α-Hydroxytropolones: A new class of potent inhibitors of inositol monophosphatase and other bimetallic enzymes

Article abstract of DOI:10.1021/ja9634278

Mono- and polyhydroxytropolones are potent competitive inhibitors of inositol monophosphatase. Modeling studies indicate that this inhibition occurs most probably through a novel mode of action involving the chelation of the two magnesium ions in the active site. This is consistent with experimental data. Inhibition occurs when at least three oxygen atoms are present on the seven-membered ring, and only if they are contiguous to one another. In addition, those oxygens should not be protected. The corresponding six-membered rings showed no activity. Other bimetallic enzymes such as alkaline phosphatase (APase) or dopamine β-monooxygenase (DBM) are also inhibited (in a competitive or uncompetitive manner) by hydroxytropolones.

Full text of DOI:10.1021/ja9634278

VOLUME 119, NUMBER 14
APRIL 9, 1997
©
Copyright 1997 by the
American Chemical Society
R-Hydroxytropolones: A New Class of Potent Inhibitors of
Inositol Monophosphatase and Other Bimetallic Enzymes
†
‡
Serge R. Piettre,* Axel Ganzhorn, Jan Hoflack, Khalid Islam, and
Jean-Marie Hornsperger
Contribution from the Marion Merrell Research Institute (presently Synth e´ labo Biomol e´ culaire),
1
6 rue d’Ankara, F-67080-Strasbourg, France
X
ReceiVed October 1, 1996
Abstract: Mono- and polyhydroxytropolones are potent competitive inhibitors of inositol monophosphatase. Modeling
studies indicate that this inhibition occurs most probably through a novel mode of action involving the chelation of
the two magnesium ions in the active site. This is consistent with experimental data. Inhibition occurs when at
least three oxygen atoms are present on the seven-membered ring, and only if they are contiguous to one another.
In addition, those oxygens should not be protected. The corresponding six-membered rings showed no activity.
Other bimetallic enzymes such as alkaline phosphatase (APase) or dopamine â-monooxygenase (DBM) are also
inhibited (in a competitive or uncompetitive manner) by hydroxytropolones.
Inositol monophosphatase (IMPase, EC 3.1.3.25) hydrolyzes
all myo-inositol monophosphates arising from the second
messenger myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the
phosphoinositide cycle, as well as from the de noVo synthesis
of L-myo-inositol 1-phosphate (L-Ins(1)P) from glucose 6-phos-
phate.1 The human enzyme has been cloned and expressed
and the side effects often associated with lithium treatment have
somewhat detracted from the value of the drug. In addition,
5
other antipsychotic drugs are often required for patients suffering
from acute mania, during the 7-10 days that it takes for lithium
to exert its antimanic effect. Thus several laboratories have
initiated the search for other inhibitors of the enzyme.6 For
example, bisphosphonic acids were described as potent competi-
tive inhibitors; however their bioavailability was low.6 We
herein report the discovery of a new class of potent inhibitors
of IMPase and other bimetallic enzymes, via a novel mode of
action.
2a
2
b-d
and was recently characterized by X-ray crystallography.
a,7
It requires at least two magnesium ions per subunit for
3
activation. The uncompetitive inhibition of IMPase by lithium
ion has led to the hypothesis that the enzyme might be the target
4
of lithium therapy. However the narrow therapeutic window
Puberulonic acid 1a is a natural product that can be isolated
from several Penicillium strains. Structurally, the compound
is characterized by a cycloheptatrienone flanked by three
*
To whom correspondence should be addressed.
Current address: Astra H a¨ ssle AB, S-431 83 M o¨ lndal, Sweden.
Current address: Roussel-Uclaf, 102 route de Noisy, F-93235 Ro-
8
†
‡
mainville Cedex, France.
X
Abstract published in AdVance ACS Abstracts, March 15, 1997.
(3) (a) Greasley, P. J.; Gore M. G. FEBS Lett. 1993, 331, 114-118. (b)
Pollack, J. S.; Atack, J. R.; Knowles, M. R.; McAllister, G.; Ragan, C. I.;
Baker, R.; Fletcher, S. R.; Iversen, L. L.; Broughton, H. B. Proc. Natl.
Acad. Sci. USA 1994, 91, 5766-5770. (c) Cole, A. G.; Gani, D. J. Chem.
Soc., Perkin Trans. 1 1995, 2685-2694. (d) Ganzhorn, A. J.; Lepage, P.;
Pelton, P. D.; Strasser, F.; Vincendon, P., Rondeau, J.-M. Biochemistry 1996,
10957-10966.
(1) Billington, D. C. The inositol Phosphates. Chemical Synthesis and
Biological Significance; VCH Publisher: Wheinheim, 1991; pp 9-21.
2) (a) McAllister, G.; Whiting, P.; Hammond, E. A.; Knowles, M. R.;
Atack, J. R.; Bailey, F. J.; Maigetter, R.; Ragan, C. I. Biochem. J. 1992,
(
2
84, 749-754. (b) Bone, R.; Springer, J. P.; Atack, J. R. Proc. Natl. Acad.
Sci. USA 1992, 89, 1031-1035. (c) Bone, R.; Frank, L.; Springer, J. P.;
Pollack, S. J.; Osborn S.; Atack, J. R. ; Knowles, M. R.; McAllister, G.;
Ragan, C. I.; Broughton, H. B.; Baker, R.; Fletcher, S. R. Biochemistry
(4) (a) Berridge, M. J.; Downes, P. F.; Hanley, M. R. Biochem. J. 1982,
206, 587-595. (b) Nahorski, S. R.; Ragan, C. I.; Challiss, R. A. J. Trends
Pharmacol. Sci. 1991, 12, 297-303.
1
994, 33, 9460-9467. (d) Bone, R.; Frank, L.; Springer, J. P.; Atack, J. R.
Biochemistry 1994, 33, 9468-9476.
(5) Peet, M.; Pratt, J. P. Drugs 1993, 46, 7-17.
S0002-7863(96)03427-0 CCC: $14.00 © 1997 American Chemical Society

3202 J. Am. Chem. Soc., Vol. 119, No. 14, 1997
Piettre et al.
Figure 1. Stereoview of the active site of the inositol monophosphatase complex with Ca and D-inositol 1-phosphate.11 M1, M2, and M3 are the
2+
2
+
2c
positions of the three Ca ions. W2 is the potential nucleophilic water molecule. Glu-70, Asp-90, Asp-93, and Asp-220 are metal ion ligands. In
our model for the binding of puberulonic acid oxygen 2 (superimposed on a phosphate oxygen) chelates both M1 and M2, oxygen 3 (superimposed
on the phosphoester oxygen) chelates M2, and oxygen 1, by displacing water W2, chelates M1, and makes a hydrogen bond with Glu-70. The plane
of the molecule was estimated to be around 70° from the mean plane of the inositol ring of D-Ins(1)P, thereby precluding the third metal ion M3
from positioning itself in the active site.
hydroxyl groups and a carboxylic anhydride function. It was
recently reported as an inhibitor of IMPase with an IC50 value
in the low micromolar range, but of unknown mode of action.
The original assumption was that, as the anhydride function is
most probably hydrolyzed at physiological pH, the biscarboxylic
moiety would chelate the magnesium ions in the active site,
mimicking the binding of a phosphate moiety, and the hydroxyl
groups would play the same role as those in D-myo-inositol
puberulonic acid (1a), both the anhydride and the opened form
(diacid) were considered. Although the seven-membered ring
in tropolone and derivatives is nonaromatic as can be seen from
the different bond lengths in known X-ray structures, its
9
1
3
conformation is completely planar. This largely facilitated
the docking experiments, as only one rigid conformation had
to be considered for puberulonic acid, while the opened form
contained only two restricted rotational bonds linking the ring
with the carboxylic acid groups. Docking was done manually
within the active site of the X-ray structure of IMPase/D-Ins(1)P
after removal of the ligand. No satisfactory results could be
obtained when attempting to obtain direct interactions between
the anhydride function (puberulonic acid) or the diacid (the
opened form), and the metallic centers in the enzyme. Position-
ing of the hydroxyl groups on the seven-membered ring in a
similar way as the cyclitol hydroxyl groups in the enzyme
substrate also led to steric clashes. However, a nearly perfect
superimposition of three of the four seven-membered ring
oxygens of puberulonic acid (1a) with the ester oxygen, the
phosphate oxygen which simultaneously binds to metal ion sites
M1 and M2, and water W2 was obtained (Figures 1 and 3).
This model suggests that neither the anhydride function in
puberulonic acid nor its opened form play an important role in
its recognition by IMPase. Additionally, a mode of action is
suggested which could be general for enzymes containing a
bimetallic center with a similar arrangement as in IMPase.
1
-phosphate (D-Ins(1)P). Consequently, various biscarboxylic
acids were tested as inhibitors of IMPase, but were found to be
inactive at concentrations of 1 mM.10 To shed more light on
the precise mode of action of puberulonic acid, modeling studies
based on the X-ray structure of the human enzyme complexed
2
+
with Ca and D-Ins(1)P were carried out.
Figure 1 shows that D-myo-inositol 1-phosphate binds to the
active site in such a way that the ester oxygen interacts with
metal ion M2 and one of the three phosphate oxygens with both
metal ions M1 and M2. In addition, a third metal ion (M3)
was found.11 Additional hydrogen bonds are present between
the hydroxyl groups in position 2 and 4 on the cyclitol ring of
D-Ins(1)P and, respectively, Ala196/Asp93 and Glu213 of the
enzyme. Hydrolysis of the phosphate ester most probably
occurs through the direct attack of a water molecule (W2) which
is coordinated to the active site metal ion M1 and to Glu70,
and is ideally positioned to be in line with the leaving group
2c
during the transition state.
An alternative mechanism of
hydrolysis involving in-line attack of another water molecule
To verify this model, mono- and polyhydroxytropones were
prepared and tested as inhibitors of IMPase (Figure 2, Table
has also been discussed.3
d,12
For the docking studies with
1
). Hydroxytropolones 1c, 1d, and 1e were prepared according
(
6) (a) Atack, J. R.; Fletcher, S. R. Drugs of the Future 1994, 19, 857-
8
66, and references cited therein (review article). (b) van Steijn, A. M. P.;
to literature procedures, using a bromination-acetolysis-
hydrolysis sequence of reactions. An improved bromination
procedure was worked out for compound 1i (R ) OH, R )
R ) Br, R ) R ) H) and 1j (R ) OH, R ) R ) R ) Br,
Willems, H. A. M.; de Boer, Th.; Geurts, J. L. T.; van Boeckel, C. A. A.
Bioorg. Med. Chem. Lett. 1995, 5, 469-474. (c) Schulz, J.; Wilkie, J.;
Lightfoot, Ph.; Rutherford, T.; Gani, D. J. Chem. Soc., Chem. Commun.
14
1
2
5
3
4
1
2
4
5
1
995, 2353-2356. (d) Schnetz, N.; Gu e´ dat, Ph.; Spiess, B.; Schlewer, G.
Bull. Soc. Chim. Fr. 1996, 133, 205-208.
7) Atack, J. R.; Cook, S. M.; Watt, A. P.; Fletcher, S. R.; Ragan, C. I.
(
(12) Wilkie, J.; Cole, A. G.; Gani, D. J. Chem. Soc., Perkin Trans. 1
1995, 2709-2727.
(13) (a) Rossi, M.; Link, J.; Lee, J. C. Arch. Biochem. Biophys. 1984,
231, 470-478. (b) Gable, R. W.; Mackay, M. F.; Banwell, M. G.; Lambert,
J. N. Acta Crystallogr. C 1990, 46, 1308-1313. (c) Gulbis, J. M.; Mackay,
M. F.; Banwell, M. G.; Lambert, J. N. Acta Crystallogr. C 1992, 48, 332-
338.
J. Neurochem. 1993, 60, 652-658.
(
(
8) Nozoe, T. Fortschr. Chem. Org. Naturst. 1956, 13, 232-301.
9) Islam, K.; Stefanelli, S.; Sponga, F.; Denaro, M. World Pat. 1996,
WO9637197.
(
(
10) Including aliphatic, vinylic, and arylic derivatives; see also 6b.
11) Ganzhorn, A. J.; Rondeau, J.-M. Miami Nature Biotechnology Short
Reports (Proceedings of the 1997 Miami Nature Biotechnology Winter
Symposium, Oxford Press, Oxford, UK) 1997, 8, 61.
(14) Takeshita, H.; Mori, A.; Kusaba, T.; Watanabe, H. Bull. Chem. Soc.
Jpn 1987, 60, 4325-4333.

R-Hydroxytropolones
J. Am. Chem. Soc., Vol. 119, No. 14, 1997 3203
match of the oxygens of the two molecules. Comparison of
the pka1s of 1c (6.7) and 2 (9.3) shows that the tropolone
1
6
derivative is markedly more acidic. The net result at neutral
pH is the deprotonation of the first hydroxyl group in 1c (but
not in 2), leading to an increased chelating potency.17 In this
context it is noteworthy that neither 1g, nor 1h showed inhibition
of the enzyme at 1 mM concentration.
Figure 2.
Other bimetallic enzymes with similar arrangements of active
site metal ions may also be inhibited by tropolones, in particular
zinc and copper enzymes, since these metal ions are known to
1
8
form stable complexes with tropolones in solution. Alkaline
phosphatase contains two catalytic zinc ions that are about 4 Å
1
9
apart, and the table shows that this enzyme is also inhibited
by hydroxytropolones. Again, inhibition by 1d was competitive
with respect to the substrate (data not shown). It is noteworthy
that with IMPase inhibition becomes more efficient with an
increasing number of oxygens, 1d being the most active
compound. In contrast, 1c has the highest affinity for alkaline
phosphatase and inhibitory activity decreases when more
hydroxyl groups are added. This may reflect the difference in
size of the active sites of the two enzymes. IMPase has a large
water-filled cavity with many possibilities for hydrophilic
Figure 3. Chelation of two metal ions by the dianion of 7-hydroxy-
tropolone as it might happen in the active site of IMPase. The distance
(d) between the two metal ions is 3.73 Å as measured from the IMPase/
D-InsP X-ray structure.
Table 1. Inhibition of IMPase, Alkaline Phosphatase (APase), and
DBM by Tropolones
IC50(µM)
IMPase
APase
DBM
2b-d
R1
R2
R3
R4
R5
interactions,
whereas the active site of alkaline phosphatase
(Mg-Mg) (Zn-Zn) (Cu-Cu)
consists of a small surface pocket, just large enough to
accommodate a phosphate moiety.
1
1
1
1
1
1
1
1
a
b
c
d
e
f
OH OH
CO-O-CO OH
10 ( 2
nda
>1000
nd
2
2
1
9
H
OH
H
H
H
H
H
H
H
OH >1000
H
H
OH
OH
OH
OH
75 ( 10
8 ( 1
17 ( 2
15
60
nd
nd
nd
nd
Dopamine â-monoxygenase (DBM) has a requirement for
OH OH
OH OH
3
20a
two copper ions, and its inhibition by unsubstituted tropolone
nd
nd
nd
nd
20b
(1b) has been described.
This effect on DBM was confirmed
H
OH
CO-O-CO OH >1000
and extended to hydroxytropolones 1c and 1d. IC50 values of
2-3 µM were obtained. Inhibition was uncompetitive versus
dopamine, indicating that, in this case, the tropolone may take
the place of the cofactor ascorbate. The inhibitory potency is
independent of the number of hydroxyl groups which may
suggest that the metal chelating properties alone are responsible
for inhibition and tropolones bind to the active-site copper ions
g
h
OMe OMe
OAc OAc
H
H
H
H
OMe >1000
OAc >1000
a
nd: not determined.
3
R ) H) (see Experimental Section). 2,3,7-Trimethoxytropone
(1g) was prepared by reacting 3,7-dihydroxytropolone with
freshly prepared diazomethane.
Tropolone (1b) was found to be inactive at concentrations
of 1 mM while 7-hydroxytropolone (1c) inhibited the enzyme
with an IC50 value of 75 µM. The addition of a third, contiguous
hydroxyl group produced compound 1d characterized by an IC50
value essentially identical to that of puberulonic acid, as is 3,5,7-
trihydroxytropolone 1e. The results thus obtained clearly
demonstrate that the carboxylate functions of puberulonic acid
21
only with no additional interactions to enzymic residues.
A more detailed interpretation of these results will have to
wait until X-ray crystallography data of enzyme-tropolone
complexes become available. However, they show that, in
principle, selectivity toward a given target enzyme may be
achieved through rational inhibitor design.
Conclusion. Tropolones substituted by at least one hydroxyl
function constitute a new class of efficient competitive inhibitors
of IMPase on the necessary condition that the array of oxygen
(
1a) play little role, if any, in the inhibitory properties of the
compound. This is confirmed by the fact that stipitatonic acid
1f) does not inhibit IMPase. It can be concluded that inhibition
(
occurs when at least three oxygen atoms are present on the
seven-membered ring, and only if they are contiguous to one
another. The difference in potency between 1c and 1d probably
results from an interaction of the additional hydroxyl group (OH
in position 7) with the carbonyl group of amino acid residue
Leu42 (distance 2.4 Å, Figure 1).
(16) (a) Abichandani, C. T.; Jaktar, S. K. K. J. Indian Inst. Sci. 1938,
A21, 417-441. (b) Yui, N. Sci. Repts T oˆ hoku UniV. First Ser. 1956, 40,
114-120.
(
17) Experiments with pyrogallol were also carried out at pH ) 9, i.e.
under deprotonation conditions (pyrogallol pK ) 9.3). No inhibition could
a
be measured at 1 mM concentration; technical problems with the enzymatic
assay and the instability of deprotonated pyrogallol, however, complicate
those experiments; a clear answer could not be obtained.
The rate of substrate hydrolysis by IMPase was measured in
a coupled spectrophotometric assay1 by varying the concentra-
tions of inositol-1-phosphate at different fixed concentrations
of 1d. A plot of 1/rate Versus 1/[inositol 1-phosphate] gave a
series of straight lines that intersected in a common point on
the y axis, indicating competitive inhibition of 1d with respect
to the substrate (data not shown). A Ki value of 5 ( 0.5 µM
(
18) Bryant, B. E.; Fernelius, W. C.; Douglas, B. E. J. Am. Chem. Soc.
5a
1953, 75, 3784-3786.
(
19) Kim, E. E.; Wyckoff, W. W. J. Mol. Biol. 1991, 218, 449-464.
(20) (a) Klinman, J. P.; Krueger, M.; Brenner, M.; Edmondson, D. E. J.
Biol. Chem. 1984, 259, 3399-3402. (b) Goldstein, M.; Lauber, E.;
McKereghan, M. R. Biochem. Pharmacol. 1964, 13, 1103-1106.
(21) Given their properties as complexing agents, tropolones may just
reduce the free metal ion concentration in the assay mixture, thereby causing
inhibition in a nonspecific fashion. However, inhibition of IMPase is
observed in the presence of a large excess of Mg (2 mM) and APase and
15b
was determined with the use of the computer program COMP.
2+
This result is consistent with the model depicted in Figure 1,
where the hydroxytropolone unit takes the place of the phosphate
moiety.
Pyrogallol (2) at 1 mM did not inhibit the enzyme despite
the fact that superimposition with 1c indicated an almost perfect
DBM contain tightly bound metal ions that are difficult to remove by
complexing agents.2
2,20b
In addition, the competitive or uncompetitive nature
of inhibition with respect to the substrate argues against depletion of free
metal ions, which is expected to give rise to noncompetitive inhibition.
Finally, the different selectivity profiles, at least with IMPase and APase,
clearly show that inhibition occurs through binding at the enzyme active
site.
(
15) (a) Strasser, F.; Pelton, P. D.; Ganzhorn, A. J. Biochem. J. 1995,
(22) Bosron, W. F.; Anderson, R. A.; Falk, M. C.; Kennedy, F. S.; Vallee,
B. L. Biochemistry 1977, 16, 610-614.
3
07, 585-593. (b) Cleland, W. W. Methods Enzymol. 1979, 63, 103-138.

3
204 J. Am. Chem. Soc., Vol. 119, No. 14, 1997
Piettre et al.
6
OD) 8.26 (s, 2H); (DMSO-d )
mp 122-123 °C);27 1H-NMR δ (CD
atoms branched on the cycle be contiguous. In addition, the
hydroxyl groups of the seven-membered ring must not be
protected as deprotonation occurs under physiological conditions
leading to more efficient chelators. Potentially, hydroxytropo-
lones may also find applications as inhibitors of other bimetallic
enzymes, in particular those that recognize phosphoesters such
as retroviral or bacterial RNase H, DNA polymerases or serine/
3
-
8
.11 (s, 2H); MS (TSP ) 355, 357, 359, 361 (M - H). Recrystallization
from DME gave large yellow crystals as a 1:0.33 mixture of product/
DME: 1H-NMR δ (500 MHz, DMSO-d
) 3.23 (s, 6H), 3.42 (s, 4H),
.14 (s, 2H); 13C-NMR δ (500 MHz, DMSO-d
) 58.0, 71.0, 103.9,
22.7, 140.0, 172.9. Anal. Calcd for C Br ‚0.33DME: C, 25.73;
6
8
1
6
H
7 3
3 2
O
H, 1.64. Found: C, 25.76; H, 1.58.
,3,7-Triacetoxytropone (1g) and 2,3,5,7-tetraacetoxytropone (1k)
were obtained by acetolysis of 1i and 1j, respectively, as a pale yellow
2
23
threonine protein phosphatases.
14
oil (1g) and a creamy solid [1k, mp 131-132 °C (lit. mp 133-134
Experimental Section
14
°
C)], identical in every respect with literature data.
3
,7-Dihydroxytropolone (1d) was obtained as creamy crystals by
Unless otherwise stated, starting materials and solvents were obtained
from commercial sources and used without further purification.
Diazomethane was freshly prepared according to the procedure of de
14
14
hydrolysis of 1g: mp 236-237 °C (lit. mp 237-238 °C). Anal.
Calcd for C : C, 54.55; H, 3.92. Found: C, 54.54; H, 3.89.
7 6 4
H O
Boer and Backer and was used immediately.24 Tetrahydrofuran and
3,5,7-Trihydroxytropolone (1e) was obtained as colorless crystals
by hydrolysis of 1g:14 mp 266-267 °C (lit. mp 263-266 °C). Anal.
Calcd for C
14
diethyl ether were distilled under nitrogen from sodium/benzophenone
immediately prior to use. Drying of the organic extract was carried
7
6 5 2
H O ‚H O: C, 44.68; H, 4.29. Found: C, 44.56; H, 4.20.
out using Na
2
SO
4
. Chromatography was performed using Merck 60
2,3,7-Trimethoxytropone (1h). To a slurry of the 3,7-dihydroxy-
tropolone (462 mg, 3 mmol) in THF (15 mL) at 0 °C was added slowly
a freshly prepared solution of diazomethane (CAUTION) (56 mL of
(
230-400 ASTM) silica gel according to the procedure published by
25
24
Still. Melting points were determined on a B u¨ chi 535 apparatus and
1
are uncorrected. Unless otherwise stated, H-NMR spectra were
a 0.4 M solution in diethyl ether, 18.09 mmol), and stirring was
continued overnight at room temperature. Acetic acid (2 mL) was
added slowly to the stirring mixture cooled at 0 °C. Evaporation of
the volatiles, chromatography of the residue, and elution (ethyl acetate)
delivered the desired compound as a pale yellow oil (288 mg, 49%
yield):28 H-NMR δ (500 MHz) 3.84 (s, 3H), 3.89 (s, 3H), 3.94 (s,
3H), 6.61 (d, 1H, J ) 9.3), 6.84 (d, 1H, J ) 10.9), 6.93 (dd, 1H, J )
9.3, 10.9); 13C-NMR δ (125 MHz) 56.4, 57.9, 59.4, 109.6, 118.3, 127.9,
recorded in deuterated chloroform at 200 MHz and proton-decoupled
13
C-NMR spectra were recorded at 50 MHz; chemical shifts are
expressed in ppm downfield from internal or external tetramethylsilane
and deuterated chloroform, respectively; coupling constants (J) are
expressed in Hertz. Low-resolution mass spectra were recorded using
the positive or negative ions thermospray method.
1
3
-Hydroxytropolone (1c). Prepared in three steps from tropolone
1
4
+
+
by the method of Takeshita. A colorless crystalline material was
obtained whose analytical data were in accordance with those pub-
153.1, 160.0, 164.8, 173.7; MS (TSP ) 197 (MH ). Anal. Calcd for
1
C
10
H
12
4
O ‚ /
2 2
H O: C, 58.53; H, 6.38. Found: C, 58.61; H, 6.05.
1
4
lished: mp 89-90 °C (lit. mp 90-91 °C). Anal. Calcd for
: C, 60.87; H, 4.38. Found: C, 60.75; H, 4.25.
,7-Dibromotropolone (1i). Tropolone (1.22 g, 10 mmol) was
dissolved in a 1:1 mixture of MeOH/CH Cl (200 mL). Calcium
Enzyme Assays. Recombinant human IMPase was produced in
3d
C
7
H
6
O
3
Escherichia coli and purified to homogeneity as previously described.
3
IC50 values were determined at 37 °C and pH ) 7.5 with 0.2 mM (≈2
× K DL-Ins(1)P using a colorimetric assay, which detects the
formation of a phosphomolybdate complex in the presence of malachite
2
2
m
)
carbonate (2.052 g, 20.5 mmol, 2.05 equiv) was added while stirring,
followed by benzyltrimethylammonium tribromide (7.8 g, 20 mmol,
green,3d or a radiochemical assay measuring the release of [ H]inositol.
3
30
2
.0 equiv). The solution was stirred at room temperature for 24 h and
Similar results were obtained with both assay systems. Alkaline
filtered, and the filtrate was evaporated. The residue was taken up in
water (180 mL) and heated at 65 °C for 3 h while stirring to dissolve
the ammonium salts. Filtration and washing with hot (65 °C) water
yielded a crystalline, yellow material. Absence of ammonium salts
should be checked at this point, and the last step can be repeated if
needed. The solid (2.8 g), when free from ammonium salt, is then
dissolved in ethylene glycol dimethyl ether (DME) (14 mL) and stirred
overnight at room temperature. Filtration and drying under reduced
pressure gave the compound as a yellow, crystalline solid (2.46 g (64%);
phosphatase from calf intestine was assayed at 37 °C and pH 8.0 in
0.5 M Tris-Cl, with 50 µM (≈5 × K
m
) 4-nitrophenyl phosphate.
Release of 4-nitrophenolate was measured at 405 nm. Dopamine
â-monooxygenase purified from beef adrenals was assayed as previ-
ously described.31
Modeling Experiments. Modeling was done using SYBYL 5.32.32
No energy optimization was performed since neither the conformation
of tropolones nor interactions with metal ions are well handled by
standard force field. This is not a problem in this case as the docked
compounds are conformationally rigid and metal binding distances could
1
a 1:1 mixture with DME by H-NMR spectroscopy): mp 157-158 °C
2
6
1
3
(
7
lit. mp 157-158 °C); H-NMR δ (CD
3
OD) 6.18 (t, 1H, J ) 10.6),
11
be taken from the known IMPase complexes X-ray structures.
3
.97 ppm (d, 2H, J ) 10.6). This compound was used as such in the
14
acetolysis reaction.
Acknowledgment. This is dedicated to Clayton H. Heath-
cock on the occasion of his 60th birthday.
3
,5,7-Tribromotropolone (1j). The hereabove procedure was
followed with 3 equiv of benzyltrimethylammonium tribromide (11.7
g, 30 mmol) to give the title compound in 98% yield as a yellow,
JA9634278
1
26
crystalline powder, pure by H-NMR spectroscopy: mp >320 °C (lit.
(
27) All the analytical data obtained from our compound were in
(
23) (a) Starnes, M. C.; Cheng, Y.-c. J. Biol. Chem. 1989, 264, 7073-
accordance with the structure of 3,5,7-tribromotropolone (1j); furthermore,
it was successfully transformed into 3,5,7-trihydroxytropolone (1e). Hence
we do not explain the discrepancy in the melting points.
7
077. (b) Davies, J. F.; Hostomska, Z.; Hostomsky, Z.; Jordan, S. R.;
Matthews, D. A. Science, Washington D.C. 1991, 252, 88-95. (c)
Derbyshire, V.; Freemont, P. S.; Sanderson, M. R.; Beese, L.; Friedman, J.
M.; Joyce, C. M.; Steitz, T. A. Science, Washington D.C. 1988, 240, 199-
(28) Isomeric 2,3,4-trimethoxytropone was also isolated in 40% yield.
1
13
Assignment of the correct structure was achieved by using H and C NMR
in 1-D and 2-D modes at 500 MHz and 125 MHz. Heteronuclear multiple
quantum correlation (HMQC) and Heteronuclear multiple bond correlation
2
01. (d) Freemont, P. S.; Friedman, J. M.; Beese, L.; Sanderson, M. R.;
Steitz, T. A. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 8924-8928. (e)
Goldberg, J.; Huang, H.; Kwon, Y.; Greengard, P.; Nairn, A. C.; Kuriyan,
J. Nature 1995, 376, 745-753. (f) Kissinger, C. R.; Parge, H. E.; Knighton,
D. R.; Lewis, C. T.; Pelletier, L. A.; Tempczyk, A.; Kalish, V. J.; Tucker,
K. D.; Showalter, R. E.; Moomaw, E. W.; Gastinel, L. N.; Habuka, N.;
Chen, X.; Maldonado, F.; Barker, J. E.; Bacquet, R.; Villafranca, J. E. Nature
13
1
(HMBC) were run to establish direct and long-range C- H connectivi-
ties.29
(29) (a) Davis, A. L.; Keeler, J.; Lane, E.; Moskau, D. J. Magn. Reson.
1992, 98, 207-216. (b) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986,
108, 2093-2094.
1
1
2
995, 378, 641-644.
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963; Collect. Vol. IV, pp 250-253.
25) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923-
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26) Cook, J. W.; Gibb, A. R.; Rapha e¨ l, R. A.; Somerville, A. R. J. Chem.
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(30) Ragan, C. I.; Watling, K. J.; Gee, N. S.; Aspley, S.; Jackson, R. G.;
Reid, G. G.; Baker, R.; Billington, B. C.; Barnaby, R. J.; Leeson, P. D.
Biochem. J. 1988, 249, 143-148.
(
(
(31) Bargar, T. M.; Broersma, R. J.; Creemer, L. C.; McCarthy, J. R.;
Hornsperger, J. M.; Palfreyman, M. G.; Wagner, J.; Jung, M. J. J. Med.
Chem. 1986, 29, 315-317.
(
(32) Tripos Ass., 6548 Clayton Road, St Louis, MO 62177.