Synthesis and enzymic evaluation of 4-mercapto-6-oxo-1,4- azaphosphinane-2-carboxylic acid 4-oxide as an inhibitor of mammalian dihydroorotase

Article abstract of DOI:10.1021/jm970814z

The design, synthesis, and enzymic evaluation of cis- and trans-4- mercapto-6-oxo-1,4-azaphosphinane-2-carboxylic acid 4-oxide 5 against mammalian dihydroorotase is presented. The design strategy for 5 was based on the strong affinity of phosphinothioic a

Full text of DOI:10.1021/jm970814z

4550
J . Med. Chem. 1998, 41, 4550-4555
Syn th esis a n d En zym ic Eva lu a tion of
4-Mer ca p to-6-oxo-1,4-a za p h osp h in a n e-2-ca r boxylic Acid 4-Oxid e a s a n In h ibitor
of Ma m m a lia n Dih yd r oor ota se
Michael K. Manthey,* Danny T. C. Huang, William A. Bubb, and Richard I. Christopherson
Department of Biochemistry, University of Sydney, Sydney, NSW 2006, Australia
Received December 3, 1997
The design, synthesis, and enzymic evaluation of cis- and trans-4-mercapto-6-oxo-1,4-
azaphosphinane-2-carboxylic acid 4-oxide 5 against mammalian dihydroorotase is presented.
The design strategy for 5 was based on the strong affinity of phosphinothioic acids for zinc
and that 5 also resembles the postulated tetrahedral transition state for the enzyme-catalyzed
reaction. The synthesis of 5 utilized a novel protection/deprotection sequence upon 4-hydroxy-
6-oxo-1,4-azaphosphinane-2-carboxylic acid 4-oxide 4, followed by incorporation of R-phenyl
benzenemethanethiol and exhaustive deprotection to afford 5 in 40% overall yield from 4. The
activities of both isomers of 5 as inhibitors of mammalian dihydroorotase were marginally
greater than that of the parent phosphinic acid 4, indicating a weak binding enhancement
due to the phosphinothioic acid moiety.
In tr od u ction
binding, would be expected to have a greater probability
of yielding a potent inhibitor of DHOase. Consequently
the phosphinothioic acid 5, which not only mimics the
transition state of the enzyme-catalyzed reaction but
may also provide a strong chelation effect via a strong
S-Zn interaction,11 was designed as a transition-state
inhibitor for DHOase. We report herein the synthesis
and enzymic evaluation of cis and trans-5 as inhibitors
against mammalian DHOase.
Dihydroorotase (DHOase) catalyzes the third reaction
of the pathway for de novo biosynthesis of pyrimidine
nucleotides, the reversible cyclization of N-carbamyl-L-
aspartate (CA-asp) 1 to L-dihydroorotate 2 (Figure 1).
A zinc atom bound at the active site of the enzyme has
been implicated in the catalytic mechanism.1 It has
been proposed that the mechanism is similar to that of
a zinc protease in which the zinc atom stabilizes the
tetrahedral transition state of the reaction by formation
of a coordination complex to two oxygen atoms at C-6
of the dihydropyrimidine ring (Figure 1).2
Inhibitors of DHOase have potential use as anti-
cancer3 or antimalarial drugs.4 Several inhibitors of
DHOase have been designed as chelators of the active
site zinc atom.5-7 To date these have principally relied
on the strong affinity of sulfur for zinc, with the most
potent inhibitor, the cis-mercaptomethyl analogue 3,
Ch em istr y
The synthetic strategy employed for the phosphi-
nothioic acids 5a ,b is outlined in Scheme 1. The
starting material for the synthesis, the parent phos-
phinic acid 4, has previously been synthesized by the
cyclization of the amino acid 6 with 1 equiv of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (DEPC) at
pH 5.0 in a yield of 21%.10 We have increased the yield
of 4 in the cyclization reaction to 66% by raising the
pH of the reaction medium to 5.6 and by employing a
1-fold excess of DEPC. Selective protection of the
carboxylic acid moiety in 4 was required prior to
functionalization of the phosphinic acid. A direct pro-
tection strategy proved troublesome. For example,
esterification of 4 with CH3OH/H+ under mild condi-
tions resulted in extensive ring opening to yield the
amino acid ester 7. Consequently, an indirect approach
exhibiting an apparent Ki of 140 nM at pH 7.4 (Km of 2
is 19 µM at pH 7.4). However 3 only has a relatively
short half-life in vitro, thereby making its clinical use
limited.
A number of transition-state analogues based on
sulfone, sulfoxide,8 carboxylate,9 and phosphinate10
groups have also been developed. One of these, the
phosphinic acid 4, was designed as a stable transition-
state analogue which closely resembles the tetrahedral
geometry of the transition state while at the same time
being relatively nonimposing with respect to steric
constraints.11
was employed for the selective protection of the car-
boxylic acid in 4. The diphenylmethyl group was
selected to protect both the carboxylic and phosphinic
acid groups in 4 to give a UV-active product that could
be purified chromatographically and easily deprotected
under mild conditions at the end of the synthesis.
An enzyme inhibitor incorporating multiple structural
attributes, which individually are known to promote
10.1021/jm970814z CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/17/1998

Synthesis of an Inhibitor of Mammalian DHOase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4551
F igu r e 1. The reaction catalyzed by dihydroorotase.
Sch em e 1a
a
Reagents: (a) 1-(3-Dimethylaminopropyl)-3-ethylcarbodimide, pH 5.6, 45-60 °C; (b) Ph2CN2; (c) CH3Ch2OH; (d) Ph2CHSH, DECP,
Et3N; (e) TFA.
Reaction of 4 with a slight excess of diphenyldiazo-
between the cis proton at C-3 and one of the protons
attached to C-5. Given the constraints already noted
for the bulky substituent at C-2, the requisite W
geometry for coupling across four single bonds14 fixes
the conformation of 8a and the assignments therein of
H-3 and H-5. The stereochemistry of the substituents
attached to the phosphorus atom follows from observa-
tion of a weak nuclear Overhauser effect (NOE) between
the benzyl proton attached to the phosphinate group,
designated H-A, and both H-3 and H-5 (Figure 2).
There was some sharpening of the H-3 resonance upon
spin-decoupling of H-1, indicating a quasi-W pathway
beween each of these protons, consistent with the
conformation assigned.
Vicinal coupling constants between H-2 and H-3/H-
3′ in 8b are of similar magnitude to those in 8a but there
was only a very weak additional coupling (evidenced by
resonance sharpening on spin-decoupling) between H-3′
and the lower frequency resonance of the H-5/H-5′ pair.
Rather, a clearly resolved 1.2 Hz splitting in the
resonance of the same C-5 proton was shown to be
associated with coupling to the NH proton, designated
H-1. Further, the C-5 resonance involved in this
putative W-coupling and the C-3 proton with the larger
vicinal coupling show NOE cross-peaks to H-A. These
methane yielded the diasteromeric bisdiphenylmethyl
esters 8a and 8b which were readily separable by
column chromatography.
The configurations of 8a and 8b were established by
assignment of their 1H NMR spectra, with the aid of
spin-decoupling to clarify relationships characterized by
relatively small J values, together with nuclear Over-
hauser effect spectrometry (NOESY) experiments. For
the latter, application of a weak pulsed field gradient
during the mixing time12 provided a significant im-
provement in spectral quality which permitted observa-
tion of the relatively small enhancements which were
critical to establishing the stereochemistry. NMR data
for the pair of isomers, 8a and 8b, and their thio
analogues, 11a and 11b, are presented in Table 1. The
data support assignment of the relationship between the
substituents bearing the dibenzyl groups as trans in 8a
and 11a but cis in 8b and 11b.
Thus in both 8a and 8b, H-2 has the axial configu-
ration as evidenced by the relative magnitudes of the
vicinal coupling constants to H-3 and H-3′. Conse-
quently, the bulky diphenylmethylcarboxylate moiety
at C-2 must have the equatorial configuration in both
isomers. In addition, 8a has a clearly resolved coupling

4552 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Manthey et al.
Ta ble 1. 1H NMR Data for Compounds Used for Stereochemical Assignmentsa
Chemical Shifts
compd
H1
H2
H3
H3′
H5
H5′
HA
HB
Harom
8a
8b
11a
11b
6.39
6.42
6.44
6.49
4.07
4.39
4.12
4.49
2.21
2.41
2.20
2.56
1.95
1.90
2.11
1.92
2.72
2.77
2.83
2.93
2.78
2.59
3.01
2.56
6.55
6.48
5.94
5.89
6.80
6.80
6.89
6.89
7.06-7.36
7.08-7.35
7.06-7.54
7.14-7.50
Coupling Constants
compd
J 1,2
J 1,5′
J 2,3
J 2,3′
J 2,P
J 3,3′
J 3,5
J 3,P
J 3′,P
J 5,5′
J 5,P
J 5′,P
J A,P
8a
8b
11a
11b
1.9
2.7
2.0
2.7
3.5
3.7
3.4
3.4
11.8
11.8
11.9
11.9
10.7
12.7
11.8
12.7
15.0
14.8
14.8
15.0
1.5
20.1
17.7
15.3
18.3
9.8
13.5
11.8
6.1
17.4
16.5
17.4
16.5
18.4
17.4
12.8
18.7
14.8
16.8
17.2
11.9
9.2
8.8
9.4
9.2
1.2
1.2
1.3
Observed NOE
H3 H3′
compd
intb
H1
H2
H2
H5
H5′
HA
HB
8a
s
H1, H3
H5
H3′, HB
H1, H3, H5
H2, H3′
H3
H5′
H2
H1, HA
H2, H5′
H5
m
w
s
m
w
s
m
w
s
m
w
H5, H5′
H2
HA
H2, H3′
H2, H5′
H3
H1, H3′
H5
H3, H5
H3′, H5′
H3
H2
H2
8b
11a
11b
H3′
H2
H3′, HB
H1, H3
H5
H1, H2, HA
H3
HA
H5
H2, H3′
H5′
H2
H3′
HA
H2, H3′
H2
H3
H2
H3, H5
H1
H2, H5′
H5
H3′
HB
H1, H2
HA
H5′
H2
a
Chemical shifts and coupling constants were determined by first-order analysis for all spin systems where ∆υ/J >10 and otherwise
by ABX calculation13 following simplification of the spectra by spin-decoupling. Relative intensity of NOE cross-peak: s, strong; m,
b
medium; w, weak.
9 were formed exclusively. Reflux of 8b under the same
conditions also resulted in the formation of 9 and
diphenylmethylethyl ether. To investigate the general
applicability of the reaction, the diphenylmethyl phos-
phinate 10 was synthesized and incubated in ethanol
at reflux. No reaction was evident after 1 h with
unreacted 10 being recovered. Hence it would seem that
de-esterification of 8 in ethanol is a consequence of
specific steric and/or electronic factors. We tentatively
propose that ethanol serves as a general acid/base
catalyst in the deprotonation of the acidic H-5ax proton
in 8, with loss of the relatively stable diphenylmethyl
data can be rationalized by the structure depicted for
cation, which is captured by ethanol driving the reac-
8b. The 1,3-diaxial interaction between the PdO and
tion. (The H-2 protons in 4 are gradually exchanged
CdO bonds, which would accompany the change in
stereochemistry of the phosphorus substituents in a full
with deuterium in D2O solution, indicating their acidic
nature.) A postulated reaction mechanism for the depro-
chair conformation, is evidently sufficient to cause a
tection of 8a is shown in Figure 3. A similar reaction
conformational change to a half-chair. This conforma-
mechanism with a chair-like transition state can be
tional change removes the W-pathway between H-3 and
drawn for 8b after a conformational change which
H-5 which is evident in 8a . The observation of a weak
brings the diphenylmethyl phosphinate ester into the
unresolved coupling between H-3′ and H-5′ may reflect
equatorial configuration.
some population of a boat conformation; further evi-
dence for conformational mobility in both 8a and 8b is
seen in the observation of weak NOE enhancements
Syntheses of phosphinothioic esters from phosphinic
acids have generally been carried out via the interme-
diacy of phosphinyl chlorides generated by the action
of either thionyl chloride or phosphorus pentachloride
on the phosphinic acid.15 These classical methods would
be unsuitable in the present case due to the concomitant
formation of HCl which would rapidly decompose 8.
Several thioesterification methods were tested on the
dibenzylphosphinic acid 12 which served as a model
compound. For example, incubation of 12 with thionyl
chloride or phosphorus pentachloride, in the presence
of triethylamine to scavenge HCl, followed by reaction
with R-phenyl benzenemethanethiol failed to yield any
between H-2 and H-3′. The majority of observed
enhancements are, however, entirely consistent with the
given conformation for each isomer.
The combined yield of 8a and 8b was dependent upon
the solvent employed in the alkylation. Use of DMF as
solvent resulted in a 76% yield of 8 (8a :8b, 3:2) whereas
ethanol led to a marked reduction to a 44% combined
yield. The solvent dependence was investigated further
by subjecting 8a to incubation in ethanol at reflux.
After 1 h of reflux, 8a had been completely consumed
and diphenylmethylethyl ether and the phosphinic acid

Synthesis of an Inhibitor of Mammalian DHOase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4553
F igu r e 2. Part of a NOESY spectrum obtained for the bis(diphenylmethyl) ester 8a . NOE cross-peaks are shown in darker
shading compared with the diagonal and a cross-peak due to exchange between the NH proton and H2O contained in the solvent.
For details, see the Experimental Section.
the relative intensities of weaker NOE cross-peaks were
even further attenuated in the thio compounds.
Removal of the diphenylmethyl protecting groups
from 11 was readily achieved employing trifluoroacetic
acid in the presence of anisole as cation scavenger. In
F igu r e 3. Proposed reaction mechanism for the ethanol-
induced decomposition of 8a .
this manner, the phosphinothioic acids 5a and 5b were
prepared in high yield from 11a and 11b, respectively.
En zym ic Eva lu a tion
The hamster dihydroorotase domain was overex-
pressed in the Escherichia coli strain SØ1263/pCW25
and purified as previously described.6 Compounds 5a
and 5b were tested for their ability to inhibit the
catalytic activity of hamster DHOase. Dixon plots for
inhibition of DHOase by 5a and 5b (Figure 4) showed
that both isomers were potent inhibitors of the enzyme
with inhibition constants (Ki values) of 2.9 ( 0.6 and
3.1 ( 0.3 µM, respectively, at pH 7.4 for hydrolysis of
L-dihydroorotate to N-carbamyl-L-aspartate. The in-
hibitory activities of 5a and 5b were comparable to that
of the parent phosphinic acid 4 (Ki ) 4.0 ( 1.0 µM,
Figure 4). To investigate whether DHOase was rapidly
converting 5 into 4, thereby giving rise to the similar
Ki values, the isomers 5a and 5b were incubated in
phosphate-buffered D2O (pH 7.2) at 38 °C with DHOase
phosphinothioate. Ultimately, diethylcyanophospho-
nate was found to be an efficient coupling agent for the
synthesis of hindered phosphinothioic esters from 12.
In this manner, the diphenylmethylthio ester 13 was
prepared in 56% yield from the reaction of 12 and
R-phenyl benzenemethanethiol in the presence of tri-
ethylamine. Extension of this reaction to 9 resulted in
the formation of the diastereomeric esters 11a ,b in 33%
and 31% yields, respectively, after purification by selec-
tive crystallization. Attempted purification of 11 via
column chromatography on silica or alumina (acidic,
basic, or neutral) led to virtually complete decomposition
(90-100%) of the individual isomers. Interestingly,
neither 11a nor 11b underwent extensive decomposition
in boiling ethanol. Presumably this is a consequence
of the increased P-S bond length in 11 compared to the
corresponding P-O bond length in 8 which does not
allow a favorable transition-state geometry to be at-
tained.
1
and the reaction was monitored by H NMR over a 15
min period corresponding to the time period of the
assay. No turnover of 5 to 4 was observed. In fact 5a
and 5b were stable under the above conditions over a
period of several weeks with no hydrolysis observable
1
over this time period by H NMR.
In conclusion, the phosphinothioic acids 5a and 5b
have been synthesized and were tested as inhibitors of
mammalian DHOase in vitro. Although both isomers
were potent inhibitors of DHOase, replacement of a
phosphinc acid with a phosphinothioic acid offered
marginal enhancement of binding capability.
The configurations of 11a and 11b were determined
by analysis of their NMR spectra. The J coupling
patterns and NOE enhancements for the respective
isomers (Table 1) are entirely analogous to those
observed for 8a and 8b, with the notable exception that

4554 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Manthey et al.
Data for 8a : mp 175-176 °C (ethyl acetate/hexane). IR:
698, 745, 978, 1006, 1187, 1253, 1672, 1738, 3192 cm-1. Anal.
(C31H28NO5P) C, H, N.
Data for 8b: mp 159-160 °C (ethyl acetate/hexane); IR:
698, 743, 990, 1208, 1674, 1742, 3207 cm-1. Anal. (C31H28
NO5P) C, H, N.
-
Diph en ylm eth yl 4-Hydr oxy-6-oxo-1,4-azaph osph in an e-
2-ca r boxyla te 4-Oxid e, 9. A mixture of either 8a or 8b (200
mg, 0.38 mmol) in ethanol (3-5 mL) was refluxed for 0.5 h.
Solvent was removed under vacuum, and the oily residue was
redissolved in ethyl acetate (5 mL) and treated dropwise with
stirring with ether to precipitate a white oily product. Solvent
was decanted and the residue washed with ether to afford 9
(126 mg, 92%). After isolation as such, 9 was used directly
for the preparation of 10; analytically pure 9 was obtained by
recrystallization from chloroform, mp 141-142 °C. 1H NMR
(ref CD2HSOCD3 ) δ 2.50): δ 8.29 (s, 1, OH), 7.92 (d, 1, NH,
J ) 4.3, 1.4 Hz), 7.27-7.46 (m, 10, ArH), 6.82 (s, 1, CH), 4.54
(dddd, 1, CH, J ) 4.3, 7.4, 7.5, 21.9 Hz), 2.71 (ddd, 1, CH2, J
) 1.2, 16.2, 17.0 Hz), 2.60 (dd, 1, CH2, J ) 16.2, 16.5 Hz), 2.33
(dddd, 1, CH2, J ) 1.2, 7.4, 15.0, 15.4 Hz), 2.22 (ddd, 1, CH2,
J ) 7.5, 14.8, 15.4 Hz). IR: 699, 758, 976, 1120, 1233, 1683,
1734, 3203 cm-1. Anal. (C18H18NO5P) C, H, N.
The ethyl acetate/ether supernatant contained diphenyl-
methylethyl ether which could be purified via column chro-
matography eluting with hexane/EtOAc (95:5) to afford 49 mg,
61% of the ether as a thick oil. 1H NMR: δ 7.15-7.35 (m, 10,
ArH), 6.88 (s, 1, CH), 3.52 (q, 2, CH2, J ) 7.3 Hz), 1.25 (t, 3,
CH3, J ) 7.3 Hz).
F igu r e 4. Dixon plots for inhibition of dihydroorotase by 4
(-9-), 5a (sbs), and 5b (-2-). The DHOase activity was
measured at fixed dihydroorotate concentration (20 µM) and
variable inhibitor concentrations (0-60 µM) as described in
Experimental Section. The data were fitted to eq 1. The initial
velocities are in units of µmol of N-carbamyl-L-aspartate/min/
mg of protein.
(+)-cis- a n d tr a n s-Dip h en ylm eth yl 4-Dip h en ylm eth -
ylm er capto-6-oxo-1,4-azaph osph in an e-2-car boxylate 4-Ox-
id e, 11a ,b. To a suspension of 9 (200 mg, 0.56 mmol) in
dichloromethane (5 mL) under an atmosphere of nitrogen were
added triethylamine (62 mg, 0.62 mmol) and R-phenyl ben-
zenemethanethiol17 (116 mg, 0.58 mmol) followed by diethyl-
cyanophosphonate (95 mg, 0.58 mmol). The mixture was
stirred at room temperature for 24 h, diluted with dichlo-
romethane (20 mL), and washed with water (2 × 20 mL). The
organic phase was dried (sodium sulfate) and solvent removed
under vacuum to afford a light yellow semisolid. This was
swirled with ethyl acetate (10 mL) and allowed to stand at
room temperature for 4 h. The precipitate was collected by
filtration to afford essentially pure 11b (93 mg, 31%). The
filtrate was treated with hexane (10 mL) and left at 0 °C
overnight. The precipitated material was collected and crys-
tallized from ethyl acetate/diethyl ether to afford 11a (99 mg,
33%).
Exp er im en ta l Section
Ch em istr y. Melting points were determined thermoelec-
trically on a Reichert hot stage apparatus and are uncorrected.
Infrared spectra were recorded on a Perkin-Elmer infrared
spectrophotometer model 783 as mulls in Nujol and are
reported in reciprocal centimeters (cm-1). The 1H NMR
spectra were acquired on either a Bruker AC-200 or an AMX-
600 spectrometer operating at 200 and 600 MHz, respectively.
The spectra were measured in CDCl3 unless otherwise stated.
Each signal is described in terms of chemical shift as parts
per million (ppm) relative to tetramethylsilane as the internal
standard (δ 0.00). Signals are described as s (singlet), d
(doublet), t (triplet), q (quartet), and m (multiplet). NOESY
spectra were acquired with application of a weak pulsed field
gradient during the mixing time of 1.2 s and processed with 5
Hz Gaussian enhancement in each dimension. Microanalyses
(C, H, N) were performed by the Australian National Univer-
sity Analytical Services, Canberra.
Reactions were followed by TLC on Merck F254 silica gel
plates. Merck silica gel (70-230 mesh) was used for column
chromatography. All solvents and reagents were obtained
from commercial sources and purified before use as required.
(()-4-Hyd r oxy-6-oxo-1,4-a za p h osp h in a n e-2-ca r boxyl-
ic Acid 4-Oxid e, 4. 3-[(Carboxymethyl)hydroxyphosphoryl]-
alanine 6 (10 g, 48.0 mmol) and 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (16 g, 83.5 mmol) were
dissolved in water (500 mL), and the pH was adjusted to 5.6
with 1 M NaOH. The mixture was stirred at 50 °C for 24 h
and then applied to a cation-exchange resin (AG 50W-X8) and
eluted with water. The fractions having a pH between 0.5 and
2.0 were combined and evaporated to a small volume to afford
4 (6.04 g, 66%) as a white solid, mp 214-216 °C (lit.10 mp 213
°C).
Data for 11a : mp 174-176 °C. IR: 1159, 1707, 1755, 3246
cm-1. Anal. (C31H28NO4PS) C, H, N.
Data for 11b: mp 204-205 °C (chloroform/ethyl acetate).
IR: 1165, 1682, 1750, 3223 cm-1
H, N.
. Anal. (C31H28NO4PS) C,
(+)-cis- a n d tr a n s-4-Mer ca p to-6-oxo-1,4-a za p h osp h i-
n a n e-2-ca r boxyla te 4-Oxid e, 5a ,b. To a solution of 11a or
11b (100 mg, 0.18 mmol) in dichloromethane (1 mL) was added
anisole (100 µL) followed by trifluoroacetic acid (2 mL). The
mixture was stirred at room temperature for 2 h, solvent was
removed under vacuum, and the residue was treated with
ether (3 mL). The small amount of insoluble material was
removed by filtration and the filtrate treated with hexane until
cloudiness ensued. After the mixture stood at room temper-
ature overnight, the resulting crystals of 5a or 5b respectively
were collected and washed with a little diethyl ether/hexane
(1:1).
Data for 5a : yield 32 mg (81%); mp 173-176 °C. 1H NMR
(DMSO-d6): δ 7.68 (dd, 1, NH, J ) 1.5, 3.4 Hz), 4.36 (dddd, 1,
CH, J ) 3.4, 5.6, 8.6, 19.6 Hz), 3.05 (dd, 1, CH, J ) 16.1, 16.7
Hz), 3.04 (dd, 1, CH, J ) 12.9, 16.1 Hz), 2.56 (ddd, 1, CH, J )
8.6, 14.7, 15 Hz), 2.49 (ddd, 1, CH, J ) 5.6, 11, 14.7 Hz). IR:
963, 1264, 1651, 1724, 3253 cm-1. Anal. (C5H8NO4PS) C, H,
N.
Data for 5b: yield 28 mg (72%); mp 169-172 °C. 1H NMR
(DMSO-d6): δ 7.76 (d, 1, NH, J ) 3.8 Hz), 4.34 (dddd, 1, CH,
J ) 3.8, 5.9, 8.0, 19.5 Hz), 3.02 (ddd, 1, CH, J ) 1.6, 14.8, 16.2
(()-cis- an d tr a n s-Diph en ylm eth yl 4-Diph en ylm eth oxy-
6-oxo-1,4-a za p h osp h in a n e-2-ca r boxyla te 4-Oxid e, 8a ,b.
To a solution of 4 (300 mg, 1.55 mmol) in dry DMF (10 mL)
was added a solution of diphenyldiazomethane16 in ether until
the distinctive purple/red color of diphenyldiazomethane per-
sisted for 1 h. Solvent was removed under vacuum and the
residue purified via column chromatography, eluting with
ethyl acetate/hexane (1:1 to 2:1) to afford 8a (370 mg, 45%)
followed by 8b (248 mg, 31%) as two well-resolved diastereo-
isomers.

Synthesis of an Inhibitor of Mammalian DHOase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4555
Hz), 2.96 (dd, 1, CH, J ) 11.2, 16.2 Hz), 2.65 (dddd, 1, CH, J
) 1.6, 5.9, 14.9, 17.0 Hz), 2.41 (ddd, 1, CH, J ) 7.8, 8.0, 14.9
Hz). IR: 925, 1243, 1613, 1724, 3224 cm-1. Anal. (C5H8NO4-
PS) C, H, N.
inhibitor concentration, Km is the Michaelis constant for DHO
(5.02 µM), and Ki is the inhibition constant.
VS
v )
(1)
Dip h en ylm eth yl Diben zylp h osp h in a te, 10. To a sus-
pension of 1318 (500 mg, 2.03 mmol) in benzene (20 mL) at 4
°C under an atmosphere of nitrogen was added thionyl chloride
(254 mg, 2.13 mmol), and the mixture stirred at room tem-
perature for 2 h. Solvent was removed under vacuum with
the exclusion of moisture. The white solid residue was
dissolved in dichloromethane (20 mL) and treated with diphen-
ylmethanol (392 mg, 2.13 mmol) and triethylamine (226 mg,
2.24 mmol). The solution was stirred at room temperature
overnight, washed with 5% HCl (20 mL), and then brine (20
mL). The organic phase was dried (magnesium sulfate) and
solvent removed under vacuum to afford a white solid which
was recrystallized from ethyl acetate/hexane yielding 519 mg
(62%) of 10, mp 145-146 °C. 1H NMR: δ 7.10-7.40 (m, 20,
ArH), 6.48 (d, 1, CH, J ) 9.3 Hz), 3.00 (d, 4, CH2, J ) 16.2
Hz); IR: 1208, 987, 695 cm-1. Anal. (C27H25NO2P) C, H, N.
Dip h en ylm eth yl Diben zylp h osp h in oth ioa te, 13. To a
suspension of 12 (200 mg, 0.81 mmol) in dichloromethane (5
mL) under an atmosphere of nitrogen were added triethyl-
amine (86 mg, 0.85 mmol) and R-phenyl benzenemethanethiol
(166 mg, 0.83 mmol) followed by diethylcyanophosphonate (136
mg, 0.81 mmol). The mixture was stirred at room temperature
for 24 h, diluted with dichloromethane (20 mL), and washed
with water (2 × 20 mL). The organic phase was dried (sodium
sulfate) and solvent removed under vacuum to afford crude
13 which was purified via column chromatography (ethyl
acetate/hexane, 1:4) to afford 13 (195 mg, 56%) as fine needles,
mp 154-156 °C (ethyl acetate/hexane). 1H NMR: δ 7.10-7.40
(m, 20, ArH), 5.77 (d, 1, CH, J ) 9.8 Hz), 3.05 (d, 4, CH2, J )
14.6 Hz). IR: 1495, 1204, 1195, 697 cm-1. Anal. (C27H25OPS)
C, H.
Deter m in a tion of In h ibition Con sta n ts. The hamster
dihydroorotase domain was overexpressed in E. coli strain
SØ1263/pCW25 and purified as previously described.6 The
pure recombinant DHOase was stored in 20 mM K.Hepes (pH
7.3), 0.1 mM ZnCl2, 1 mM DTT, and 30% (v/v) glycerol at -80
°C and had a specific activity for the reverse reaction (L-
dihydroorotate f N-carbamyl-L-aspartate) of 2.22 µmol N-
carbamyl-L-aspartate formed/min/mg protein.
The inhibition constants of 4 and 5 were determined using
10 concentrations of inhibitor ranging from 0 to 60 µM. The
reaction mixture contained 50 mM K.Hepes (pH 7.4), 5% (v/v)
glycerol, L-[2-14C]DHO (20 µM, 54 Ci/mol), and inhibitor (0-
60 µM) in a total volume of 25 µL.6 The reaction mixture was
preincubated at 37 °C for 5 min, the reaction was then initiated
by addition of DHOase (2 µL, 1.0 ng protein), and the enzymic
activity was assayed (DHO f CA-asp) as previously de-
scribed.6,9 The pure DHOase was diluted in enzyme buffer
[20 mM K.Hepes (pH 7.3), 10% (v/v) glycerol, and 1 mM DTT]
to achieve less than 10% conversion of DHO to CA-asp in 15
min. Duplicate determinations were made for each data point.
Initial reaction velocities were determined by linear regression
from three samples taken at 5 min intervals. Ki values with
associated standard errors were determined by nonlinear
regression to the velocity equation describing competitive
inhibition (eq 1) using the program DNRP53,19 where v is the
measured initial rate, Vmax is the maximal rate (2.22 µmol/
min/mg protein), S is the DHO concentration (20 µM), I is the
Km(1 + I/Ki) + S
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