A Novel Class of Zinc-Binding Inhibitors for the Phosphatidylcholine-Preferring Phospholipase C from Bacillus cereus

Article abstract of DOI:10.1021/jo9915731

The phospholipase C (PLC) isozymes catalyze the hydrolysis of phospholipids to provide diacylglycerol (DAG) and a phosphorylated headgroup. Because DAG has been implicated in cellular signal transduction cascades in mammalian systems, there has been considerable interest in the development of inhibitors of these enzymes. Toward this end, we have discovered that the cyclic N,N′-dihydroxyureas 6-10 inhibit the phosphatidylcholine preferring PLC from Bacillus cereus (PLC_Bc). This class of inhibitors is believed to function by the bidentate chelation of the N,N′-dihydroxyurea array to one or more of the zinc ions at the active site of the enzyme. Because the affinities of these compounds correlate with the pK_as of the N-OH hydroxyl groups, it is apparent that one or both of the hydroxyl groups must be ionized for effective coordination to the zinc ions. It is also apparent that there may be rather strict steric requirements for these inhibitors.

Full text of DOI:10.1021/jo9915731

J . Org. Chem. 2000, 65, 4509-4514
4509
A Novel Cla ss of Zin c-Bin d in g In h ibitor s for th e
P h osp h a tid ylch olin e-P r efer r in g P h osp h olip a se C fr om Ba cillu s
cer eu s
Stephen F. Martin,* Bruce C. Follows, Paul J . Hergenrother, and Christopher L. Franklin
Department of Chemistry and Biochemistry, The University of Texas, Austin, Texas 78712
Received October 8, 1999
The phospholipase C (PLC) isozymes catalyze the hydrolysis of phospholipids to provide diacyl-
glycerol (DAG) and a phosphorylated headgroup. Because DAG has been implicated in cellular
signal transduction cascades in mammalian systems, there has been considerable interest in the
development of inhibitors of these enzymes. Toward this end, we have discovered that the cyclic
N,N′-dihydroxyureas 6-10 inhibit the phosphatidylcholine preferring PLC from Bacillus cereus
(PLCBc). This class of inhibitors is believed to function by the bidentate chelation of the N,N′-
dihydroxyurea array to one or more of the zinc ions at the active site of the enzyme. Because the
affinities of these compounds correlate with the pKas of the N-OH hydroxyl groups, it is apparent
that one or both of the hydroxyl groups must be ionized for effective coordination to the zinc ions.
It is also apparent that there may be rather strict steric requirements for these inhibitors.
In tr od u ction
which either the bridging or the nonbridging oxygens
have been replaced.7 For example, modest inhibitors of
PLCBc activity have been identified wherein the phos-
phate oxygen of the scissile P-O bond was replaced with
CH2, CF2, or NR groups to provide nonhydrolyzable
phosphonate or phosphoramidate analogues as generally
shown in 1. Replacement of the nonbridging phosphate
oxygens with sulfur also leads to inhibitors. Although a
number of several monovalent anions and Tris have been
found to be weak inhibitors of PLCBc,8 the only potent
inhibitor of PLCBc that is not a substrate analogue is the
xanthate derivative D609 (2).9,10
Phospholipase C isozymes catalyze the hydrolysis of
phospholipids to provide diacylglycerol (DAG) and a
phosphorylated headgroup (Scheme 1). The products from
the action of mammalian PLCs have been implicated in
the cellular signal transduction cascade.1 In particular,
DAG and inositol triphosphate are important for their
respective activation of protein kinase C and release of
intracellular calcium.2 The phosphatidylcholine-prefer-
ring phospholipase C from Bacillus cereus (PLCBc) is a
bacterial enzyme with a known antigenic similarity to
its mammalian counterpart.3 This similarity has made
studies of the PLCBc mechanism of action4-6 and its
selective inhibition7 important biological pursuits.
Sch em e 1
To design novel, nonsubstrate analogues as inhibitors
of PLCBc, it is helpful to examine the X-ray crystal
structures of PLCBc in its native state11 as well as in its
complexes with various ions12,13 and a phosphonate
inhibitor.14 For example, the structure of the PLCBc-
inhibitor complex reveals PLCBc to be a monomeric,
highly R-helical enzyme that contains three zinc ions in
its active site (Figure 1). The nonbridging phosphate
In the context of developing inhibitors of PLCBc, it is
noteworthy that most have been substrate analogues in
(8) Aakre, S. E.; Little, C. Biochem. J . 1982, 203, 799-801.
(9) Muller-Decker, K. Biochem. Biophys. Res. Commun. 1989, 162,
198-205.
(1) Exton, J . H. Eur. J . Biochem. 1997, 243, 10-20.
(2) Bell, R. Cell 1986, 45, 631-632.
(3) Clark, M. A.; Shorr, R. G. L.; Bomalaski, J . S. Biochem. Biophys.
Res. Comm. 1986, 140, 114-119.
(10) Amtmann, E. Drugs Exp. Clin. Res. 1996, 22, 287-294.
(11) Hough, E.; Hansen, L. K.; Birkness, B.; J ynge, K.; Hansen, S.;
Hordvik, A.; Little, C.; Dodson, E.; Derewenda, Z. Nature 1989, 338,
357-360.
(4) Martin, S. F.; Spaller, M. R.; Hergenrother, P. J . Biochemistry
1996, 35, 12970-12977.
(5) Martin, S. F.; Hergenrother, P. J . Biochemistry 1998, 37, 5755-
5760.
(6) Martin, S. F.; Hergenrother, P. J . Biochemistry 1999, 38, 4403-
4408.
(12) Hansen, S.; Hansen, L. K.; Hough, E. J . Mol. Biol. 1992, 225,
543-549.
(13) Hansen, S.; Hansen, L. K.; Hough, E. J . Mol. Biol. 1993, 231,
870-876.
(7) Martin, S. F.; Wong, Y.-L.; Wagman, A. S. J . Org. Chem. 1994,
59, 4821-4831.
(14) Hansen, S.; Hough, E.; Svensson, L. A.; Wong, Y.-L.; Martin,
S. F. J . Mol. Biol. 1993, 234, 179-187.
10.1021/jo9915731 CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/24/2000

4510 J . Org. Chem., Vol. 65, No. 15, 2000
Martin et al.
having the general structure of 4 might be interesting
candidates as possible inhibitors of PLCBc. These com-
pounds contain an N,N′-dihydroxyurea functional group
in place of the polyhydroxylated framework of the
tropolone ring. While the dihydroxyurea moiety of 4 is
technically not a hydroxamic acid, it seemed likely that
it would retain the known zinc-chelating ability of
hydroxamic acids. In a fashion similar to that proposed
for 3, the carbonyl group and the two N-hydroxyl groups
on 4 would serve as Lewis basic sites for coordination to
two of the zinc ions in the active site of PLCBc; this
possibility was supported by preliminary docking studies
using SYBYL. The synthesis and evaluation of a series
of compounds for the general structure 4 as inhibitors of
PLCBc is detailed herein.
F igu r e 1. Interaction of phosphonate inhibitor with three zinc
ions in the active site of PLCBc (adapted from ref 14).
Resu lts a n d Discu ssion
Design a n d Syn th esis of Cyclic N,N′-Dih yd r ox-
yu r ea s. In deciding what analogues of 4 should be
evaluated as possible inhibitors of PLCBc, we wanted
especially to probe two factors that might affect bind-
ing: (1) What role do the pKas of the two hydroxyl groups
play in determining inhibitor potency? and (2) Are there
possible steric interactions that might interfere with
binding? Toward addressing these questions, we focused
upon the series of N,N′-dihydroxyurea derivatives 5-10.
Upon the basis of electronic effects, we predicted that the
pKas of 5 and 9 would be comparable to 3, but 10 and
6-8 should be less acidic. The N,N′-dihydroxybarbituric
acid 5 held the greatest potential for incurring unfavor-
able steric interactions on binding, whereas 9, 10, and
6-8 presenting correspondingly less likelihood for such
interactions at the active site of PLCBc.
oxygens of the substrate analogue are tightly coordinated
with the three metal ions, suggesting a catalytic role for
all three zincs. Moreover, Zn1 and Zn3 of PLCBc appear
to comprise a ‘bimetallic center’ because the internuclear
distance is only 3.5 Å, and they are bridged by a car-
boxylate residue.15
It occurred to us that a promising strategy for the
discovery of inhibitors would be to identify bidentate
ligands that would bind to this bimetallic site. For
example, alkaline phosphatase, dopamine â-monooxyge-
nase, and inositol monophosphatase, each of which
contain similar bimetallic sites, are strongly inhibited by
polyhydroxy tropolones containing three contiguous oxy-
gen groups on the seven membered ring.16,17 In these
cases, the polyhydroxy tropolone presumably chelates to
both metal ions at the active site.16 When we tested the
most active inhibitor, 2,7-dihydroxytropolone (3) against
PLCBc, we found that it exhibited an inhibition constant
(Ki) of 16 µM at pH 7.3, a potency comparable to the best
of the previously described substrate analogues.7 Al-
though this was an interesting discovery, we recognized
the problems associated with optimizing enzyme affinity
by derivatizing the tropolone ring, which is known to
have limited stability under certain conditions.17-20
A
stable zinc binding entity that could be easily derivatized
into a variety of structures for optimal inhibition was
needed.
The barbituric acid 5 was known,22 but it was neces-
sary to devise tactics for synthesizing 6-10 (Schemes
2-4). Compounds 6 and 7 were prepared in a straight-
forward fashion by a sequence that commenced with the
alkylation of O-benzylhydroxylurea with 1,2-dibromoet-
hane or 1,3-dibromopropane in the presence of 2 equiv
of KH in tetrahydrofuran (THF) at 0 °C to produce the
cyclized products 12 and 13 (Scheme 2).23 Initial attempts
to remove the O-benzyl groups by hydrogenolysis using
Pearlman’s catalyst, Pd/C, and PtO2 were unselective,
and cleavage of the N-O bond was a significant side
Because hydroxamic acids are well-known zinc chela-
tors,21 we reasoned that heterocyclic analogues of 3
(15) Wilcox, D. Chem. Rev. 1996, 96, 2435-2458.
(16) Piettre, S. R.; Ganzhorn, A.; Hoflack, J .; Islam, K.; Hornsperger,
J .-M. J . Am. Chem. Soc. 1997, 119, 3201-3204.
(17) Piettre, S. R.; Andre, C.; Chanel, M.-C.; Ducep, J .-B.; Lesur,
B.; Piriou, F.; Raboisson, P.; Rondeau, J .-M.; Schelcher, C.; Zimmer-
mann, P.; Ganzhorn, A. J . Med. Chem. 1997, 40, 4208-4221.
(18) Takeshita, H.; Mori, A. Synthesis 1986, 579-580.
(19) Takeshita, H.; Mori, A.; Kusaba, T.; Watanabe, H. Bull. Chem.
Soc. J pn. 1987, 60, 4325-4333.
(21) Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1420-1436.
(22) Cowden, W.; J acobsen, N. W. Aust. J . Chem. 1982, 35, 795-
797.
(23) Sulsky, R.; Demers, J . P. Synth. Commun. 1989, 19, 1871-
1874.
(20) Davies, H.; Clark, T. J . Tetrahedron 1994, 50, 9883-9892.

Zinc-Binding Inhibitors for Phospholipase C
J . Org. Chem., Vol. 65, No. 15, 2000 4511
Sch em e 2
Sch em e 4
Sch em e 3
14 was first monoalkylated with allyl bromide in the
presence of KH to give 19 in 65% yield. Acylation of 19
was best achieved by reaction with acrylic anhydride,
which was generated in situ, in the presence of LiCl to
give 20 (37%).28 The RCM of 20 to provide 21 using
Grubbs’s catalyst required the addition of Ti(O-i-Pr)4 (2
equiv);29,30 when the RCM reaction was performed in the
absence of Ti(O-i-Pr)4, only starting material was recov-
ered. Removal of the THP groups of 21 under acidic
conditions using AcOH in aqueous THF provided 10 in
80-90% yield.
N,N′-Dih yd r oxyu r ea s 5-10 a s In h ibitor s of P LCBc
.
With the N,N′-dihydroxyureas 5-10 in hand, the stage
was set to evaluate their potencies as inhibitors of PLCBc
using an assay we had previously developed for deter-
mining the kinetic parameters of PLCBc and mutants
thereof. Briefly, the assay involves quantitation of the
inorganic phosphate that is produced by the reaction of
alkaline phosphatase with the phosphorylcholine that is
liberated by the PLCBc-catalyzed hydrolysis of 1,2-di-n-
hexanoyl-sn-glycero-3-phosphocholine (see Experimental
Section).31 The Kis of 3, 5-10 were determined at pHs
7.3 and 9.5, and the results are summarized in Table 1.
The pKas of compounds 316 and 532 had been previously
determined, so the pKas of 8 and 10 were determined
experimentally by alkaline titration.33 Because of its
rather low solubility, it was not possible to obtain reliable
values for the pKas of 9, but they should be similar to
those reported for 5.
reaction. However, the O-benzyl groups were cleanly
removed using Pd/BaSO4 as the catalyst in MeOH (40
psi H2) to afford the desired N,N′-dihydroxyureas 6 and
7 in good yields.
In initial experiments, we found that the reaction of
11 with 1,4-dibromobutane (15) in the presence of KH
gave only low yields (<10%) of the seven-membered ring.
However, when the THP-protected urea 14 was used as
the reactant, 17 was obtained in 31% yield (Scheme 3).
Removal of the THP groups under acidic conditions using
AcOH-THF-H2O provided 8 (76%). The tricarbonyl
analogue 9 was also most efficiently prepared from 14.
Thus, when 14 was treated with succinyl chloride 16 and
2 equiv of 4-(N,N-dimethylamino)pyridine (DMAP) in
THF, 18 was obtained in 45% yield. When bases such as
Et3N and i-Pr2EtN were employed instead of DMAP, 16
appeared to decompose, perhaps by polymerization, and
unreacted 14 was recovered. It was therefore crucial to
use DMAP to activate the acid chloride toward acylation
of 14. Removal of the THP groups from 18 using standard
conditions (AcOH-THF-H2O) gave an inseparable mix-
ture of the desired 9 together with a side product that
The N,N′-dihydroxybarbituric acid derivative 5 showed
no detectable inhibition of PLCBc at concentrations up to
1 mM and a pH of either 7.3 or 9.5. Although the simple,
monocarbonyl derivatives 6-8 did not exhibit significant
inhibition of PLCBc at pH 7.3, they were modest inhibitors
at pH 9.5. Interestingly, the potency varied with ring size
(24) Grubbs, R.; Miller, S.; Fu, G. Acc. Chem. Res. 1995, 28, 446-
452.
(25) Schuster, M.; Blechert, S. Angew. Chem., Int. Ed. Engl. 1997,
36, 2036-2056.
(26) Armstrong, S. J . Chem. Soc., Perkin Trans. 1 1998, 371-388.
(27) Grubbs, R.; Chang, S. Tetrahedron 1998, 54, 4413-4450.
(28) Ho, G.-J .; Mathre, D. J . J . Org. Chem. 1995, 60, 2271-2273.
(29) Ghosh, A. K.; Cappiello, J .; Shin, D. Tetrahedron Lett. 1998,
39, 4651-4654.
1
was tentatively identified by H NMR as being acyclic.
However, when the deprotection was conducted in the
less nucleophilic solvent isopropyl alcohol in the presence
of catalytic pyridinium p-toluenesulfonate (PPTS), 9 was
obtained in nearly quantitative yield.
The unsaturated dicarbonyl compound 10 was pre-
pared by a sequence that featured a ring closing metath-
esis (RCM) reaction (Scheme 4).24-27 The RCM substrate
was readily prepared in two steps from 14. In the event,
(30) Fu¨rstner, A.; Langemann, K. J . Am. Chem. Soc. 1997, 119,
9130-9136.
(31) Hergenrother, P. J .; Martin, S. F. Anal. Biochem. 1997, 251,
45-49.
(32) Cowden, W.; J acobsen, N. W.; Stunzi, H. Aust. J . Chem. 1982,
35, 5, 1251-1253.
(33) Harris, D. C. Quantitative Chemical Analysis; W. H. Freeman
and Company: New York, 1991.

4512 J . Org. Chem., Vol. 65, No. 15, 2000
Martin et al.
Ta ble 1. Kis of 3 a n d th e Cyclic N,N′-Dih yd r oxyu r ea s
5-10
Ki (µM)
Ki (µM)
compound
pKa1, pKa2
5.6, 7.0
at pH 7.3
at pH 9.5
3
5
6
7
8
16
23
5.6, 7.1
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
388
no inhibition
303
146
not determined
not determined
9.4, 11.2
not determined
5.0, 8.7
71
9
10
no inhibition
53
with the seven-membered analogue 8 being about twice
as potent as the six-membered ring compound 7 and four
times more effective than the five-membered ring deriva-
tive 6. Although the tricarbonyl compound 9 did not
inhibit the enzyme, its unsaturated, dicarbonyl analogue
10 exhibited modest activity at pH 7.3 and good inhibi-
tion at pH 9.5.
F igu r e 2. Interaction of ring carbonyl group of 5 with Phe66
of PLCBc
.
carbonyl group, it occurred to us that one of the carbonyl
groups in 5 and 9 might interfere with binding because
of an unfavorable steric interaction with the enzyme. This
hypothesis was supported by preliminary modeling and
docking studies (SYBYL) of a complex of 5 with PLCBc.
In this complex, which is shown in part in Figure 2, it
appears that one of these carbonyl groups is forced to tie
within 3 Å of the aromatic ring of the Phe 66 residue;
such an interaction would clearly be detrimental to
binding. Whether in fact this is true must be tested by
further experimentation.
In conclusion, a novel class of inhibitors for PLCBc has
been designed and synthesized. This class of inhibitors
is believed to function by the bidentate chelation of the
N,N′-dihydroxyurea array to one or more of the zinc ions
at the active site of the enzyme. Because the affinities of
these compounds correlate with the pKas of the N-OH
hydroxyl groups, it is apparent that one or both of the
hydroxyl groups must be ionized for effective coordination
to the zinc ions. It is also apparent that there may be
rather strict steric requirements for these inhibitors.
Finally, it is possible that the N,N′-dihydroxyurea array
might be more generally useful as a zinc binding group
in the design of inhibitors of other metalloenzymes. These
various questions are under current investigation on
several fronts.
PLCBc must retain all three active site zinc ions for full
activity,4,5 so one must consider the possibility that 3,
6-8, and 10 are inhibitors of PLCBc because they
sequester zinc ions from the enzyme active site; however,
this interpretation seems unlikely. Namely, the Ki for the
dihydroxytropolone 3 was 16 µM at pH 7.3 and 23 µM at
pH 9.5. Inasmuch as both of these values are below the
concentration of zinc ion (33 µM) in the assay, 3 cannot
be inhibiting the enzyme by sequestering zinc ions. The
structural similarity of 3 and the N,N′-dihydroxyureas
6-8 and 10 suggests that these compounds are also
unlikely to inhibit the enzyme by sequestering zinc ions.
In support of this hypothesis, we found that a sample of
PLCBc (0.5 µM) in 1.0 mM dimethyl glutarate buffer
(DMG) (pH ) 7.3) containing 0.1 mM ZnSO4 and 10
(6 mM) retained approximately three zinc ions (as
determined by flame atomic absorption) after dialysis
against 1.0 mM DMG buffer (three times, 1:1000, 3 h, 4
°C). The buffer from the last dialysis was used as a blank
and contained no zinc ion. Analyses were performed in
triplicate.
As might be expected, the pKas of the two hydroxyl
groups play a major role because the ionized N-hydroxyl
function would be expected to bind better than a neutral
hydroxyl group to an active site zinc ion. The first pKa of
8 was determined to be 9.4, so that at pH 7.3 the N,N′-
dihydroxyurea array in compounds 6-8 should be fully
protonated. Because 6-8 were inactive at pH 7.3 but
active at pH 9.5, it is apparent that at least one of the
N-hydroxyl groups must be ionized in order for the N,N′-
dihydroxyurea array to serve effectively as a ligand for
the zinc ions at the active site. Assuming that ring size
does not influence the pKa, a comparison of the pKas of
8, 10, and 5 reveals that placement of a carbonyl group
adjacent to the N-hydroxyl group lowers its pKa. Thus,
the increased potency of 10 relative to 8 may be easily
rationalized on the basis of its lower pKas. That 10 is an
order of magnitude more potent at pH 9.5 than at 7.3
suggests that both hydroxyl groups must be ionized for
optimal binding. In this context, it is noteworthy that the
dihydroxytropolone 3 would exist at least partially in its
dianionic form at pH 7.3 and is thus approximately
equipotent at pH 7.3 and 9.5.
Exp er im en ta l Section
Gen er a l. Unless otherwise noted, all starting materials
were obtained from commercial suppliers and were used
without further purification. Melting points were determined
on a Thomas-Hoover capillary melting point apparatus and
are uncorrected. Tetrahydrofuran (THF) and toluene were
distilled from potassium/benzophenone ketyl immediately prior
to use. Triethylamine and methylene chloride were distilled
from calcium hydride and stored over 4 Å molecular sieves
under nitrogen. All reactions involving organometallic reagents
or other air-sensitive reagents were executed under an atmo-
sphere of dry nitrogen or argon, using oven- or flame-dried
glassware. Spectra were recorded on compounds that were
g95% pure by HPLC or 1H NMR spectroscopy. IR spectra of
oils were recorded as thin films (NaCl plates), whereas the IR
spectra of solids were determined as solutions in CHCl3. The
1H and 13C NMR spectra were determined unless otherwise
indicated as solutions in CDCl3 at the indicated field; chemical
shifts are expressed in parts per million (δ units) downfield
from tetramethylsilane. Splitting patterns are designated as
s, singlet; d, doublet; t, triplet; q, quartet; p, pentuplet; m,
multiplet; comp, complex multiplet; br, broad.
It is at first surprising that 5 and 9, which have pKas
approximately the same as 3, are inactive as inhibitors
of PLCBc. However, it is perhaps significant that both 5
and 9 have two carbonyl groups on the ring connecting
the dihydroxyurea moiety. Because 10 has only one such
1,3-N,N′-Diben zyloxyim id a zolid -2-on e (12). A slurry of
NaH (5.4 mg, 0.14 mmol) in DMF (0.2 mL) was added to 11
(37 mg, 0.14 mmol) in DMF (0.2 mL). The reaction was stirred
for 10 min before slowly adding dibromoethane (11.7 µL, 0.14
mmol). After 30 min, NaH (5.4 mg, 0.14 mmol) in DMF (0.4

Zinc-Binding Inhibitors for Phospholipase C
J . Org. Chem., Vol. 65, No. 15, 2000 4513
mL) was added, and the mixture was stirred for 12 h. The
reaction was poured into sat. aqueous NaHCO3 (10 mL). The
mixture was extracted with CH2Cl2 (3 × 10 mL). The organic
layers were combined. The organic phase was washed with
H2O (10 mL) and dried (MgSO4). The solvents were removed
under reduced pressure. The crude material was purified by
flash chromatography eluting with 5% Et2O-CH2Cl2 to provide
10 mg of 12 (43%) as a viscous oil; 1H NMR (300 MHz, CDCl3)
δ 7.44-7.33 (comp, 10 H), 4.99 (s, 4 H), 3.07 (s, 4 H); 13C NMR
(75 MHz, CDCl3) δ 136.0, 129.3, 128.5, 128.4, 78.0, 44.5; IR
2883, 1752, 1454, 1369, 1240, 1013, 749 cm-1; mass spectrum
(CI) m/z 299.1393 [C17H19N2O3 (M + 1) requires 299.1396].
2945, 2870, 1682, 1487, 1114, 1037 cm-1; mass spectrum (CI)
m/z 261.1456 [C11H21N2O5 (M + 1) requires 261.1450].
1,3-N,N′-Bis(tetr a h yd r op yr a n yloxy)p en ta h yd r o-1,3-d i-
a zep in -2-on e (17). To a solution of 14 (138 mg, 0.53 mmol)
in THF (10 mL) at 0 °C was added KH (21 mg, 0.53 mmol).
The reaction was stirred rapidly for 15 min before adding 1,4-
dibromobutane (63 µL, 0.53 mmol) via syringe. The reaction
was allowed to warm to 23 °C in 1 h before adding KH (21
mg, 0.53 mmol). After 24 h of rapid stirring, the reaction was
diluted with EtOAc (10 mL) and then poured into sat. aqueous
NH4Cl (10 mL). The organic layer was separated. The aqueous
phase was extracted with EtOAc (3 × 10 mL). The organic
layers were combined and dried (MgSO4). The solvents were
removed under reduced pressure. The crude material was
purified by flash chromatography to provide 45 mg of 17 (27%)
as a clear oil and starting material (8 mg, 0.03 mmol); 1H NMR
(300 MHz, CDCl3) δ 4.95-4.92 (m, 1 H), 4.89-4.87 (m, 1 H),
4.16-3.93 (comp, 2 H), 3.63-3.42 (comp, 3 H), 1.90-1.48
(comp, 16 H); 13C NMR (75 MHz, CDCl3) δ 163.6, 102.6, 101.9,
62.8, 62.6, 55.6, 55.5, 28.5, 28.4, 27.0, 26.7, 25.2, 19.0, 18.8;
IR 2940, 1710, 1441, 1204, 1113, 1039 cm-1; mass spectrum
(CI) m/z 315.1912 [C15H27N2O5 (M + 1) requires 315.1912].
1,3-N,N′-Dih yd r oxyp en ta h yd r o-1,3-d ia zep in -2-on e (8).
A solution of 17 (22 mg, 0.07 mmol) in AcOH-THF-H2O (3.5
mL, 4:2:1) was stirred for 12 h at room temperature. The
solvents were removed under reduced pressure. The crude
material was purified by flash chromatography eluting with
10% MeOH-CH2Cl2 to provide 7.8 mg of 8 (76%) as a white
solid; mp ) 100-102 °C; 1H NMR (300 MHz, D2O) δ 3.39-
3.36 (comp, 4 H), 1.64-1.60 (comp, 4 H); 13C NMR (75 MHz,
D2O) δ 166.4, 56.1, 25.7; IR 3249, 2934, 1663, 1468, 1260, 1021
cm-1; mass spectrum (CI) m/z 147.0769 [C5H11N2O3 (M + 1)
requires 147.0770].
1,3-N,N′-Diben zyloxytetr a h yd r op yr im id -2-on e (13). A
slurry of NaH (8 mg, 0.19 mmol) in DMF (0.2 mL) was added
to 11 (51 mg, 0.19 mmol) in DMF (0.8 mL). The reaction was
cooled to 0 °C. After 10 min, dibromopropane (19 µL, 0.19
mmol) was added dropwise. After 30 min, NaH (5.4 mg, 0.14
mmol) in DMF (0.2 mL) was added to the reaction. The
mixture was stirred for 12 h. The reaction was poured into
sat. aqueous NaHCO3 (10 mL). The aqueous phase was
extracted with EtOAc (3 × 10 mL). The organic layers were
combined and dried (MgSO4). The solvents were removed
under reduced pressure. The residue was purified by flash
chromatography eluting with 50% EtOAc-hexanes to provide
1
30 mg of 13 (51%) as an off white solid; H NMR (300 MHz,
CDCl3) δ 7.49-7.34 (comp, 10 H), 4.99 (s, 4 H), 3.26 (t, J )
6.2 Hz, 4 H), 1.89 (p, J ) 5.9 Hz, 2 H); 13C NMR (75 MHz,
CDCl3) δ 159.5, 135.9, 129.5, 128.4, 128.3, 77.1, 49.1, 20.6; IR
1690, 1454, 1294, 1183 cm-1; mass spectrum (CI) m/z 313.1546
[C18H21N2O3 (M + 1) requires 313.5522].
1,3-N,N′-Dih yd r oxyim id a zolid -2-on e (6). A solution con-
taining 12 (10 mg, 0.06 mmol) and 5 wt % Pd/BaSO4 (2.4 mg)
in MeOH (4 mL) was evacuated under aspirator pressure and
then charged with H2 at 40 psi (repeated three times). The
reaction was shaken on a Parr shaker for 12 h. The reaction
was filtered through a plug of glass wool to remove the 5 wt
% Pd/BaSO4 catalyst. The solvents were removed under
reduced pressure. The residue was purified by flash chroma-
tography eluting with 10% MeOH-CH2Cl2 to provide 4.5 mg
of 6 (68%) as light brown oil; 1H NMR (300 MHz, CDCl3) δ
3.31 (s, 4 H); 13C NMR (75 MHz, CDCl3) δ 106.9, 46.0; mass
spectrum (CI) m/z 119.0457 [C3H7N2O3 (M + 1) requires
119.0457].
1,3-N,N′-Dih yd r oxytetr a h yd r op yr im id -2-on e (7). A so-
lution containing 13 (124 mg, 0.40 mmol) and 5 wt % Pd/BaSO4
(21 mg) in MeOH-THF (12 mL, 7:5) was evacuated under
aspirator pressure and then charged with H2 at 40 psi
(repeated three times). The reaction was shaken on a Parr
shaker for 12 h. The Pd/BaSO4 was removed by filtration
through glass wool to remove the Pd/BaSO4. The solvents were
removed under reduced pressure. The residue was purified by
flash chromatography eluting with 20% MeOH-CH2Cl2 to
provide 45 mg of 7 (86%) as a light brown solid; mp ) 122-
124 °C; 1H NMR (300 MHz, CDCl3) δ 3.37 (t, J ) 5.9 Hz, 4 H),
2.01 (p, J ) 5.9 Hz, 2 H); 13C NMR (75 MHz, CDCl3) δ 161.5,
49.9, 19.9; IR 3303, 2870, 1650, 1534, 1310 cm-1; mass
spectrum (CI) m/z 133.0611 [C4H9N2O3 (M + 1) requires
133.0613].
N,N′-Bis(tetr a h yd r op yr a n yloxy)u r ea (14). To a solution
of THPONH234 (2.97 g, 25.4 mmol) in THF (60 mL) was added
a solution of phosgene (35.6 mmol) in toluene (5 mL) at -5 °C
over 15 min. The reaction was then allowed to warm slowly
to room temperature over 1 h while continuously stirring
vigorously via mechanical stirring and then the reaction was
stirred for an additional 6 h. The reaction was poured into sat.
aqueous NH4Cl (50 mL). The aqueous phase was extracted
with EtOAc (3 × 50 mL). The organic phase was dried (MgSO4)
and the solvents were removed under reduced pressure. The
crude material was purified by flash chromatography eluting
with 75% EtOAc-hexanes to provide 3.09 g of 14 (94%) as a
white foam; 1H NMR (300 MHz, CDCl3) δ 8.14 (s, 2 H), 4.89-
4.86 (comp, 2 H), 4.03-3.91 (comp, 2 H), 3.64-3.56 (comp, 2
H), 1.81-1.56 (comp, 12 H); 13C NMR (75 MHz, CDCl3) δ 160.3,
160.0, 103.6, 103.3, 63.5, 28.3, 24.9, 19.5, 19.4; IR 3478, 3250,
N,N′-Bis(tetr a h yd r op yr a n yloxy)d ih yd r o-1,3-d ia zep in -
2,4,7-tr ion e (18). To a solution of 14 (188 mg, 0.72 mmol) and
succinyl dichloride (80 µL, 0.72 mmol) in THF (8 mL) at room
temperature was added DMAP (176 mg, 1.5 mmol). The
reaction immediately turned light purple in color, and a dark
purple precipitate was observed in the solution. After 48 h,
the reaction was poured into sat. aqueous NH4Cl (15 mL). The
aqueous phase was extracted with EtOAc (3 × 15 mL). An
insoluble dark purple precipitate was present in both phases.
The organic layers were dried (MgSO4), and the solvents were
removed under reduced pressure. The crude material was
purified by flash chromatography eluting with 75% EtOAc-
hexanes to provide 111 mg of 18 (45%) as a viscous oil and 81
1
mg of starting material; H NMR (300 MHz, CDCl3) δ 5.00-
4.93 (comp, 2 H), 4.13-4.01 (comp, 2 H), 3.60-3.51 (comp, 2
H), 3.06-2.84 (comp, 4 H), 2.06-1.48 (comp, 12 H); 13C NMR
(75 MHz, CDCl3) δ 167.9, 147.5, 102.4, 102.3, 62.7, 31.1, 28.3,
27.7, 24.8, 17.7; IR 2945, 1731, 1711, 1303, 1205, 1141, 1036
cm-1; mass spectrum (CI) m/z 343.1505 [C15H23N2O7 (M + 1)
requires 343.1505].
1,3-N,N′-Dih yd r oxy-5,6-d ih yd r o-1,3-d ia zep in -2,4,7-tr i-
on e (9). To a solution of 18 (22 mg, 0.064 mmol) in THF-i-
PrOH (1:1) at 50 °C was added PPTS (5 mg, 0.02 mmol). After
2 h, PPTS (5 mg, 0.02 mmol) was added to the reaction. After
2 h, the reaction was complete by TLC, and the flask was
cooled to -78 °C for 5 h. The product crystallized into tiny
white crystals that were isolated by first transferring the
reaction mixture to an epindorf tube. The tube was briefly
centrifuged at high speed. The mother liquor was decanted.
The crystals were washed with cold i-PrOH. The excess solvent
was removed under reduced pressure to provide 3.5 mg of 9
in quantitative yield; 1H NMR (300 MHz, CD3OD) δ 2.96 (s, 4
H); 13C NMR (75 MHz, CD3OD) δ 170.7, 150.8, 31.4; mass
spectrum (CI) m/z 175.0361 [C5H7N2O5 (M + 1) requires
175.0355].
N-(2-P r op en yl)-N,N′-b is(t et r a h yd r op yr a n yloxy)u r ea
(19). To a solution of 14 (740 mg, 2.9 mmol) and allyl bromide
(295 µL, 3.4 mmol) in THF (18 mL) was added KH (111 mg,
2.9 mmol) at -60 °C. The reaction was warmed to room
temperature over 1 h and then stirred at room temperature
for an additional 12 h. The reaction was quenched by pouring
it into sat. aqueous NaHCO3 (50 mL). The layers were

4514 J . Org. Chem., Vol. 65, No. 15, 2000
Martin et al.
separated, and the aqueous phase was extracted with CH2Cl2
(3 × 100 mL). The organic layers were dried (MgSO4). The
solvents were removed under reduced pressure. The crude
material was purified by flash chromatography eluting with
75% EtOAc-hexanes to provide 556 mg of 19 (65%) as a white
foam; 1H NMR (300 MHz, CDCl3) δ 9.12 (s, 0.5 H), 9.07 (s, 0.5
H), 5.89 (m, 1 H), 5.31-5.19 (comp, 2 H), 4.95 (dt, J ) 11.8,
3.4 Hz, 1 H), 4.75 (m, 1 H), 4.36-4.27 (m, 1 H), 4.05-3.91
(comp, 3 H), 3.66-3.48 (comp, 2 H), 1.90-1.46 (comp, 12 H);
13C NMR (75 MHz, CDCl3) δ 161.7, 161.5, 132.0, 132.9, 119.7,
119.6, 106.3, 106.0, 103.2, 102.4, 66.3, 66.1, 63.2, 62.9, 54.2,
29.7, 29.6, 28.8, 28.7, 25.7, 25.3, 21.7, 21.6, 19.6, 19.2; IR 3308,
2944, 2868, 1701, 1458, 1037 cm-1; mass spectrum (CI) m/z
301.1768 [C14H25N2O5 (M + 1) requires 301.1763].
N-(Acr yloxyca r b a m oyl)-N′-(2-p r op en yl)-N,N′-b is(t et -
r a h yd r op yr a n yloxy)u r ea (20). To a solution of acrylic acid
(252 µL, 3.68 mmol) and Et3N (994 µL, 7.08 mmol) in THF
(10 mL) at -20 °C was added acryloyl chloride (252 µL, 3.40
mmol) dropwise via syringe.35 The mixture was stirred for a 1
h, and LiCl (132 mg, 3.11 mmol) was added followed by
dropwise addition of a solution of 19 (849 mg, 2.83 mmol) in
THF (9 mL). The reaction was allowed to warm to room
temperature and stirred for an additional 3.5 h. The reaction
was diluted with EtOAc (50 mL) and poured into sat. aqueous
NH4Cl (70 mL). The aqueous phase was extracted with EtOAc
(3 × 150 mL). The organic layers were combined and dried
(MgSO4). The solvents were removed under reduced pressure.
The crude material was purified by flash chromatography to
provide 369 mg of 20 (37%) as a light brown oil; 1H NMR (300
MHz, CDCl3) δ 6.68 (dd, J ) 17.1, 10.3 Hz, 0.5 H), 6.60 (dd, J
) 17.1, 9.8 Hz, 0.5 H), 6.48 (dd, J ) 16.9, 1.8 Hz, 1 H), 6.06-
5.92 (m, 1 H), 5.81 (dd, J ) 10.3, 1.6 Hz, 0.5 H), 5.80 (dd, J )
10.0, 2.1 Hz, 0.5 H), 5.36-5.14 (comp, 3 H), 5.09-5.03 (m, 1
H), 4.59-4.49 (comp, 0.5 H), 4.46-4.36 (comp, 0.5 H), 4.31-
4.18 (comp, 1 H), 4.06-3.92 (comp, 2 H), 3.66-3.52 (comp, 2
H), 1.96-1.50 (comp, 12 H); 13C NMR (75 MHz, CDCl3) δ 165.3,
154.5, 132.0, 130.8, 126.80, 126.81, 126.75, 118.6, 118.4, 63.1,
62.7, 62.5, 54.4, 53.4, 28.6, 28.5, 28.2, 25.0, 24.8, 18.7, 18.6,
18.5, 18.3; IR 2946, 1698, 1403, 1206, 1115, 1038 cm-1; mass
spectrum (CI) m/z 355.1869 [C17H27N2O6 (M + 1) requires
355.1859].
6.91-6.82 (m, 1 H), 6.28-6.23 (m, 1 H), 5.13-5.01 (comp, 2
H), 4.58-3.97 (comp, 4 H), 3.70-3.57 (comp, 2 H), 2.12-1.51
(comp, 12 H); 13C NMR (75 MHz, CDCl3) δ 161.0, 140.9, 128.4,
128.0, 104.6, 103.9, 102.6, 102.1, 63.6, 62.4, 48.5, 28.3, 27.8,
27.7, 24.9, 19.1, 17.8; IR 2945, 1728, 1690, 1205, 1115, 1035,
938 cm-1; mass spectrum (CI) m/z 327.1554 [C15H23N2O6 (M
+ 1) requires 327.1556].
1,3-N ,N ′-Dih yd r oxy-5,6-d ih yd r o-1,3-d ia ze p in -2,4-d i-
on e (10). A solution of 21 (31 mg, 0.095 mmol) in AcOH/THF/
H2O (3.5 mL, 4:2:1) was heated to 40 °C for 24 h. The solvents
were removed under reduced pressure. The crude material was
purified by flash chromatography eluting with CH2Cl2-MeOH
(7.5:2) to provide 9 mg of 10 (60%) as a colorless glass; 1H NMR
(300 MHz, CD3OD) δ 6.91 (dt, J ) 10.8, 6.6 Hz, 1 H), 6.15 (d,
J ) 10.8 Hz, 1 H), 4.24 (d, J ) 6.6 Hz, 2 H); 13C NMR (125
MHz, D2O) δ 165.1, 154.4, 143.4, 127.2, 49.5; IR (MeOH) 3409,
2874, 1711, 1680, 1632, 1242, 828, 670; mass spectrum (CI)
m/z 159.0399 [C5H7N2O4 (M+1) requires 159.0406], 159 (base),
143, 102.
Deter m in a tion of Kis for Com p ou n d s 5-10. The inhibi-
tion constants (Ki) for compounds 3 and 5-10 were determined
with a PLCBc assay based on the quantitation of inorganic
phosphate, performed in 96-well plates.31 In this procedure,
the PLCBc reaction was allowed to proceed for a defined period
of time, at which point the activity was quenched with the
biological buffer Tris, a known PLCBc inhibitor.13 The phos-
phorylcholine product of the PLCBc reaction was then converted
to inorganic phosphate (Pi) and choline through the action of
alkaline phosphatase (APase). A blue-colored complex with Pi
was then formed by the addition of solutions containing
ammonium molybdate, ascorbic acid, trichloroacetic acid, and
sodium metaarsenite; this “molybdenum blue” complex had a
λmax of 700 nm and was read with a microplate reader. To
determine Kis, the assay was performed in an analogous
fashion, except the synthesized organic compounds were added
at the following concentrations: 3 at 10 µM; 5 at 1 mM, 6 at
1 mM; 7 at 40 µM; 8 at 40 µM; 9 at 80 µM; 10 at 60 µM. The
data obtained in the presence of the inhibitor was then
compared to data gathered in its absence. The Vint vs concen-
tration of phosphatidylcholine plots of these two data sets
(from a minimum of eight substrate concentrations) were fit
to the Michaelis-Menten curves using the program Kaleida-
Graph. A Ki was then deduced from the apparent Vmax and
Km values in the presence of the inhibitor. Because 2,7-
dihydroxytropolone (3) is a known inhibitor of alkaline phos-
phatase, a great excess of APase (>10-fold relative to a typical
assay) was added to all the kinetic assays. Control experiments
indicated that under these conditions of high APase concentra-
tion phosphorylcholine was rapidly and quantitatively con-
verted to Pi and choline, even in the presence of 3.
1,3-N,N′-Bis(tetr a h yd r op yr a n yloxy)-5,6-d ih yd r o-1,3-d i-
a zep in -2,4-d ion e (21). A solution of 20 (84 mg, 0.24 mmol)
was stirred rapidly in degassed, dry CH2Cl2 (24 mL) at 0 °C,
and Ti(O-i-Pr)4 (140 µL, 0.48 mmol) was added dropwise. After
10 min, a solution of Grubbs’s catalyst36 (78 mg, 0.095 mmol)
in degassed, dry CH2Cl2 (6 mL) was added dropwise via syringe
over 30 min. The reaction was rapidly stirred for 30 min at 0
°C. The ice bath was then removed, and the solution was
stirred for 4 h at room temperature. The ruthenium catalyst
was removed by filtration through a short column of SiO2 using
EtOAc to elute the product. The solvents were removed under
reduced pressure, and the brown residue was purified by flash
chromatography eluting with EtOAc to provide 58 mg of 21
(74%) as a very dark brown oil; 1H NMR (300 MHz, CDCl3) δ
Ack n ow led gm en t. We thank the National Insti-
tutes of Health and the Robert A. Welch Foundation for
their generous support of this research. We also thank
Ms. Nina M. Antikainen and Hilary R. Plake for assis-
tance in preparing the cover graphic art.
(34) Hitchcock, S. A.; Boyer, S. H.; Chumoyer, M. Y.; Olson, S. H.;
Danishefsky, S. J . Angew. Chem., Int. Ed. Engl. 1994, 33, 858-862.
(35) Ho, S. N.; Hunt, H. D.; Horton, R. M.; Pullen, J . K.; Pease, L.
R. Gene 1989, 77, 51-59.
(36) Dias, E. L.; Nguyen, S. T.; Grubbs, R. H. J . Am. Chem. Soc.
1997, 119, 3887-3897.
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
spectra of all new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O9915731