rigid hydrogen bonding signals. For this reason we prepared the 9,3Ј-di-O-acetyl-
system, respectively. A selective esterification of 9-OH was 21-deoxyconcanamycin A (7) by treatment of 5 with acetic
only possible with prior protection of 23-OH. Nosylate pro- anhydride/pyridine at room temperature. Extensive NMR
tection of the latter appeared appropriate, because nosyl- studies with different solvents (CDCl3 and C6D6) and 2D
4526
Eur. J. Org. Chem. 2001, 4525Ϫ4532

A Doubly Labelled Concanamycin Derivative for Binding Studies
FULL PAPER
1
methods [1H,1H COSY, Delay-COSY and J(C,H)-COSY]
allowed all proton signals and coupling constants to be as-
signed. The magnitude of the diagnostically important
J21,22ax (11.5 Hz) clearly indicates the axial orientation of
21-H (Table 1).
Table 1. Selected 1H NMR (500 MHz) data of the diacetates 7 and
8 (J in Hz)
H atom
7[a]
7[b]
8[a]
Coupling pattern
3
9
6.39
4.84
3.83
5.17
3.60
3.42
2.10
3.49
3.29
5.34
5.58
1.62
4.65
2.22
4.98
4.60
3.42
6.68
5.04
3.88
5.65
4.12
3.71
2.24
3.57
3.34
5.37
5.44
1.38
4.53
2.30
5.24
4.93
3.29
6.39
4.83
3.80
5.16
3.59
3.56
2.15
4.64
3.40
5.43
5.60
1.62
-
s
d (11.0)
dd (9.0, 9.0)
dd (9.0, 1.5)
Figure 2. Structure of diacetates generated from 5 and 6
16
17
19
21
22eq
23
25
26
27
28
1Ј
the 1H NMR spectrum of 9 the new aromatic protons were
clearly visible at δH ϭ 8.12 and 8.40 and the 23-H signal
was shifted downfield from its position in 6 (∆δH ϭ 1.66).
ddd (10.0, 5.0, 1.5)
ddd (11.0, 9.0, 0.5)
dd (12.0, 5.0)
ddd (10.0, 10.0, 5.0)
dd (10.0, 7.0)
ddq (15.0, 7.0, 1.0)
ddq (15.0, 6.0, 1.0)
dd (6.0, 1.0)
dd (10.0, 2.0)
ddd (12.0, 5.0, 2.0)
ddd (11.0, 9.0, 5.0)
dd (9.0, 9.0)
2Јeq
3Ј
4Ј
-
-
-
-
5Ј
dd (9.0, 6.0)
[a]
[b]
In CDCl3; In C6D6.
Because of the lack of hemiacetal activation, deglycosyl-
ation of 5 turned out to be more difficult than that de-
scribed for 1:[14] the use of large amounts of p-toluenesul-
fonic acid in MeOH/1  HCl and a long reaction time gave
the desired 21-deoxyconcanolide A (6) in moderate yield.
After modification of the reaction conditions, the yield
could be increased to 34% by use of AcCN/H2O (5:1) at 38
°C for 12 hours. In contrast to the deglycosylation of 1, no
23-O-methylation was observed in the presence of meth-
anol.[14] This showed that the reaction mechanism was dif-
ferent from that of the methanolysis of the hemiacetal-con-
taining concanamycin A (1). Finally, to obtain better re-
solved NMR spectra (Table 1), 6 was transformed into the
corresponding 9,23-di-O-acetyl-21-deoxyconcanolide A (8;
1
Scheme 2. Synthesis of 11a/11b
Figure 2). The H NMR spectra of 6 and 8 indicated that
the aglycone had been obtained, since the characteristic de-
oxyrhamnose signals seen in 5 could not be observed. De-
tailed analysis of the coupling constants of 8 established
that the deglycosylation proceeded with retention of config-
uration. It should be mentioned that all deoxygenated con-
canamycins, especially 6, have much better stability against
acids and bases.
For the subsequent esterification of 9 at 9-OH with p-
(trifluoroethyldiazirinyl)benzoic acid numerous methods
were available. On the basis of our previous experience with
several concanamycin derivatives we used DCC for the re-
action.[21] Treatment of 9 with DCC/DMAP and p-(trifluor-
oethyldiazirinyl)benzoic acid in dichloromethane resulted in
the formation of 10 within 18 h (yield: 46%). The ester-
ification of only one hydroxy group was confirmed by the
ESI mass spectrum, with a peak at m/z ϭ 1096 [M ϩ Na]ϩ.
23-Iodo(125I)-9-O-[p-(trifluoroethyldiazirinyl)benzoyl]-21,23-
dideoxyconcanolide A (11b)
1
The H NMR spectrum revealed additional signals due to
As described above, the first step towards the target mo- the new aromatic ring (δH ϭ 7.30, 8.08) and a downfield
lecule 11b was the protection of 23-OH by nosylation. shift of 9-H to δH ϭ 5.08 (∆δH ϭ 1.86 compared with 9),
Compound 6 was treated with p-nitrobenzenesulfonyl which indicated the esterification of 9-OH. The 13C NMR
chloride at 0 °C in the presence of DMAP/NEt3 to yield signals, especially the quaternary carbon atom at δC
ϭ
9 (89%; Scheme 2). The ESI mass spectrum confirmed the 121.2 for the diazirine residue, were clearly attributable to
molecular mass, with a peak at m/z ϭ 884 [M ϩ Na]ϩ. In structure 10.
Eur. J. Org. Chem. 2001, 4525Ϫ4532
4527

A. Zeeck et al.
FULL PAPER
Table 3. Selected NMR spectroscopic data of 11a and its 23-epi diastereomer
11a
'
23-epi-11a[13]
Atom
1H NMR[a]
13C NMR[a]
1H NMR[b]
13C NMRba]
21
3.38 (m)
80.5
45.4
3.97 (ddd, 10.0, 7.0, 1.5)
1.85 (m)
2.30 (m)
4.74 (ddd, 2.5, 2.5, 3.0)
0.53 (m)
75.1
40.6
22ax
22eq
23
2.05Ϫ2.14 (m)
2.57 (ddd, 13.0, 4.5, 1.5)
4.08 (ddd, 12.0, 12.0, 4.5)
1.68 (m)
38.7
46.3
46.0
40.0
24
24-Me
25
26
27
28
0.96 (d, 6.5)
3.40 (m)
5.31 (m)
5.56 (dq, 15.0, 6.5)
1.59 (m)
19.5
84.2
131.2
129.0
17.8
0.78 (d, 6.5)
3.66 (m)
5.34 (ddq, 15.0, 7.5, 2.0)
5.61 (dq, 15.0, 6.5)
1.61 (dd, 6.5, 1.5)
20.8
80.9
130.3
129.7
18.1
[a]
[b]
500 MHz, CD2Cl2, J in Hz; 500 MHz, CDCl3, J in Hz.
In the last step, the nucleophilic substitution of the nosyl- activity. To achieve this, we treated 10 with only 2.5 equiva-
ate moiety with sodium iodide was achieved in good yields lent NaI in addition to 10 mCi Na125I. Because of the lower
(92%) by stirring 10 in the presence of seven equivalents of concentration of NaI, the reaction time was increased.
NaI in DMF at 40 °C for 3 h.[22] The 13C NMR spectrum After 48 h we obtained 23-iodo(125I)-9-O-[p-(trifluoroethyl-
of the product showed the signal of C(23) at δC ϭ 38.7, diazirinyl)benzoyl]-21,23-dideoxyconcanolide A (11b) in a
with an expected upfield shift from that in 10 (δC ϭ 87.7). yield of 50% and with a specific radioactivity of 438.7
1
From the coupling constants in the H NMR spectrum of mCi/mmol.
1
11a, in conjunction with its H,1H-COSY spectrum, this
reaction proceeds with retention of the stereochemistry of
C(23), indicating either an SN1 mechanism or a repeated
SN2 process with neighbouring group participation (e.g.,
Discussion
19-OH). The vicinal coupling constant J23,24 ϭ 12 Hz (10:
The photoreactive derivative of concanamycin 11a was
J23,24 ϭ 11 Hz) clearly indicates the axial orientation of 23-
tested as an inhibitor against Manduca sexta V-type ATPase
H, which represents the configuration of lower energy.[23]
and E. coli Kdp-ATPase.[15,25] As shown in Table 3, com-
1
An impurity (about 10%) could be detected in the H
pound 11a inhibited the V-ATPase half maximally at a con-
NMR spectrum of 11a. In particular, the signal of 23-H
centration of 15Ϫ20 µ, equivalent to an I50 value of ap-
had shifted from δH ϭ 4.08 to 4.74 and its coupling con-
proximately 0.8 µmol per mg of protein and the Kdp-AT-
stants had changed, indicating the equatorial orientation of
Pase with a Ki value of 40 µ. Unfortunately, 11a is not as
23-H in the by-product (Table 2).[13] The data for 11a and
potent an inhibitor as concanamycin A (1) and 21-deoxy-
its 23-epi-iodo diastereomer (Figure 3) are in agreement
concanamycin A (6), although its remaining activity for the
with those of iodocyclohexane derivatives.[24]
V-type ATPase is high enough for subsequent binding stud-
The inhibitory efficacy of 11a against V- and P-type
ies.
ATPases is not as high as that of 6 (Table 3), but it still
Labelling experiments with the purified Kdp-ATPase
is an effective inhibitor and therefore suitable for labelling
showed reactions between 11b and subunits KdpA and
experiments. Furthermore, it was necessary that the re-
KdpB, as already observed with 9-O-[(3,5-3H)-4-azidoben-
quired radioactive 11b should exhibit a high specific radio-
zoyl]-21-deoxyconcanamycin A (3).[16] However, neither
concanamycin A (1) nor bafilomycin B1 quenched this co-
valent modification.[15] Thus, the specificity of this covalent
modification remains ambiguous.
UV irradiation of the purified M. sexta V-ATPase in the
presence of 11b resulted in a specific labelling of the V-AT-
Pase subunit c.[25] The specificity of this reaction is corrob-
Figure 3. Partial structure of 11a and its epimer
orated by the fact that the covalent modification can be
Table 3. Inhibitory activity of 1, 6 and 11a against purified E. coli Kdp-ATPase and the V-type ATPases of N. crassa and M. sexta
Kdp-ATPase
Ki (µ)/I50 (µmol/mg)
V-ATPase (N. crassa)
Ki (n)/I50 (µmol/mg)
V-ATPase (M. sexta)
Ki (µ)/I50 (µmol/mg)
1
6
11a
1.2/0.11
15/1.36
40/4.1
1.2/0.12·10Ϫ3
0.9/0.09·10Ϫ3
not tested
0.01/0.5·10Ϫ3
not tested
15Ϫ20/0.8
4528
Eur. J. Org. Chem. 2001, 4525Ϫ4532

A Doubly Labelled Concanamycin Derivative for Binding Studies
FULL PAPER
ε) ϭ 244 (4.55), 282 (4.41); [α]2D0 ϭ ϩ46 (c ϭ 0.4 in MeOH); CD
(MeOH): λextr. ([Θ]22) ϭ 211 nm (sh) (7700), 242 (84800), 267
(Ϫ49200); 1H NMR (500 MHz, CD2Cl2/CD3OD, 2:1): δ ϭ 0.85 (d,
J ϭ 7.0 Hz, 3H 24-CH3), 0.85 (m, 3 H, 8-Et-CH3), 0.86 (d, J ϭ
7.0 Hz, 3 H, 18-CH3), 0.98 (d, J ϭ 7.0 Hz, 3 H, 20-CH3), 1.02 (d,
J ϭ 7.0 Hz, 3 H, 6-CH3), 1.04 (m, 3 H, 10-CH3), 1.22 (m, 2 H, 8-
Et-CH2), 1.24 (d, J ϭ 6.0 Hz, 3 H, 6Ј-H3), 1.26 (m, 1 H, 24-H),
1.42 (m, 1 H, 8-H), 1.47 (m, 1 H, 22-Hax), 1.62 (m, 1 H, 2Ј-Hax),
1.70 (dd, J ϭ 6.5, 1.5 Hz, 3 H, 28-H), 1.81 (s, 3 H, 12-CH3), 1.89
(m, 2 H, 11-H2), 1.97 (m, 1 H, 18-H), 1.99 (s, 3 H, 4-CH3), 2.05
quenched by an excess of concanamycin A (1) or bafilomy-
cin B1. This labelling experiment provided the first direct
evidence that subunit c, which forms the major part of the
proton-translocating V0 complex of V-type ATPases, con-
tains the binding site for plecomacrolides.
In summary, 11b represents an important derivative for
the determination of the binding site of concanamycin-type
compounds in V-type ATPases. Further experiments with
V-type ATPases to define the site of covalent modification
more precisely are in progress. Compound 11b was not suit- (m, 1 H, 20-H), 2.22 (ddd, J ϭ 12.0, 5.0, 2.0 Hz, 1 H, 2Ј-Heq), 2.29
(m, 1 H, 10-H), 2.33 (dd, J ϭ 13.0, 5.0 Hz, 1 H, 22-Heq), 2.75 (m,
1 H, 6-H), 3.02 (s, 3 H, 21-OCH3), 3.09 (m, 1 H, 9-H), 3.23 (s, 3
H, 16-OCH3), 3.35Ϫ3.44 (m, 2 H, 19-H and 5Ј-H), 3.48 (dd, J ϭ
10.0, 8.0 Hz, 1 H, 25-H), 3.58 (m, 1 H, 7-H), 3.63 (m, 1 H, 23-H),
3.60 (m, 1 H, 3Ј-H), 3.64 (s, 3 H, 2-OCH3), 3.80 (dd, J ϭ 9.0,
9.0 Hz, 1 H, 16-H), 4.28 (dd, J ϭ 9.0, 9.0 Hz, 1 H, 4Ј-H), 4.56 (dd,
J ϭ 10.0, 1.0 Hz, 1 H, 1Ј-H), 5.07 (dd, J ϭ 9.0, 0.5 Hz, 1 H, 17-
H), 5.23 (dd, J ϭ 15.0, 9.0 Hz, 1 H, 15-H), 5.40 (ddq, J ϭ 15.0,
8.0, 1.5 Hz, 1 H, 26-H), 5.66 (dq, J ϭ 15.0, 6.5 Hz, 1 H, 27-H),
5.69 (m, 1 H, 5-H), 5.78 (d, J ϭ 11.0 Hz, 1 H, 13-H), 6.45 (s, 1 H,
able for the identification of the plecomacrolide binding site
within the Kdp-ATPase. A specific labelling of this ATPase
should probably be achievable with a derivative of a higher
specific radioactivity and especially with a better specific
inhibitory potential.
To modify multifunctional antibiotics for binding studies
with target enzymes, detailed knowledge about the chemical
behaviour of the parent compounds should be available in
order that appropriate derivatives may be synthesised. In
the case of the concanamycins there seems to be a relatively 3-H), 6.50 (dd, J ϭ 15.0, 11.0 Hz, 1 H, 14-H); 13C NMR (125 MHz,
CD2Cl2/CD3OD, 2:1, Ϫ20 °C): δ ϭ 7.2 (q, 20-CH3), 10.0 (q, 18-
CH3), 11.6 (q, 8-Et-CH3), 13.2 (q, 24-CH3), 13.9 (q, 4-CH3), 16.0
(q, 12-CH3), 16.7 (q, 6-CH3), 17.4 (q, C-6Ј), 17.8 (q, C-28), 21.7
(q, 10-CH3), 22.5 (t, 8-Et-CH2), 34.7 (d, C-6), 34.9 (d, C-10), 35.7
(t, C-22), 37.8 (d, C-20), 38.1 (d, C-18), 39.6 (t, C-2Ј), 40.3 (d, C-
24), 43.6 (d, C-8), 45.3 (t, C-11), 46.4 (q, 21-OCH3), 55.7 (q, 16-
OCH3), 60.0 (q, 2-OCH3), 69.0 (d, C-3Ј) 69.5 (d, C-19), 69.8 (d, C-
5Ј), 73.2 (d, C-23), 75.2 (d, C-7), 75.7 (d, C-17), 76.9 (d, C-25), 78.6
(d, C-4Ј), 79.4 (d, C-9), 82.3 (d, C-16), 95.9 (d, C-1Ј), 103.6 (s, C-
21), 122.8 (d, C-13), 126.5 (d, C-15), 130.1 (d, C-27), 130.3 (d, C-
26), 131.5 (s, C-4), 132.2 (d, C-3), 133.8 (d, C-14), 141.4 (d, C-5),
141.5 (s, C-2), 142.7 (s, C-12), 157.9 (s, CONH2), 166.6 (s, C-1);
FAB MS: m/z ϭ 879 [M Ϫ H] (neg.); 903 [M ϩ Na] (pos.);
C47H77NO14 (880.12).
high tolerance to modification without loss of the specific
ATPase inhibitor activity. In particular, 9-OH and 23-OH
of 6 can be modified widely, and these are fortunately the
most reactive functional groups of the molecule, while the
others are so deactivated that they seem to be protected by
internal conformational constraints. Thus, the plecomacrol-
ides are good examples of the value and potential of semi-
synthetic studies of multifunctional compounds.
Experimental Section
General Remarks: Melting points: Reichert hot stage microscope
(not corrected); IR spectra: PerkinϪElmer 298 spectrometer; UV
spectra: Varian Cary 3E spectrophotometer. Optical rotation:
PerkinϪElmer 241; CD spectra: JASCO J 500 A spectrometer;
FAB-MS: Finnigan MAT 8230 with nitrobenzyl alcohol as matrix;
21-Deoxyconcanamycin A (5): A solution of 4 (710 mg, 0.81 mmol)
and NaBH3CN (361 mg, 4.86 mmol, 6 equiv.) in EtOH (70 mL)
was treated with HCl (0.5 , 7 mL) and stirred at room temp. After
4 h, phosphate buffer (60 mL) was added, EtOH was removed in
vacuo and the resulting aqueous solution was extracted with
CH2Cl2. The usual workup procedure followed by CC (RP-8,
MeOH/H2O, 8:2) gave 5 (302 mg, 46%) as a white, amorphous
powder. M.p. 94 °C (dec.); IR (KBr): ν˜ ϭ 3443 cmϪ1, 2927, 1712,
1620 w, 1455 w, 1381, 1252, 1102; UV (MeOH): λmax (log ε) ϭ 244
(4.46), 282 (4.11); [α]2D0 ϭ Ϫ46 (c ϭ 1 in MeOH); CD (MeOH):
1
ESI-MS: Finnigan LQC; H and 13C NMR spectra: Varian VXR
500 (500 MHz). Chemical shifts are expressed in δ values (ppm)
using the solvent as internal reference. The multiplicities of the 13
C
NMR values were assigned by attached proton test (APT). TLC:
silica gel plates (60 F245 silica gel on aluminium foil, Merck), RP-
8
(F254S, Merck). LC: silica gel ICN Sili Tech 32Ϫ63
(0.032Ϫ0.063 mm, ICN Biomedicals GmbH), Sephadex LH-20
(Pharmacia), RP-8 (Merck, Lobar, size B). Staining reagent: vanil-
lin/sulfuric acid: 1 g of vanillin in 100 mL of sulfuric acid. Concan-
amycin: Isolated from the mycelium of Streptomyces strain Gö 22/
15 and purified as described elsewhere.[18] Usual workup procedure:
threefold extraction of the reaction mixture with the indicated solv-
ent, washing with water, drying with Na2SO4 and removal of the
solvent in vacuo.
1
λextr. ([Θ]22) ϭ 210 nm (sh) (7500), 242 (69600), 267 (Ϫ43100); H
NMR (500 MHz, CD2Cl2/CD3OD, 2:1): δ ϭ 0.81 (d, J ϭ 7.0 Hz,
3 H, 18-CH3), 0.83 (d, J ϭ 7.0 Hz, 3H 24-CH3), 0.84 (t, J ϭ 7.0 Hz,
3 H, 8-Et-CH3), 0.85 (d, J ϭ 7.0 Hz, 3 H, 20-CH3), 0.96 (m, 3 H,
6-CH3), 1.07 (d, J ϭ 7.0 Hz, 3 H, 10-CH3), 1.08 (m, 1 H, 22-Hax),
1.09 (m, 2 H, 8-Et-CH2), 1.19 (m, 1 H, 24-H), 1.21 (d, J ϭ 6.0 Hz,
3 H, 6Ј-H3), 1.41 (m, 1 H, 8-H), 1.56 (m, 1 H, 2Ј-Hax), 1.61 (dd,
J ϭ 6.0, 1.5 Hz, 3 H, 28-H), 1.64 (m, 1 H, 20-H), 1.83 (m, 2 H,
21-O-Methylconcanamycin A (4): A mixture of concanamycin A (1) 11-H2), 1.96 (s, 3 H, 12-CH3), 1.99 (m, 1 H, 18-H), 2.00 (s, 3 H, 4-
(800 mg, 0.92 mmol) in MeOH (20 mL) was treated with FeCl3 CH3), 2.09 (m, 1 H, 2Ј-Heq), 2.13 (m, 1 H, 22-Heq), 2.26 (m, 1 H,
(15 mg, 0.09 mmol) and stirred for 15 min. After addition of phos- 10-H), 2.71 (m, 1 H, 6-H), 3.20 (m, 1 H, 9-H), 3.23 (s, 3 H, 16-
phate buffer (pH ϭ 7, 20 mL), MeOH was removed in vacuo and OCH3), 3.30 (dd, J ϭ 10.0, 8.0 Hz, 1 H, 25-H), 3.34 (m, 1 H, 5Ј-
the aqueous residue was extracted with ethyl acetate. The usual H), 3.38 (m, 1 H, 21-H), 3.48 (ddd, J ϭ 11.0, 11.0, 4.5 Hz, 1 H,
workup procedure was followed by a short filtration through silica 23-H), 3.54 (s, 3 H, 2-OCH3), 3. 57 (dd, J ϭ 10.0, 1.5 Hz, 1 H, 19-
gel (acetone/cyclohexane, 2:3) to yield 4 (780 mg, 96%) as a white H), 3.67 (m, 1 H, 3Ј-H), 3.70 (m, 1 H, 7-H), 3.80 (dd, J ϭ 9.0,
foam. M.p. 128 °C (dec.); IR (KBr): ν˜ ϭ 3455 cmϪ1, 2966, 1709, 9.0 Hz, 1 H, 16-H), 4.20 (dd, J ϭ 9.0, 9.0 Hz, 1 H, 4Ј-H), 4.59 (dd,
1680 sh, 1621 w, 1454, 1380, 1250, 1102; UV (MeOH): λmax (log J ϭ 10.0, 1.5 Hz, 1 H, 1Ј-H), 5.11 (m, 1 H, 17-H), 5.19 (dd, J ϭ
Eur. J. Org. Chem. 2001, 4525Ϫ4532
4529

A. Zeeck et al.
FULL PAPER
15.0, 9.0 Hz, 1 H, 15-H), 5.32 (ddq, J ϭ 15.0, 8.0, 1.5 Hz, 1 H, 26- (4.06); [α]2D0 ϭ Ϫ23 (c ϭ 0.6 in MeOH); CD (MeOH): λextr.
H), 5.57 (dq, J ϭ 15.0, 6.0 Hz, 1 H, 27-H), 5.69 (m, 1 H, 5-H), ([Θ]22) ϭ 212 nm (sh) (11600), 242 (99500), 267 (Ϫ65100); 1H
5.80 (d, J ϭ 11.0 Hz, 1 H, 13-H), 6.39 (s, 1 H, 3-H), 6.55 (dd, J ϭ NMR (500 MHz, CD2Cl2/CD3OD, 2:1): δ ϭ 0.81 (d, J ϭ 7.0 Hz,
15.0, 11.0 Hz, 1 H, 14-H); 13C NMR (125 MHz, CD2Cl2/CD3OD, 3 H, 18-CH3), 0.82 (m, 3 H, 8-Et-CH3), 0.85 (d, J ϭ 7.0 Hz, 3 H,
2:1, Ϫ20 °C): δ ϭ 7.9 (q, 20-CH3), 9.3 (q, 18-CH3), 11.7 (q, 8-Et-
20-CH3), 0.87 (d, J ϭ 6.5 Hz, 3H 24-CH3), 1.02 (m, 3 H, 6-CH3),
CH3), 13.2 (q, 24-CH3), 14.1 (q, 4-CH3), 16.1 (q, 12-CH3), 16.8 (q, 1.04 (d, J ϭ 7.0 Hz, 3 H, 10-CH3), 1.11 (m, 2 H, 8-Et-CH2), 1.17
6-CH3), 17.3 (q, C-6Ј), 17.8 (q, C-28), 21.7 (q, 10-CH3), 22.6 (t, 8- (m, 1 H, 24-H), 1.20 (m, 1 H, 22-Hax), 1.46 (m, 1 H, 8-H), 1.61
Et-CH2), 34.8 (d, C-6), 34.8 (t, C-22), 35.6 (d, C-10), 37.2 (d, C-
(dd, J ϭ 6.0, 1.5 Hz, 3 H, 28-H), 1.64 (m, 1 H, 20-H), 1.84 (s, 3 H,
18), 39.1 (d, C-20), 39.6 (t, C-2Ј), 41.2 (d, C-24), 43.5 (d, C-8), 45.2 12-CH3), 1.80Ϫ1.90 (m, 2 H, 11-H2), 1.96 (s, 3 H, 4-CH3), 2.03 (m,
(t, C-11), 55.7 (q, 16-OCH3), 59.2 (q, 2-OCH3), 69.1 (d, C-3Ј) 69.2 1 H, 22-Heq), 2.04 (m, 1 H, 18-H), 2.26 (m, 1 H, 10-H), 2.71 (m, 1
(d, C-19), 69.9 (d, C-5Ј), 73.3 (d, C-7), 75.8 (d, C-17), 76.2 (d, C-
H, 6-H), 3.20 (m, 1 H, 9-H), 3.23 (s, 3 H, 16-OCH3), 3.27 (m, 1 H,
21), 78.1 (d, C-23), 78.5 (d, C-4Ј), 79.4 (d, C-9), 82.1 (d, C-16), 82.9 23-H), 3.31 (m, 1 H, 25-H), 3.43 (dd, J ϭ 11.0, 8.0, 1.5 Hz, 1 H,
(d, C-25), 95.6 (d, C-1Ј), 122.9 (d, C-13), 126.7 (d, C-15), 128.7 (d,
C-27), 130.6 (d, C-26), 131.6 (s, C-4), 132.1 (d, C-3), 133.8 (d, C-
21-H), 3.54 (s, 3 H, 2-OCH3), 3. 59 (dd, J ϭ 10.0, 1.5 Hz, 1 H, 19-
H), 3.61 (m, 1 H, 7-H), 3.80 (dd, J ϭ 9.0, 9.0 Hz, 1 H, 16-H), 5.14
14), 140.9 (d, C-5), 141.7 (s, C-2), 142.7 (s, C-12), 158.1 (s, (br. d, J ϭ 6.0 Hz, 1 H, 17-H), 5.19 (dd, J ϭ 15.0, 9.0 Hz, 1 H, 15-
CONH2), 166.2 (s, C-1); FAB MS: m/z ϭ 849 [M Ϫ H] (neg.); 873 H), 5.32 (ddq, J ϭ 15.0, 8.0, 1.5 Hz, 1 H, 26-H), 5.57 (dq, J ϭ
[M ϩ Na] (pos.); C46H75NO13 (850.09).
15.0, 6.0 Hz, 1 H, 27-H), 5.69 (d, J ϭ 10.0 Hz, 1 H, 5-H), 5.80 (d,
J ϭ 11.0 Hz, 1 H, 13-H), 6.39 (s, 1 H, 3-H), 6.55 (dd, J ϭ 15.0,
11.0 Hz, 1 H, 14-H); 13C NMR (125 MHz, CD2Cl2/CD3OD 2:1,
Ϫ20 °C): δ ϭ 7.9 (q, 20-CH3), 9.3 (q, 18-CH3), 11.7 (q, 8-Et-CH3),
13.2 (q, 24-CH3), 14.1 (q, 4-CH3), 16.1 (q, 12-CH3), 16.8 (q, 6-
CH3), 17.8 (q, C-28), 21.7 (q, 10-CH3), 22.6 (t, 8-Et-CH2), 34.9 (d,
C-6), 35.6 (d, C-10), 37.2 (d, C-18), 38.8 (t, C-22), 39.1 (d, C-20),
43.5 (d, C-24), 43.7 (d, C-8), 45.4 (t, C-11), 55.7 (q, 16-OCH3), 59.2
(q, 2-OCH3), 69.2 (d, C-19), 73.3 (d, C-23), 73.3 (d, C-7), 75.8 (d,
C-17), 76.5 (d, C-21), 79.4 (d, C-9), 82.1 (d, C-16), 82.9 (d, C-25),
122.9 (d, C-13), 126.7 (d, C-15), 128.7 (d, C-27), 130.6 (d, C-26),
131.1 (d, C-3), 131.6 (s, C-4), 133.8 (d, C-14), 140.9 (d, C-5), 141.7
(s, C-2), 142.7 (s, C-12), 166.2 (s, C-1); FAB MS: m/z ϭ 676 [M Ϫ
H] (neg.); 699 [M ϩ Na] (pos.); C39H64O9 (676.93).
3Ј,9-Di-O-acetyl-21-deoxyconcanamycin A (7): A solution of 5
(36 mg, 0.042 mmol) in pyridine (0.6 mL) was treated with acetic
anhydride (0.5 mL) and stirred for 3 h at room temp. The mixture
was poured into ice-water and extracted with CHCl3. After the
usual workup procedure, traces of acid were removed in vacuo by
addition of toluene. Purification by CC (EtOAc/n-hexane, 3:2) gave
7 (40 mg, 77%). M.p. 133 °C (dec.); IR (KBr): ν ϭ 3500 cm
Ϫ1
˜
,
2970, 1740, 1720sh, 1690sh, 1620 w, 1450, 1380, 1240, 1110; UV
(MeOH): λmax (log ε) ϭ 245 (4.56), 284 (4.20); [α]2D0 ϭ Ϫ45 (c ϭ
0.5 in MeOH); 1H NMR (500 MHz, CDCl3) see Table 2; 1H NMR
(500 MHz, [D6]benzene) see Table 2 and δ ϭ 0.93 (t, J ϭ 7.0 Hz,
3 H, 8-Et-CH3), 0.95 (d, J ϭ 7.0 Hz, 3 H, 6-CH3, 18-CH3), 0.95
(m, 1 H, 8-Et-CH2), 1.08 (d, J ϭ 7.0 Hz, 3 H, 20-CH3), 1.10 (m, 1
H, 8-Et-CH2), 1.11 (d, J ϭ 7.0 Hz, 3 H, 24-CH3), 1.33 (d, J ϭ 9,23-Di-O-acetyl-21-deoxyconcanolide
A
(8): Compound
6
6.0 Hz, 3 H, 6Ј-H3), 1.35 (ddd, J ϭ 12.0, 11.0, 10.0 Hz, 1 H, 22- (12.8 mg, 0.019 mmol) was acetylated as in the acetylation of 5. CC
Hax), 1.55 (ddq, J ϭ 10.0, 10.0, 7.0 Hz, 1 H, 24-H), 1.65 (m, 1 H, (EtOAc/n-hexane, 1:1) gave 8 (7.4 mg, 77%); UV (MeOH): λmax
1
8-H), 1.78 (s, 6 H, 12-CH3, H3C-COO), 1.89 (ddd, J ϭ 12.0, 11.0, (log ε) ϭ 245 (4.59), 284 (4.23); H NMR (500 MHz, CDCl3) see
10.0 Hz, 1 H, 2Ј-Hax), 1.93 (dq, J ϭ 9.0, 7.0 Hz, 1 H, 20-H), 1.97
(m, 2 H, 11-H2), 2.07 (s, 6 H, 4-CH3, H3C-COO), 2.55 (m, 2 H, 6-
Table 2 and δ ϭ 0.77 (d, J ϭ 7.0 Hz, 3 H, 24-CH3), 0.80 (d, J ϭ
7.0 Hz, 3 H, 18-CH3), 0.86 (d, J ϭ 7.0 Hz, 3 H, 20-CH3), 0.90 (d,
H, 18-H), 2.71 (m, 1 H, 10-H), 3.10 (s, 3 H, 16-OCH3), 3.64 (s, 3 J ϭ 7.0 Hz, 3 H, 10-CH3), 1.08 (d, J ϭ 7.0 Hz, 3 H, 6-CH3), 1.27
H, 2-OCH3), 5.18 (dd, J ϭ 15.0, 9.0 Hz, 1 H, 15-H), 5.75 (d, J ϭ (ddd, J ϭ 12.0, 11.0, 11.0 Hz, 1 H, 22-Hax), 1.43 (ddd, J ϭ 11.0,
10.0 Hz, 1 H, 13-H), 5.82 (d, J ϭ 10.0 Hz, 1 H, 5-H), 6.50 (dd, J ϭ 10.0, 6.5 Hz, 1 H, 24-H), 1.52 (m, 1 H, 8-H), 1.68 (m, 1 H, 20-H),
15.0, 10.0 Hz, 1 H, 14-H); 13C NMR (125 MHz, CDCl3): δ ϭ 8.2 1.86 (s, 3 H, 12-CH3), 1.94 (s, 3 H, 4-CH3), 1.9Ϫ2.0 (m, 2 H, 11-
(q, 20-CH3), 9.6 (q, 18-CH3), 11.9 (q, 8-Et-CH3), 14.1 (q, 4-CH3),
H2), 2.04 (s, 3 H, H3C-COO), 2.05 (m, 1 H, 18-H), 2.10 (s, 3 H,
16.2 (q, 12-CH3), 16.9 (q, 6-CH3), 17.5 (q, C-6Ј),17.6 (q, C-28), H3C-COO), 2.56 (m, 1 H, 10-H), 2.67 (ddd, J ϭ 10.0, 7.0, 2.5 Hz,
21.5 (q, 10-CH3), 21.5 (t, 8-Et-CH2), 33.9 (d, C-6), 34.8 (t, C-22),
1 H, 6-H), 3.23 (s, 3 H, 16-OCH3), 3.57 (s, 3 H, 2-OCH3), 3.66 (m,
35.0 (d, C-10), 37.0 (t, C-2Ј), 37.3 (d, C-18), 39.5 (d, C-20), 41.4
1 H, 7-H), 5.25 (dd, J ϭ 15.0, 9.0 Hz, 1 H, 15-H), 5.55 (d, J ϭ
(d, C-24), 43.9 (d, C-8), 45.3 (t, C-11), 55.7 (q, 16-OCH3), 59.2 (q, 10.0 Hz, 1 H, 5-H), 5.78 (d, J ϭ 10.5 Hz, 1 H, 13-H), 6.54 (dd, J ϭ
2-OCH3), 69.6 (d, C-19), 71.0 (d, C-3Ј), 70.1 (d, C-5Ј),75.0 (d, C- 15.0, 10.5 Hz, 1 H, 14-H); ESI MS: m/z ϭ 784 [M ϩ Na] (pos);
7), 75.3 (d, C-4Ј), 76.1 (d, C-17), 76.8 (d, C-21), 78.8 (d, C-23), 79.7 C43H68O11 (760.96).
(d, C-9), 82.0 (d, C-16), 82.9 (d, C-25), 95.5 (d, C-1Ј),123.8 (d, C-
23-O-(p-Nitrobenzylsulfonyl)-21-deoxyconcanolide A (9): DMAP
(179.5 mg, 1.5 mmol, 12.5 equiv.), NEt3 (1.6 mL) and p-nitroben-
zenesulfonyl chloride (334 mg, 1.5 mmol, 12.5 equiv.) were added
to a solution of 6 (82 mg, 0.012 mmol) in CH2Cl2 (5 mL). After
2 h stirring at 0 °C, the mixture was poured into ice-water and
13), 128.0 (d, C-15), 128.2 (d, C-27), 130.8 (d, C-26), 130.0 (d, C-
3), 132.4 (s, C-4), 133.0 (d, C-14), 138.6 (d, C-5), 141.2 (s, C-2),
142.4 (s, C-12), 155.9 (s, CONH2), 165.9 (s, C-1); ESI MS: m/z ϭ
957 [M ϩ Na] (pos); C50H79NO15 (934.11).
21-Deoxyconcanolide A (6): A mixture of 5 (302 mg, 0.37 mmol) extracted with CH2Cl2. The usual workup procedure and CC
and p-toluenesulfonic acid (164 mg, 0.85 mmol, 2.3 equiv.) in (EtOAc/cyclohexane, 2:3) furnished 9 (94 mg, 89%) . M.p. 75 °C
AcCN/H2O (30 mL, 5:1) was stirred for 12 h at 38 °C. After cooling
to 0 °C and addition of 30 mL sat. NaHCO3 solution, the organic
solvent was removed in vacuo and the resulting aqueous solution
(dec.); IR (KBr): ν˜ ϭ 3446 cmϪ1, 2968, 1694, 1619 w, 1534, 1454
w, 1351, 1250, 1187, 1102; UV (MeOH): λmax (log ε) ϭ 245 (4.63),
282 (4.29); [α]2D0 ϭ Ϫ34 (c ϭ 0.6 in MeOH); CD (MeOH): λextr.
was extracted with CH2Cl2. The usual workup procedure, followed ([Θ]22) ϭ 210 nm (sh) (7600), 242 (80800), 268 (Ϫ49200); 1H NMR
by CC (EtOAc/cyclohexane, 2:3), gave 6 (82 mg, 34%) as a white,
amorphous powder and 90 mg (30%) starting material. M.p. 97 °C
(dec.); IR (KBr): ν˜ ϭ 3456 cmϪ1, 2965, 1696, 1650 w, 1623 w, 1454,
(500 MHz, CD2Cl2/CD3OD, 2:1): δ ϭ 0.65 (d, J ϭ 6.5 Hz, 3H 24-
CH3), 0.76 (d, J ϭ 6.0 Hz, 3 H, 20-CH3), 0.78 (d, J ϭ 6.0 Hz, 3
H, 18-CH3), 0.82 (m, 3 H, 8-Et-CH3), 0.96 (m, 3 H, 6-CH3), 1.04
1361, 1250, 1104; UV (MeOH): λmax (log ε) ϭ 243 (4.40), 281 (m, 2 H, 8-Et-CH2), 1.14 (d, J ϭ 7.0 Hz, 3 H, 10-CH3), 1.45 (m, 1
4530 Eur. J. Org. Chem. 2001, 4525Ϫ4532

A Doubly Labelled Concanamycin Derivative for Binding Studies
FULL PAPER
H, 8-H), 1.49 (m, 1 H, 22-Hax), 1.50 (m, 1 H, 24-H), 1.60 (dd, J ϭ Et-CH2), 21.5 (q, 10-CH3), 34.0 (d, C-10), 35.3 (d, C-6), 37.2 (t, C-
6.0, 2.0 Hz, 3 H, 28-H), 1.63 (m, 1 H, 20-H), 1.84 (s, 3 H, 12-CH3), 22), 37.3 (d, C-18), 39.7 (d, C-20), 41.6 (d, C-24), 44.5 (d, C-8),
1.97 (s, 3 H, 4-CH3), 1.98 (m, 2 H, 11-H2), 2.03 (m, 1 H, 18-H), 45.9 (t, C-11), 55.9 (q, 16-OCH3), 59.4 (q, 2-OCH3), 69.7 (d, C-
2.16 (ddd, J ϭ 11.0, 5.0, 1.5 Hz, 1 H, 22-Heq), 2.29 (m, 1 H, 10-
H), 2.72 (m, 1 H, 6-H), 3.22 (m, 1 H, 9-H), 3.23 (s, 3 H, 16-OCH3), 81.4 (d, C-9), 82.3 (d, C-16), 82.6 (d, C-25), 121.2 (s, C-CF3), 123.4
3.34 (dd, J ϭ 10.0, 8.0 Hz, 1 H, 25-H), 3.44 (dd, J ϭ 11.0, 8.0, (s, CF3), 124.2 (d, C-13), 124.8 (d, 2 ϫ C-3Ј), 126.8 (d, 2 ϫ C-3ЈЈ),
1.5 Hz, 1 H, 21-H), 3.50 (m, 1 H, 7-H), 3.54 (s, 3 H, 2-OCH3), 3.59 128.1 (d, C-15), 129.5 (d, 2 ϫ C-2Ј),129.7 (d, C-27), 130.1 (d, C-
(m, 1 H, 19-H), 3.82 (dd, J ϭ 9.0, 9.0 Hz, 1 H, 16-H), 4.43 (ddd, 26), 130.2 (d, 2 ϫ C-2ЈЈ) 130.5 (d, C-3), 132.1 (s, C-4), 132.6 (s, C-
19), 87.7 (d, C-23), 75.1 (d, C-7), 76.1 (d, C-17), 76.5 (d, C-21),
J ϭ 11.0, 11.0, 5.0 Hz, 1 H, 23-H), 5.08 (br. d, J ϭ 7.0 Hz, 1 H, 1ЈЈ), 133.8 (s, C-4ЈЈ), 133.8 (d, C-14), 139.2 (d, C-5), 142.0 (s, C-2),
17-H), 5.19 (dd, J ϭ 15.0, 9.0 Hz, 1 H, 15-H), 5.27 (ddq, J ϭ 15.0, 142.6 (s, C-12), 143.4 (s, C-1Ј), 151.0 (s, C-4Ј), 165.7 (s, COO) 166.2
8.0, 2.0 Hz, 1 H, 26-H), 5.57 (dq, J ϭ 15.0, 6.0 Hz, 1 H, 27-H), (s, C-1); ESI MS: m/z ϭ 1096 [M ϩ Na] (pos.); C54H70F3N3O14S
5.68 (d, J ϭ 10.0 Hz, 1 H, 5-H), 5.80 (d, J ϭ 11.0 Hz, 1 H, 13-H),
6.38 (s, 1 H, 3-H), 6.55 (dd, J ϭ 15.0, 11.0 Hz, 1 H, 14-H), 8.12
(d, J ϭ 9.0 Hz, 2 H, 2 ϫ 2ЈH), 8.40 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ 3ЈH);
13C NMR (125 MHz, CD2Cl2/CD3OD, 2:1, Ϫ20 °C): δ ϭ 7.7 (q,
20-CH3), 9.2 (q, 18-CH3), 11.6 (q, 8-Et-CH3), 13.2 (q, 24-CH3),
14.0 (q, 4-CH3), 16.0 (q, 12-CH3), 16.8 (q, 6-CH3), 17.7 (q, C-28),
21.6 (t, 8-Et-CH2), 22.5 (q, 10-CH3), 34.8 (d, C-6), 35.6 (d, C-10),
36.8 (t, C-22), 37.1 (d, C-18), 39.1 (d, C-20), 41.0 (d, C-24), 43.6
(d, C-8), 45.3 (t, C-11), 55.7 (q, 16-OCH3), 59.1 (q, 2-OCH3), 73.2
(d, C-19), 87.2 (d, C-23), 69.0 (d, C-7), 75.6 (d, C-17), 75.8 (d, C-
21), 79.4 (d, C-9), 82.0 (d, C-16), 82.4 (d, C-25), 122.8 (d, C-13),
124.7 (d, C-3Ј), 126.6 (d, C-15), 128.2 (d, C-26), 129.3 (d, C-2Ј),
129.9 (d, C-27), 131.2 (d, C-3), 131.6 (s, C-4), 133.8 (d, C-14), 141.0
(d, C-5), 141.6 (s, C-2), 142.4 (s, C-12), 142.8 (s, C-1Ј), 150.6 (s, C-
4Ј), 166.3 (s, C-1); ESI MS: m/z ϭ 884 [M ϩ Na] (pos.);
C45H67NO13S (862.09).
(1074.22).
23-Iodo-9-O-[p-(trifluoroethyldiazirinyl)benzoyl]-21,23-dideoxy-
concanolide A (11a): A solution of 10 (9 mg, 0.01 mmol) and NaI
(10 mg, 0.07 mmol, 7 equiv.) in DMF (3 mL) was stirred for 3 h at
40 °C. Phosphate buffer (pH ϭ 7, 4 mL) was added and the mixture
was extracted with CH2Cl2. After the usual workup procedure,
traces of DMF were removed in vacuo by addition of toluene. Puri-
fication by CC (EtOAc/cyclohexane, 1:3) gave 11a (9 mg, 92%).
M.p. 97 °C (dec.); IR (KBr): ν ϭ 3448 cmϪ1, 2968, 1719, 1697,
˜
1623 w, 1605 (sh) w, 1344, 1274, 1194, 1159, 1108; UV (MeOH):
λmax (log ε) ϭ 243 (4.75), 281 (4.28); [α]2D0 ϭ ϩ36 (c ϭ 0.2 in
MeOH); CD (MeOH): λextr. ([Θ]22) ϭ 213 nm (Ϫ6700), 224
(Ϫ10000), 246 (84700), 267 (Ϫ21400)- 1H NMR (500 MHz,
CD2Cl2): δ ϭ 0.79 (d, J ϭ 7.0 Hz, 3 H, 18-CH3), 0.82 (d, J ϭ
7.0 Hz, 3 H, 20-CH3), 0.83 (m, 3 H, 10-CH3), 0.89 (m, 3 H, 8-Et-
CH3), 0.96 (d, J ϭ 6.5 Hz, 3H 24-CH3), 1.00 (d, J ϭ 7.0 Hz, 3 H,
6-CH3), 1.23 (m, 2 H, 8-Et-CH2), 1.59 (m, 3 H, 28-H), 1.61 (m, 1
H, 20-H), 1.68 (m, 1 H, 24-H), 1.75 (m, 1 H, 8-H), 1.89 (s, 3 H,
12-CH3), 1.92 (s, 3 H, 4-CH3), 2.02 (m, 1 H, 18-H), 2.05Ϫ2.14 (m,
2 H, 11-H2), 2.05Ϫ2.14 (m, 1 H, 22-Hax), 2.57 (ddd, J ϭ 13.0, 4.5,
1.5 Hz, 1 H, 22-Heq), 2.65 (m, 1 H, 6-H), 2.86 (m, 1 H, 10-H), 3.22
(s, 3 H, 16-OCH3), 3.38 (m, 1 H, 21-H), 3.40 (m, 1 H, 25-H), 3.54
(m, 1 H, 19-H), 3.53 (s, 3 H, 2-OCH3), 3.77 (br. d, J ϭ 10.0 Hz, 1
H, 7-H), 3.81 (dd, J ϭ 9.0, 9.0 Hz, 1 H, 16-H), 4.08 (ddd, J ϭ 12.0,
12.0, 4.5 Hz, 1 H, 23-H), 5.05 (m, 1 H, 9-H), 5.07 (m, 1 H, 17-H),
5.21 (dd, J ϭ 15.0, 9.0 Hz, 1 H, 15-H), 5.31 (m, 1 H, 26-H), 5.56
(dq, J ϭ 15.0, 6.5 Hz, 1 H, 27-H), 5.62 (d, J ϭ 10.0 Hz, 1 H, 5-H),
5.82 (br. d, J ϭ 11.0 Hz, 1 H, 13-H), 6.39 (s, 1 H, 3-H), 6.59 (dd,
J ϭ 15.0, 11.0 Hz, 1 H, 14-H), 7.28 (d, J ϭ 8.5 Hz, 2 H, 2 ϫ 3ЈЈH),
8.06 (d, J ϭ 8.5 Hz, 2 H, 2 ϫ 2ЈЈH); 13C NMR (125 MHz, CD2Cl2):
δ ϭ 8.2 (q, 20-CH3), 9.6 (q, 18-CH3), 12.1 (q, 8-Et-CH3), 14.3 (q,
4-CH3), 16.1 (q, 12-CH3), 16.9 (q, 6-CH3), 17.8 (q, C-28), 19.5 (q,
24-CH3), 21.7 (q, 10-CH3), 29.4 (t, 8-Et-CH2), 34.1 (d, C-10), 35.3
(d, C-6), 37.3 (d, C-18), 38.7 (d, C-23), 39.5 (d, C-20), 45.4 (t, C-
22), 46.3 (d, C-24), 44.5 (d, C-8), 45.9 (t, C-11), 55.9 (q, 16-OCH3),
59.4 (q, 2-OCH3), 69.6 (d, C-19), 75.1 (d, C-7), 76.1 (d, C-17), 80.5
(d, C-21), 81.4 (d, C-9), 82.4 (d, C-16), 84.2 (d, C-25), 121.4 (s, C-
CF3), 123.4 (s, CF3), 124.2 (d, C-13), 126.9 (d, 2 ϫ C-3ЈЈ),128.1 (d,
C-15), 129.0 (d, C-27), 131.2 (d, C-26), 130.2 (d, 2 ϫ C-2ЈЈ), 130.3
(d, C-3), 132.1 (s, C-4), 132.7 (s, C-1ЈЈ), 133.8 (d, C-14), 133.9 (s,
C-4ЈЈ), 139.2 (d, C-5), 142.0 (s, C-2), 142.6 (s, C-12), 165.7 (s, COO)
23-O-(p-Nitrobenzylsulfonyl)-9-O-[p-(trifluoroethyldiazirinyl)-
benzoyl]-21-deoxyconcanolide A (10): Compound
9
(40 mg,
0.05 mmol) in dry CH2Cl2 (3 mL) was treated with DMAP
(24.3 mg, 0.2 mmol, 4 equiv.), DCC (41.3 mg, 0.2 mmol, 4 equiv.)
and p-(trifluoroethyldiazirinyl)benzoic acid (29.7 mg, 0.15 mmol, 3
equiv.). The resulting mixture was stirred for 18 h at room temp.
and the organic solvent was removed in vacuo. The crude extract
was purified on silica gel (EtOAc/cyclohexane, 1:3) and then on
LH-20 (acetone) to yield 10 (17 mg, 46%). M.p. 100 °C (dec.); IR
(KBr): ν ϭ 3442 cmϪ1, 2970, 1716, 1698, 1628 w, 1609 w, 1535,
˜
1454, 1349, 1275, 1188, 1158, 1106; UV (MeOH): λmax (log ε) ϭ
244 (4.83), 281 (4.47); [α]2D0 ϭ ϩ36 (c ϭ 0.2 in MeOH); CD
(MeOH): λextr. ([Θ]22) ϭ 210 nm (Ϫ2020), 225 (Ϫ10300), 246
(120500), 267 (Ϫ27400); 1H NMR (500 MHz, CD2Cl2): δ ϭ 0.66
(d, J ϭ 6.5 Hz, 3H 24-CH3), 0.77 (d, J ϭ 7.0 Hz, 3 H, 20-CH3),
0.78 (d, J ϭ 7.0 Hz, 3 H, 18-CH3), 0.90 (m, 3 H, 10-CH3), 0.91 (m,
3 H, 8-Et-CH3), 1.02 (d, J ϭ 7.0 Hz, 3 H, 6-CH3), 1.10 (m, 2 H,
8-Et-CH2), 1.48 (m, 1 H, 22-Hax), 1.48 (m, 1 H, 24-H), 1.60 (dd,
J ϭ 7.0, 1.5 Hz, 3 H, 28-H), 1.64 (m, 1 H, 20-H), 1.70 (m, 1 H, 8-
H), 1.91 (s, 3 H, 12-CH3), 1.94 (s, 3 H, 4-CH3), 2.05 (m, 1 H, 18-
H), 2.09Ϫ2.14 (m, 2 H, 11-H2), 2.17 (ddd, J ϭ 12.0, 5.0, 2.0 Hz, 1
H, 22-Heq), 2.67 (m, 1 H, 6-H), 2.87 (m, 1 H, 10-H), 3.23 (s, 3 H,
16-OCH3), 3.35 (dd, J ϭ 9.0, 8.0 Hz, 1 H, 25-H), 3.45 (dd, J ϭ
11.0, 8.0, 2.0 Hz, 1 H, 21-H), 3.51 (m, 1 H, 19-H), 3.54 (s, 3 H, 2-
OCH3), 3.77 (dd, J ϭ 10.0, 1.5 Hz, 1 H, 7-H), 3.82 (dd, J ϭ 9.0,
9.0 Hz, 1 H, 16-H), 4.44 (ddd, J ϭ 11.0, 11.0, 5.0 Hz, 1 H, 23-H),
5.04 (dd, J ϭ 9.0, 1.5 Hz, 1 H, 17-H), 5.08 (d, J ϭ 11.0 Hz, 1 H,
9-H), 5.22 (dd, J ϭ 15.0, 9.0 Hz, 1 H, 15-H), 5.27 (ddq, J ϭ 15.0,
8.0, 1.5 Hz, 1 H, 26-H), 5.57 (dq, J ϭ 15.0, 7.0 Hz, 1 H, 27-H),
166.2 (s, C-1); ESI MS: m/z
C48H66F3IN2O9 (998.98).
ϭ 1021 [M ϩ Na] (pos.);
23-Iodo(125I)-9-O-[p-(trifluoroethyldiazirinyl)benzoyl]-21,23-di-
5.64 (d, J ϭ 10.0 Hz, 1 H, 5-H), 5.84 (d, J ϭ 11.0 Hz, 1 H, 13-H), deoxyconcanolide A (11b): Na125I (10 mCi) was purchased as a so-
6.42 (s, 1 H, 3-H), 6.61 (dd, J ϭ 15.0, 11.0 Hz, 1 H, 14-H), 7.30 lution in water. The water was removed in vacuo and 10 (13 mg,
(d, J ϭ 8.5 Hz, 2 H, 2 ϫ 3ЈЈH), 8.08 (d, J ϭ 8.5 Hz, 2 H, 2 ϫ 0.012 mmol), NaI (4.5 mg, 0.030 mmol, 2.5 equiv.) and DMF (2
2ЈЈH), 8.13 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ 2ЈH), 8.40 (d, J ϭ 9.0 Hz, 2 mL) were added to the residue. The resulting mixture was stirred
H, 2 ϫ 3ЈH); 13C NMR (125 MHz, CD2Cl2): δ ϭ 8.0 (q, 20-CH3), for 48 h at 40 °C and the workup was carried out as described for
9.6 (q, 18-CH3), 12.1 (q, 8-Et-CH3), 13.5 (q, 24-CH3), 14.2 (q, 4- 11a to yield 11b (5.9 mg, 50%) with a specific radioactivity of 438.7
CH3), 16.1 (q, 12-CH3), 16.8 (q, 6-CH3), 17.8 (q, C-28), 21.6 (t, 8- mCi/mmol.
Eur. J. Org. Chem. 2001, 4525Ϫ4532
4531

A. Zeeck et al.
FULL PAPER
[11]
B. P. Crider, X.-S. Xie, D. K. Stones, J. Biol. Chem. 1994,
269, 17379Ϫ17381.
Acknowledgments
[12]
[13]
[14]
[15]
We thank Prof. Dr. J. Brunner (ETH Zürich) for providing us with
p-(trifluoroethyldiazirinyl)benzoic acid, M. Huss and Prof. Dr. H.
Wieczorek (Universität Osnabrück) for the V-type ATPase tests
and R. Machinek and C. Zolke for recording the NMR spectra.
Part of this work was supported by grants from the Deutsche For-
schungsgemeinschaft (SFB 416 and SFB 431).
K. U. Bindseil, PhD Dissertation 1993, Universität Göttingen.
C. Boddien, PhD Dissertation 1995, Universität Göttingen.
K. U. Bindseil, A. Zeeck, J. Org. Chem. 1993, 58, 5487Ϫ5492.
S. Dröse, C. Boddien, M. Gaßel, G. Ingenhorst, A. Zeeck, K.
Altendorf, Biochemistry 2001, 40, 2816Ϫ2825.
K. Altendorf, M. Gassel, W. Puppe, T. Möllenkamp, A. Zeeck,
C. Boddien, K. Fendler, E. Bamberg, S. Dröse, Acta Physiol.
Scand. 1998, 643, 137Ϫ146.
H. Kinashi, K. Someno, K. Sakaguchi, J. Antibiot. 1984, 37,
1333Ϫ1343.
K. U. Bindseil, A. Zeeck, Helv. Chim. Acta 1993, 76, 150Ϫ157.
J. Brunner, H. Senn, F. M. Richards, J. Biol. Chem. 1980,
255, 3313Ϫ3317.
[16]
[17]
[1]
C. Boddien, J. Gerber-Nolte, A. Zeeck, Liebigs Ann. 1996,
1381Ϫ1384.
[2]
[18]
[19]
K. U. Bindseil, A. Zeeck, Liebigs Ann. Chem. 1994, 305Ϫ312.
E. J. Bowman, A. Siebers, K. Altendorf, Proc. Natl. Acad. Sci.
[3]
USA 1988, 85, 7972Ϫ7976.
S. Dröse, K. U. Bindseil, E. J. Bowman, A. Siebers, A. Zeeck,
[4]
[20]
[21]
D. A. Horne, A. Jordan, Tetrahedron Lett. 1978, 16,
K. Altendorf, Biochemistry 1993, 32, 3902Ϫ3906.
J.-T. Woo, Y. Ohba, K. Tagami, K. Sumitani, K. Yamaguchi,
1357Ϫ1358.
[5]
B. Neises, W. Steglich, Angew. Chem. 1978, 90, 556Ϫ557; An-
gew. Chem. Int. Ed. Engl. 1978, 17, 552Ϫ553.
J.-H. Cho, C. Djerassi, J. Org. Chem. 1987, 52, 4517Ϫ4521.
In our previous publication,[15] 11b was mentioned as pos-
sessing a 23-epi configuration. This should now be changed on
the basis of better resolved NMR spectra.
E. Pretsch et. al., Strukturaufklärung organischer Verbindungen,
Springer Verlag Berlin, 1990.
M. Huss, M. Gassel, G. Ingenhorst, S. Dröse, S. König, A.
Zeeck, K. Altendorf, H. Wieczorek, Biochemistry, submitted.
Received June 18, 2001
T. Tsuji, Biol. Pharm. Bull. 1996, 19, 297Ϫ299.
S. Gagliardi, P. A. Gatti, P. Belfiore, A. Zocchetti, G. D. Clarke,
[6]
[22]
[23]
C. Farina, J. Med. Chem. 1998, 41, 1883Ϫ1893.
J. W. Westley, C. M. Liu, L. H. Sello, R. H. Evans, N. Troupe,
[7]
J. F. Blount, A. M. Chiu, L. J. Todaro, P. A. Miller, J. Antibiot.
1984, 37, 1738Ϫ1740.
T. Manabe, T. Yoshimori, N Henomatsu, Y. Tashiro, J. Cell.
[24]
[25]
[8]
Physiol. 1993, 157, 445Ϫ452.
[9]
S. Dröse, K. Altendorf, J. Exp. Biol. 1997, 200, 1Ϫ8.
[10]
J. Zhang, Y. Feng, M. Forgac, J. Biol. Chem. 1994, 269,
23518Ϫ23523.
[O01292]
4532
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