Synthesis and V-ATPase Inhibition of Simplified Lobatamide Analogues

Article abstract of DOI:10.1021/ol026391z

(Matrix Presented) Simplified analogues of the lobatamides have been synthesized and evaluated for inhibition of bovine V-ATPase. The salicylate phenol, enamide NH, and the ortho-substitution of the salicylate ester have been shown to be important for V-A

Full text of DOI:10.1021/ol026391z

ORGANIC
LETTERS
2002
Vol. 4, No. 18
3103-3106
Synthesis and V-ATPase Inhibition of
Simplified Lobatamide Analogues
Ruichao Shen,† Cheng Ting Lin,† Emma Jean Bowman,‡ Barry J. Bowman,‡ and
,†
John A. Porco, Jr.*
Department of Chemistry and Center for Streamlined Synthesis, Metcalf Center for
Science and Engineering, Boston UniVersity, 590 Commonwealth AVenue,
Boston, Massachusetts 02215, and Department of Molecular, Cell and DeVelopmental
Biology, Sinsheimer Labs, UniVersity of California, Santa Cruz, California 95064
porco@chem.bu.edu
Received June 19, 2002
ABSTRACT
Simplified analogues of the lobatamides have been synthesized and evaluated for inhibition of bovine V-ATPase. The salicylate phenol, enamide
NH, and the ortho-substitution of the salicylate ester have been shown to be important for V-ATPase inhibitory activity.
The recently reported salicylate enamide macrolides, includ-
ing the salicylihalamides,1 lobatamides,2 apicularens,3 CJ-
12950, and CJ-13357,4 and the oximidines5 comprise a
unique class of natural products with a common benzolactone
core structure and a highly unsaturated enamide side chain.
(Figure 1). Two members of the class, salicylihalamides and
lobatamides, displayed high potency in the NCI’s 60 cell-
line human tumor screen (mean panel GI50’s: ∼1.6 nM for
lobatamides and ∼15 nm for salicylihalamide A) along with
unique cellular response profiles.1,2 Recent studies have
shown that the salicylate enamide macrolides potently inhibit
† Boston University.
‡ University of California.
(1) (a) Erickson, K. L.; Beutler, J. A.; Cardellina, J. H., II; Boyd, M. R.
J. Org. Chem. 1997, 62, 8188 (b) Erickson, K. L.; Beutler, J. A.; Cardellina,
J. H., II; Boyd, M. R. J. Org. Chem. 2001, 66, 1532.
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Antibiot. 1998, 51, 1075. (b) Jansen, R.; Kunze, B.; Reichenbach, H.; Hofle,
G. Eur. J. Org. Chem. 2000, 6, 913.
(4) Dekker: K. A.; Aiello, R. J.; Hirai, H.; Inagaki, T.; Sakakibara, T.;
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Org. Chem. 1999, 64, 153.
Figure 1. Salicylate enamide natural products.
10.1021/ol026391z CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/03/2002

mammalian vacuolar-type (H+)-ATPases (V-ATPases) which
are ubiquitous proton-translocating pumps of eukaryotic
cells.6 Accordingly, these natural products are exciting new
targets for chemical synthesis, lead optimization studies, and
preparation of designed analogues to further define inter-
actions with the molecular target. The salicylihalamides have
been synthesized in several laboratories,7 and De Brabander
et al. have reported the preparation and structure-function
analysis of a number of promising derivatives.8 We recently
reported the total synthesis and stereochemical assignment
of lobatamide C using Cu(I)-catalyzed amidation for syn-
thesis of the C1-C10 fragment.9 In continuation of these
studies, we report the synthesis and biological evaluation of
simplified analogues of the lobatamides in order to clarify
the minimal core structure (pharmacophore) required for
V-ATPase inhibition.
TBAF to afford enamide alcohol 4. Encouraged by literature
precedent for esterification of salicylate cyanomethyl esters,13
we prepared salicylate enamides 5 and 6 by heating
compound 4 with cyanomethyl ester 7 or 814 in the presence
of a catalytic amount of K2CO3 in DMA (Scheme 1).15 On
the basis of literature reports and lack of acylation using
cyanomethyl benzoate, we initially suspected that keto-
ketenes (cf. Scheme 1, inset) were the active acylating
agents.16 However, trapping experiments with N,N-dimethyl
cyanamide, ethyl vinyl ether, ethoxyacetylene, and N-
benzylmaleimide failed to provide the corresponding Diels-
Alder adducts.17 An alternative mechanistic pathway was
further suggested by conformational analysis of the sodium
salt of 7.18 As shown in Figure 2, the ester carbonyl in the
Our initial objective was to prepare acyclic lobatamide
analogues and determine their ability to inhibit V-ATPase.
Since previous studies have shown that the enamide side
chain is important for the potent bioactivities of salicyli-
halamides1,8 and apicularen A,10 we initiated studies employ-
ing the C1-C8 subunit of the lobatamides (Scheme 1). TIPS-
Scheme 1a
Figure 2. Transesterification using intramolecular general base-
catalysis.
conformation shown is out of planarity, which is expected
to increase the reactivity of the carbonyl toward transesteri-
fication. In addition, the o-hydroxyl is suitably oriented to
act as a general base-catalyst and direct attack of the alcohol
to the carbonyl π*. This mechanism is further substantiated
by several literature reports.19
Two related methylated analogues 10 and 11 were
prepared in order to determine the biological importance of
the phenol and enamide hydrogens of the lobatamides,
respectively (Scheme 2). Enamide 3 was methylated with
NaH/MeI and desilylated to afford N-methyl enamide alcohol
12, which was acylated using cyanomethyl ester 7 to afford
analogue 10. Unfortunately, esterification of enamide alcohol
4 with o-anisic acid using modified Keck esterification20
a Reagents and conditions: (a) CuTC, Cs2CO3, 1, 10-phenan-
throline, dba, DMA, 50%; (b) TBAF, THF, 80%; (c) 7 or 8, K2CO3
(5 mol %), DMA, 90 °C, 1 h, 91% (5), 70% (6).
(10) Bhattacharjee, A.; Seguil, O. R.; De Brabander, J. K. Tetrahedron
Lett. 2001, 42, 1217.
(11) Posner, G. H.; Weitzberg, M.; Hamill, T. G.; Asirvatham, E.
Tetrahedron 1986, 42, 2919
protected (E)-4-iodo-3-buten-1-ol11 (1) underwent copper(I)
thiophenecarboxylate (CuTC)-mediated cross coupling with
amide 212 to furnish enamide 3, which was desilylated using
(12) Shen, R.; Porco, J. A., Jr. Org. Lett. 2000, 2, 1333.
(13) Sherlock, M. H. S. African Pat. 1968 ZA 6802187; CAN 70: 106224
(14) (a) Byers, J. H.; Baran, R. C.; Craig, M. E.; Jackman, J. T. Org.
Prep. Proced. Int. 1991, 23, 373 (b) Hugel. H. M.; Bhaskar, V.; Longmore,
R. W. Synth. Commun. 1992, 22, 693. For the preparation of 8, see ref 9.
(15) Analogue 9 was synthesized from (E,E)-2,4-hexadienamide (Pel-
legata, R.; Italia, A.; Villa, M. Synthesis 1985, 517) according to the general
procedure used to prepare analogue 5.
(16) For a recent review on keto-ketenes, see: Simion, C.; Costea, I.;
Badea, F.; Iordache, F. Roum. Chem. Quart. ReV. 2001, 8, 131.
(17) (a) Kaneko, C.; Sato, M.; Sakaki, J-i.; Abe, Y. J. Heterocycl. Chem.
1990, 27, 25. (b) Stadler, A.; Zangger, K.; Belaj, F.; Kollenz, G. Tetrahedron
2001, 57, 6757.
(6) Boyd, M. R.; Farina, C.; Belfiore, P.; Gagliardi, S.; Kim, J. W.;
Hayakawa, Y.; Beutler, J. A.; McKee, T. C.; Bowman, B. J.; Bowman, E.
J. J. Pharm. Exp. Ther. 2001, 297, 114.
(7) (a) Wu, Y.; Esser, L.; De Brabander, J. K. Angew. Chem., Int. Ed.
2000, 39, 4308. (b) Labrecque, D.; Charron, S.; Rej, R.; Blais, C.; Lamothe,
S. Tetrahedron Lett. 2001, 42, 2645. (c) Snider, B. B.; Song, F. Org. Lett.
2001, 3, 1817. (d) Smith, A. B., III; Zheng, J. Synlett 2001, 1019. (e)
Furstner, A.; Dierkes, T.; Thiel, O. R.; Blanda, G. Chem. Eur. J. 2001, 7,
5286.
(8) Wu, Y.; Liao, X.; Wang, R.; Xie, X-s.; De Brabander, J. K. J. Am.
Chem. Soc. 2002, 124, 3245.
(18) A conformational search was performed using PC Spartan Pro
version 1.0.7 (Wavefunction, Irvine, CA).
(9) Shen, R.; Lin, C. T.; Porco, J. A., Jr. J. Am. Chem. Soc. 2002, 124,
5650.
(19) (a) Bender, M. L.; Kezdy, F. J.; Zerner, B. J. Am. Chem. Soc. 1963,
85, 3017. (b) Khan, M. N. Int. J. Chem. Kinet. 1988, 20, 443.
3104
Org. Lett., Vol. 4, No. 18, 2002

alcohol 17 exhibited significantly lower reactivity in acylation
reactions with cyanomethyl ester 7 to afford 15 (25%). In
addition, 15% of a â-elimination product as well as consider-
able amounts of recovered 17 were obtained. Under similar
conditions, acylation of 17 with cyanomethyl ester 8 did not
afford the desired salicylate product. However, acid 18, after
neutralization with Bu4NOH and heating with 8 and Na2-
CO3 (1.0 equiv), furnished the desired salicylate ester. In
this reaction, a number of carbonate bases (Li, K, Rb, and
Cs) were also evaluated, but interestingly only stoichiometric
levels of Na2CO3 gave useful results. To facilitate purifica-
tion, the unstable enamide acid product was methylated with
trimethylsilyl diazomethane to afford methyl ester 16 (34%
from 18).
Scheme 2a
We have also initiated studies employing the C1-C10
fragment of the lobatamides to prepare simplified and
potentially more chemically stable macrocyclic analogues
(Scheme 4). Our initial target, macrodilactone 19, replaces
a Reagents and conditions: (a) NaH, DMF, MeI, 98%; (b) TBAF,
THF, 90%; (c) 7, K2CO3, DMA, 81%; (d) 2, CuTC, Cs2CO3, DMA,
29%; (e) CSA (50 mol %), CH2Cl2, rt, 1 h (78%).
Scheme 4a
employing DMAP-CSA as a cocatalyst did not afford the
desired product 11. Since we suspected side reactions of the
enamide under acidic conditions, enamide 6 was treated with
CSA in CH2Cl2 to cleanly afford dimer 13 (78%).21 Dimer-
ization of enamides by protonation to an N-acyliminium ion
and reaction with a second equivalent of enamide is
precedented in the literature22 and represents a competing
side reaction in the presence of anhydrous acid. Analogue
11 was ultimately prepared using Cu-mediated enamide bond
construction using amide 2 and vinyl iodide 14.23
Additional acyclic, salicylate enamide analogues 15 and
16 were prepared using the C1-C10 fragment of lobatamide
C (Scheme 3, 17 and 18)9 to determine the effect of the C8
a Reagents and conditions: (a) Pd2(dba)3CHCl3, AsPh3, THF,
70 °C, 60%; (b) TBAF, THF, 0 °C, 73%; (c) 18, Bu4NOH, MeOH;
azeotrope water; Na2CO3, 23, DMF/2-butanone, 80 °C, 2 h; (d)
HF-pyridine/pyridine, THF, 36% (2 steps); (e) DIAD, PPh3, THF,
65%.
Scheme 3a
the fragile divinylcarbinol moiety with a Z-olefin and
simplified three-carbon segment. Stille coupling24 of vinyl
stannane 2025 and benzylic bromide 219 afforded salicylate
22. Selective desilylation (TBAF) provided cyanomethyl
(20) Sunazuka, T.; Hirose, T.; Harigaya, Y.; Takamatsu, S.; Hayashi,
M.; Komiyama, K.; Omura, S.; Sprengeler, P. A.; Smith, A. B. J. Am. Chem.
Soc. 1997, 119, 10247.
(21) Dimer 13 has a hydrogen-bonded amide proton (Ha) and a non-
hydrogen-bonded NH proton (Hb); cf. Gardner, R.; Liang, G.-B.; Gellman,
S. J. Am. Chem. Soc. 1999, 121, 1806.
(22) (a) Ben-Ishai, D.; Giger, R. Tetrahedron Lett. 1965, 6, 4523. (b)
Schoemaker, H. E.; Boer-Terpstra, T.; Dijkink, J.; Speckamp, W. N.
Tetrahedron 1980, 36, 143.
a Reagents and conditions: (a) 7, K2CO3, DMA, 25%, 60%
recovered 17; (b) (i) Bu4NOH, MeOH; azeotrope water; Na2CO3,
8, DMF/2-butanone, 80 °C, 2 h, (ii) TMSCHN2; 34% (two steps).
(23) Prepared by DIC coupling of o-anisic acid and 4-iodo-3-buten-1-
ol. See Supporting Information for details.
(24) Kamlage, S.; Sefkow, M.; Peter, M. G. J. Org. Chem. 1999, 64,
2938.
(25) Lipshutz, B. H.; Keil, R.; Barton, J. C. Tetrahedron Lett. 1992, 33,
5861.
stereogenic center on V-ATPase inhibition. Although satis-
factory yields were obtained for primary alcohols in the
synthesis of analogues such as 5 and 6 (Scheme 1), secondary
Org. Lett., Vol. 4, No. 18, 2002
3105

ester 23, which was coupled with enamide acid 18 to furnish
the salicylate 24. Desilylation (HF-pyridine/pyridine) pro-
vided hydroxy acid 25, which underwent Mitsunobu mac-
rolactonization9 (0.005 M) to afford the target macrocyclic
analogue 19 (65%).
substituted compound 16 (0.1 µM), which was 180 times
more active than unsubstituted compound 15. Conformational
analysis18 of 15 and 16 (Figure 3A) indicates that ortho-H-
The simplified lobatamide analogues were evaluated for
activity against bovine V-ATPase (Table 1).26 Enamide 4
Table 1. Effect of Simplified Analogues of Lobatamides
against Bovine V-ATPasea
analogue
IC50 (µM)
analogue
IC50 (µM)
lobatamide C
5
6
9
0.002
b
1.3
c
11
13
15
16
19
no effect
d
18
0.1
1.2
Figure 3. Representative minimum energy conformations of 15
(A) and 16 (B) (Chem 3D, enamide side chain omitted for clarity).
10
200
substituted salicylate 15 maintains near planarity of the
carbonyl with the aromatic ring. In contrast, the o-methyl
substituent forces the carbonyl out-of-plane by approximately
60°, presumably because of steric hindrance effects.27 This
sterically hindered resonance effect is also seen in the X-ray
crystal structure of apicularen A3b (salicylate ester carbonyl
out of planarity by 80°) and may be an important configu-
ration of the salicylate moiety in potent V-ATPase inhibitors.
In summary, a series of simplified analogues of the
lobatamides have been prepared and evaluated as mammalian
V-ATPase inhibitors. Although simplified derivatives are not
as potent as the natural products, a number of useful
structure-reactivity relationships have been uncovered,
including enhancement of V-ATPase inhibition in acyclic
analogues by ortho-substitution of the salicylate ring. Con-
tinued studies on the synthesis of salicylate enamides and
analogues are ongoing.
a ATPase activity determined as described in the Supporting Information
using 20 µg of membrane protein and the indicated amount of inhibitor.
b 25% inhibition at 20 µm, higher concentrations not soluble. c 25%
inhibition at 30 µm, higher concentrations not soluble. d 35% inhibition at
30 µm, higher concentrations not soluble.
was found to be inactive, which indicates the requirement
for both the salicylate ester and enamide for inhibition. The
minimal salicylate enamide compounds 5 and 9 were found
to inhibit bovine V-ATPase (25% inhibition at 20 and 30
mm, respectively). Interestingly, permutation of the ortho
hydrogen to a methyl group substantially increased the
activity against bovine ATPase (6, IC50 ) 1.3 µM). Dimeric
compound 13 was considerably less active. Methylated
analogues 10 and 11 showed relatively weak inhibition of
V-ATPase, which indicates the importance of a free phenol
and enamide NH. Macrodilactone 19 showed good V-
ATPase inhibition (1.2 µm), but not at the nanomolar potency
of the lobatamides, which indicates that the ring size and
substitution of the macrolactone are important for potent
V-ATPase inhibition. The most potent simplified salicylate
enamide analogue to emerge from this study was o-methyl-
Acknowledgment. Financial support was provided by the
NIH [GM62842 (J.A.P, Jr.) and GM28703, GM58903
(B.J.B.)]. C-T.L. was supported in part by the Boston
University Undergraduate Research Opportunities Program.
We thank Profs. J. Panek and J. Snyder (Boston University)
for helpful comments, and Waters, Inc. for providing HPLC
equipment.
(26) ATPase activity was determined as described in Supporting
Information. The assay measures turnover of ATP, which includes ATPase
activity of the vacuolar ATPase and other potential ATPase activity present
in the membrane preparations. In these assays more than 90% of the activity
was inhibited by lobatamide C or by concanamycin A, specific inhibitors
of the vacuolar ATPase.
(27) Cf. (a) Pinkus, A. G.; Lin, Ellen Y. J. Mol. Struct. 1975, 24, 9. (b)
Decouzon, M.; Ertl, P.; Exner, O.; Gal, J. F.; Maria, P. C. J. Am. Chem.
Soc. 1993, 115, 12071.
Supporting Information Available: Experimental pro-
cedures and characterization data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL026391Z
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