Synthesis and biological evaluation of a water-soluble derivative of the potent V-ATPase inhibitor archazolid

Article abstract of DOI:10.1016/j.bmcl.2012.09.081

The water-solubility of the highly potent V-ATPase inhibitors archazolid A and the glucosylated derivative archazolid C was studied in the presence of a wide range of cosolvents, revealing very low solubilites. The first water-soluble analogue was then designed, synthesized, and evaluated for V-ATPase inhibitory activity in vitro.

Full text of DOI:10.1016/j.bmcl.2012.09.081

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7735–7738
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a water-soluble derivative
of the potent V-ATPase inhibitor archazolid
Elke Persch a, Teodora Basile b, Svenja Bockelmann c, Markus Huss c, Helmut Wieczorek c,
Teresa Carlomagno b, Dirk Menche a,d,
⇑
a Institut für Organische Chemie, Ruprecht-Karls-Universität Heidelberg, INF 270, 69120 Heidelberg, Germany
b EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
c Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Tierphysiologie, 49069 Osnabrück, Germany
d Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The water-solubility of the highly potent V-ATPase inhibitors archazolid A and the glucosylated deriva-
tive archazolid C was studied in the presence of a wide range of cosolvents, revealing very low solubilites.
The first water-soluble analogue was then designed, synthesized, and evaluated for V-ATPase inhibitory
activity in vitro.
Received 27 August 2012
Revised 19 September 2012
Accepted 22 September 2012
Available online 13 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Archazolid
Derivative
V-ATPase
Water-solubility
Structure–activity data
Antibiotics
In 1993 the group of Höfle reported the archazolids A and B (1,
2, Fig. 1)1 as the first examples of a structurally novel family2 of
secondary metabolites from the myxobacterium Archangium ge-
phyra.3–5 Biological evaluation of the complex 24-membered mac-
rolides revealed potent growth-inhibition of a number of murine
and human cancer cell lines at subnanomolar concentrations. This
was later attributed to the selective inhibition of vacuolar-type
ATPases (V-ATPases), both in vitro6 and in vivo.1 V-ATPases are
implicated in several diseases, such as renal tubular acidosis,7 oste-
opetrosis,8 and cancer,9 and therefore represent an attractive target
for inhibitor development from the perspective of medicinal chem-
istry. Very recently, it has been shown that archazolid abrogates
tumor metastasis via inhibition of endocytic activation of the
Rho-GTPase Rac1.10 The potency of the archazolid family as V-ATP-
ase inhibitors was revealed due to the similarity of their inhibitory
efficacy on a wide range of mammalian cell lines to the well known
plecomacrolidic type V-ATPase inhibitors concanamycin and
bafilomycin, and their prevention of the lysosomal acidification
in cells.1,6 An enzymatic assay with purified V-ATPase extracted
from the midgut of the tobacco hornworm confirmed these
observations.6
A competition assay showed that the binding site of the
archazolids is located at the VO proteolipid subunit c and shares
at least some parts with the binding site of the plecomacrolides.6
Cross-linking studies with the radioactive photoactivatable
14C-diazirinyl derivative 4 confirmed these findings (Fig. 1).11
Further investigations employing site directed mutagenesis
provided a more precise picture of the binding site and suggest
that the archazolids do not bind directly between two adjacent c
subunits, as concanamycin and bafilomycin do,12 but mainly
within one c subunit.11
In order to further develop the potential of archazolids as drug
candidates and to gain a better understanding of the binding mode
by more accurate molecular modeling of the archazolids in com-
plex with the V-ATPase,13 it is crucial to know the bioactive confor-
mation of the bound macrolides under physiological conditions.
Such analysis requires
a water-soluble derivative of these
inhibitors.14
Herein, we report the solubility of archazolids in water in differ-
ent solvent mixtures, the synthesis of the first water-soluble arc-
hazolid derivative (5) and the V-ATPase inhibitory effect of this
compound.
First, solvent mixtures that allow a concentration of archazolid
in the range of 0.1–1 mM for NMR studies were examined (Table 1).
The solutions were made up of either an organic solvent or a deter-
gent with a concentration equal or larger than the critical micelle
⇑
Corresponding author. Tel.: +49 228 732653; fax: +49 228 735813.
E-mail address: dirk.menche@uni-bonn.de (D. Menche).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.081

7736
E. Persch et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7735–7738
O
O
P
O
O
O
P
O
O
c
a, b
HO
MeO
HO
OH MeO
Br
O
O
O
O
OH
OR
7
6
7
8
2
O
O
R
O
P
O
O
O
O
O
O
O
O
N
N
P
2,
R =
H
N
H
N
O
d
O
S
S
O
O
HO
HO
15
O
O
O
MeO
O
HO
7
OH
1: Archazolid A (R = Me)
2: Archazolid B (R = H)
3: Archazolid C
e
O
RO
RO
O
15
5
15
N
MeO
OR
MeO
OR
H
N
7
7
O
S
10
O
O
O
Scheme 1. Synthesis of phosphonate
8 and of the water-soluble archazolid
derivative 5. Reagents and conditions: (a) NaI, acetone, 25 °C, 2.5 h, 85%; (b)
P(OAllyl)3, 110 °C, 3 h, 85%; (c) HCOOH, 25 °C, 24 h, 94%; (d) 8, 2,4,6-trichloroben-
zoyl chloride, NEt3, toluene, 25 °C, 1.5 h, 4-dimethylaminopyridine 0 °C, 45 min,
75%; (e) NaI, acetone, 56 °C, 3 h, 98%.
O
O
N
N
R =
R =
O
H
N
H
N
O
S
O
S
14C
O
O
P
O
O
O
CF3
OH
interactions under these conditions of archazolid A (1) with the
V-ATPase has been investigated. This choice was motivated by a
literature report, which illustrates that the subunit c of the F1FO-
ATPase adopts its native conformation in this solvent mixture
N
N
4: 14
5: Phosphonate-Derivative
C-Diazirinyl-Derivate
of Archazolid A
of Archazolid B
Figure 1. Archazolid A (1), B (2) and C (3) of the archazolid family and their 14C-
diazirinyl (4) and phosphonate derivatives (5).
(+50 m
M
NaCl).15 V-ATPases are structurally and functionally re-
lated to the F-ATP synthase: the subunit c of the V-ATPase shows
particularly high sequence homology and is therefore likely to be-
have natively as well.
Table 1
After characterizing archazolid A (1) and C (3) in this solvent
mixture, several samples of the subunit c of the V-ATPase were ex-
tracted with CHCl3/MeOH (2:1) according to a previously reported
procedure,16 and tested for interactions with archazolids. However,
no interaction could be detected and NMR analysis revealed that
the subunit c remained unfolded in the solvent mixture CDCl3/
MeOH-d4/D2O (4:4:1). Thus, structural studies of the inhibitor with
a functional V-ATPase could not be performed, due to the insolubil-
ity of the archazolids in an appropriate solvent mixture.
We therefore turned our attention to the development of a
water-soluble archazolid derivative. Inspired by various phosphate
derivatives synthesized for enhanced water-solubility and featur-
ing a prodrug strategy,17 we initiated a synthetic program towards
the development of a phosphate derivative of archazolid.
Solubility studies of archazolid
solvents or detergents.
A (1) in water with different additives, organic
Solvent mixtures/
detergents
[Archazolid A (1)]
in m
Solvent ratio/
Soluble
M
[detergent] in mM
DMSO-d6/D2O
DMSO-d6/D2O
CNCD3-d6/D2O
EtOH-d6/D2O
MeOH-d4/D2O
EtOH-d6/D2O
EtOH-d6/CNCD3/D2O
CDCl3/MeOH-d4/D2O
LDAO
0.5
1
1
10:90
50:50
5:95
5:95
5:95 and 10:90
5:95
42:9:49
40:40:1
4
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
1
0.5
0.5
<0.5
1
1
DHPC
1
17
DPC
LMPC
DM
OG
0.5
0.5
0.5
0.5
3a,b
2a,b
Derivatization of the archazolid scaffold can be easily achieved
by modifying the hydroxyl group in positions 7 and 15 as was re-
ported before.3a,5 It is also known that derivatization of 7-OH led to
only small changes in the 3D structure; the influence on the con-
formation by modification of the 15-OH substituent and preserva-
tion of the tetrahedral hybridization at the same time has not yet
been studied. The inhibitory efficiency on the purified V-ATPase
of O-substituted archazolids is distinctly reduced in comparison
to their parent structures, but still in the low micromolar range,
0.4a,b
50a,b
a
20 mm pH 8 NaPi, 20 mm NaCl.
Double the CMC.
b
concentration (CMC) as additives in water. Except for one solvent
mixture, none of the additives could induce sufficient solubility
of the macrolides. We obtained similar results for archazolid C
(3): the highest concentration could be reached in a solution of
detergents and water (40
cient for structural studies.
as illustrated by the IC50 value of 0.6
l
M
of the 14C-diazirinyl
derivative 4. Therefore, it was anticipated that the introduction
of a polar substituent in 7-O and 15-O position should increase
the water-solubility and still conserve sufficient biological activity.
The first attempt to obtain a water-soluble derivative of
archazolid B (2) was focused on the synthesis of a phosphate
lM in 20 mM; pH 8) and was not suffi-
As CDCl3/MeOH-d4/D2O (4:4:1) was found to be the only sol-
vent mixture that allowed for good solubility of archazolids, the

E. Persch et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7735–7738
7737
Figure 2. 1H NMR of the water-soluble archazolid derivative 5 in D2O with a concentration of 4.1 mm.
As shown in Figure 2, the mono deprotected phosphonate
groups in position 7 and 15 introduced sufficient polarity to gain
water-solubility as shown by a 1H NMR spectra in D2O. The con-
100
80
60
40
20
0
centration used amounted to 4.1 m
M and is high enough to allow
NMR experiments for structural data analysis provided that this
compound retains biological potency.
For biological evaluation of the water-soluble derivative 5, the
inhibitory efficacy on purified V-ATPase holoenzyme from the mid-
gut of the tobacco hornworm was evaluated. For this purpose,
V-ATPase was purified according to published procedures21 and a
standard V-ATPase assay was employed with or without the
water-soluble derivative in direct comparison with known inhibi-
tors 1–4 at various concentrations.6 The conentration of inorganic
phosphate, produced in the V-ATPase assay, was measured accord-
ing to the protocol of Wieczorek et al.22 However, no inhibitory ef-
fect of the water-soluble derivative 5 was observed, even at very
high concentrations (Fig. 3).
Archazolid A (1)
Archazolid B (2)
Archazolid C (3)
Diazirinyl archazolid A (4)
Phosphonate archazolid B (5)
0.1
1
10
100
1000
10000
Inhibitor concentration [nM]
A reason for this complete lack of activity is as yet unknown.
However, the retained biological potency of the diazirinyl com-
pound 4, which bears the substituents in the same positions as
the phosphonate derivative 5, suggest that the water-soluble ana-
logue 5 may be inactive for pharmacokinetic reasons, possibly be-
cause it may not reach the V-ATPase due to its polarity or
hydrophilicity. This explanation suggests that the introduction of
a spacer between the archazolids and a polar group might be ben-
eficial in designing a promising water-soluble derivative.
In conclusion, we have revealed very low degrees of water-
solubilities of the potent V-ATPase inhibitors archazolid A (1)
and the glucosylated derivative archazolid C (3), even in the
presence of various cosolvents. Based on these findings, a first
water-soluble derivative was designed, synthesized in an efficient
manner by derivatization of the parent natural compound, and
evaluated in vitro for its V-ATPase inhibitory effects. The lack of
biological activity of this derivative will be helpful in devising
new types of soluble archazolid analogues, for example, by
installment of a spacer between the polar group and the parent
natural products.
Figure 3. Inhibition of the V-ATPase activity by archazolid A (1), B (2) and C (3), the
non radioactive version of the diazirinyl archazolid (4) and the phosphonate
derivative (5). Values are the average of three independent V-ATPase preparations.
Error bars are standard deviations.
derivative via the formation of a phosphine with subsequent oxi-
dation. However, the commonly used tetrazole activated phospho-
ramidite (AllylO)2PN(i-Pr)2 procedure,18 and the more reactive
diethyl chlorophosphite in combination with Hünig’s base, did
not lead to any conversion and therefore, the application of penta-
valent phosphorous reagents was investigated. Besides diphenyl
phosphoryl chloride and the diphosphate tetrabenzyl pyrophos-
phate, phosphorus(V) oxychloride was utilized as reagent, but
either no conversion or decomposition was observed. Finally, the
introduction of polar elements into archazolid B (2) was achieved
by phosphonate esters that can be linked to the natural product
by esterification as previously reported.3a,5
Phosphonate 8 was synthesized from the commercially avail-
able tert-butyl bromoacetate (6) and converted, in a Finkelstein
reaction, to tert-butyl iodoacetate in good yield. Arbuzov reaction
with triallylphosphite provided 7 and subsequent saponification
gave 8 (Scheme 1).
Acknowledgments
Esterification of archazolid B (2) with 8 under Yamaguchi con-
ditions resulted in bis-O-substituted archazolid B 10 which could
be mono deprotected by sodium iodide in acetone to yield 5 almost
quantitatively.19,20
Financial support from the Volkswagenstiftung (Funding Initia-
tive: ‘Interplay between Molecular Conformation and Biological
Function’) is gratefully acknowledged. We further thank Dr. Adam
Richard Lacy for proofreading the manuscript,

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E. Persch et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7735–7738
13. Dreisigacker, S.; Latek, D.; Bockelmann, S.; Huss, M.; Wieczorek, H.; Filipek, S.;
Supplementary data
Gohlke, H.; Menche, D.; Carlomagno, T. J. Chem. Inf. Model. 2012, 52, 2265.
14. Ni, F. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 517.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
09.081.
15. Girvin, M. E.; Rastogi, V. K.; Abildgaard, F.; Markley, J. L.; Fillingame, R. H.
Biochemistry 1998, 37, 8817.
16. Arai, H.; Berne, M.; Forgac, M. J. Biol. Chem. 1987, 262, 11006.
17. Stella, V. J.; Nti-Addae, K. W. Adv. Drug Delivery. Rev. 2007, 59, 677.
18. (a) Shibahara, S.; Fujino, M.; Tashiro, Y.; Okamoto, N.; Esumi, T.; Takahashi, K.;
Ishihara, J.; Hatakeyama, S. Synthesis 2009, 2009, 2935; (b) Shimada, K.;
Kaburagi, Y.; Fukuyama, T. J. Am. Chem. Soc. 2003, 125, 4048.
References and notes
19. All new compounds had spectroscopic data in full support of the assigned
structures. 1H NMR spectroscopic data: Compound 5: 1H NMR (600.130 MHz,
CDCl3): d = 0.80 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 7.0 Hz,
3H), 0.99 (d, J = 6.9 Hz, 3H), 1.05 (d, J = 6.9 Hz, 3H), 1.58 (s, 3H), 1.71–1.80 (m,
1H), 1.71–1.76 (m, 1H), 1.73 (s, 3H), 1.73–1.73 (m, 1H), 1.76–1.81 (m, 1H), 1.77
(s, 3H), 1.85–1.91 (m, 1H), 1.88 (s, 3H), 2.45 (ddq, J = 9.5, 9.3, 7.0 Hz, 1H), 2.63–
2.79 (m, 1H), 2.68–2.84 (m, 4H), 2.70 (s, 3H), 2.79–2.87 (m, 1H), 2.95–3.04 (m,
1H), 2.99–3.05 (m, 1H), 3.08 (s, 3H), 3.22 (d, J = 9.6 Hz, 1H), 4.38 (dd, J = 6.4,
5.7 Hz, 2H), 4.43 (dd, J = 6.6, 5.7 Hz, 2H), 5.07–5.14 (m, 2H), 5.10 (d, J = 10.0 Hz,
1H), 5.16 (d, J = 9.6 Hz, 1H), 5.23 (t, J = 9.6 Hz, 1H), 5.31 (dd, J = 13.1, 1.5 Hz,
1H), 5.34 (dd, J = 13.2, 1.6 Hz, 1H), 5.50 (d, J = 7.8 Hz, 1H), 5.51 (dd, J = 15.0,
8.5 Hz, 1H), 5.77 (dd, J = 16.0, 8.2 Hz, 1H), 5.82 (s, 1H), 5.85 (d, J = 10.1 Hz, 1H),
5.89 (d, J = 15.3 Hz, 1H), 5.92 (d, J = 5.5 Hz, 1H), 5.93–6.05 (m, 4H), 6.40 (d,
J = 16.0 Hz, 1H), 6.95 (ddd, J = 15.3, 7.4, 6.8 Hz, 1H), 7.26 (s, 1H)); 13C NMR
(150.90 MHz, CDCl3): d = 12.4, 14.6, 17.0, 17.3, 18.2, 18.5, 20.1, 22.5, 23.5, 24.7,
26.0, 27.7, 36.3, 37.0, 39.8, 42.6, 44.3, 45.4, 46.2, 66.9, 66.9, 73.4, 76.4, 77.2,
78.8, 88.0, 116.1, 116.3, 117.7, 123.1, 126.4, 126.5, 127.1, 128.2, 128.3, 128.8,
130.3, 131.2, 131.3, 131.5, 131.9, 132.1, 132.6, 133.7, 135.1, 135.6, 136.5, 136.6,
137.0, 139.1, 149.4, 155.9, 158.4, 167.0, 169.9, 174.2; HR-MS (ESI): found m/
z = 1047.4207 (C51H73N2O15P2S), calculated m/z = 1047.4207.
1. (a) Höfle, G.; Reichenbach, H.; Sasse, F.; Steinmetz, H. German Patent DE 41 42
951 C1, 1993.; (b) Sasse, F.; Steinmetz, H.; Höfle, G.; Reichenbach, H. J. Antibiot.
2003, 56, 520.
2. (a) Menche, D.; Hassfeld, J.; Steinmetz, H.; Huss, M.; Wieczorek, H.; Sasse, F.
Eur. J. Org. Chem. 2007, 1196; (b) Menche, D.; Hassfeld, J.; Steinmetz, H.; Huss,
M.; Wieczorek, H.; Sasse, F. J. Antibiot. 2007, 60, 328; (c) Horstmann, N.; Essig,
S.; Bockelmann, S.; Wieczorek, H.; Huss, M.; Sasse, F.; Menche, D. J. Nat. Prod.
2011, 74, 1100.
3. For the stereochemical determination of the archazolids, see: (a) Hassfeld, J.;
Farès, C.; Steinmetz, H.; Carlomagno, T.; Menche, D. Org. Lett. 2006, 8, 4751; (b)
Farès, C.; Hassfeld, J.; Menche, D.; Carlomagno, T. Angew. Chem., Int. Ed. 2008,
47, 3722.
4. For total syntheses of the archazolids, see: (a) Menche, D.; Hassfeld, J.; Li, J.;
Rudolph, S. J. Am. Chem. Soc. 2007, 129, 6100; (b) Roethle, P. A.; Chen, I. T.;
Trauner, D. J. Am. Chem. Soc. 2007, 129, 8960; (c) Menche, D.; Hassfeld, J.; Li, J.;
Mayer, K.; Rudolph, S. J. Org. Chem. 2009, 74, 7220.
5. For first SAR studies, see: Menche, D.; Hassfeld, J.; Sasse, F.; Huss, M.;
Wieczorek, H. Bioorg. Med. Chem. Lett. 2007, 17, 1732.
6. Huss, M.; Sasse, F.; Kunze, B.; Jansen, R.; Steinmetz, H.; Ingenhorst, G.; Zeeck,
A.; Wieczorek, H. BMC Biochem. 2005, 6, 13.
7. Wagner, C. A.; Finberg, K. E.; Breton, S.; Marshansky, V.; Brown, D.; Geibel, J. P.
Physiol. Rev. 2004, 84, 1263.
8. Tolar, J.; Teitelbaum, S. L.; Orchard, P. J. N. Engl. J. Med. 2004, 351, 2839.
9. Sennoune, S. R.; Bakunts, K.; Martinez, G. M.; Chua-Tuan, J. L.; Kebir, Y.; Attaya,
M. N.; Martinez-Zaguilan, R. Am. J. Physiol. Cell Physiol. 2004, 286, C1443.
10. Wiedmann, R. M.; von Schwarzenberg, K.; Palamidessi, A.; Schreiner, L.;
Kubisch, R.; Liebl, J.; Schempp, C.; Trauner, D.; Vereb, G.; Zahler, S.; Wagner, E.;
Müller, R.; Scita, S.; Vollmar, A. M. Cancer Res. 2010. http://dx.doi.org/10.1158/
0008-5472.CAN-12-1772.
11. Bockelmann, S.; Menche, D.; Rudolph, S.; Bender, T.; Grond, S.; von Zezschwitz,
P.; Muench, S. P.; Wieczorek, H.; Huss, M. J. Biol. Chem. 2010, 285, 38304.
12. (a) Bowman, E.; Bowman, B. J. Bioenerg. Biomembr. 2005, 37, 431; (b) Bowman,
B. J.; Bowman, E. J. J. Biol. Chem. 2002, 277, 3965; (c) Bowman, E. J.; Graham, L.
A.; Stevens, T. H.; Bowman, B. J. J. Biol. Chem. 2004, 279, 33131.
20. The close similarity of the coupling constants of 5 with the parent archazolid
suggest that
5 resides in a very similar solution conformation. Small
differences are observed for the coupling constants between H14 and H15 as
well as between H20 and H21, as expected for these flexible regions of these
natural products. For a tabulated comparison of the coupling constant data, see
Supplementary data.
21. Huss, M.; Ingenhorst, G.; König, S.; Gassel, M.; Dröse, S.; Zeeck, A.; Altendorf, K.;
Wieczorek, H. J. Biol. Chem. 2002, 277, 40544.
22. Wieczorek, H.; Cioffi, M.; Klein, U.; Harvey, W. R.; Schweikl, H.; Wolfersberger,
M. G. Methods Enzymol. 1990, 192, 608.