Stereoelectronic effect on one-electron reductive release of 5-Fluorouracil from 5-fluoro-1-(2'-oxocycloalkyl)uracils as a new class of radiation-activated antitumor prodrugs

Article abstract of DOI:10.1021/jo000245u

A series of 5-fluoro-1-(2'-oxocycloalkyl)uracils (3-11) that are potentially novel radiation-activated prodrugs for the radiotherapy of hypoxic tumor cells have been synthesized to evaluate a relationship between the molecular structure and the reactivity of one-electron reductive release of antitumor 5-fluorouracil (1) in anoxic aqueous solution. All the compounds 3-11 bearing the 2'-oxo group were one-electron reduced by hydrated electrons (e(aq)^-) and thereby underwent C(1')-N(1) bond dissociation to release 5-fluorouracil 1 in 47-96% yields upon radiolysis of anoxic aqueous solution, while control compounds (12, 13) without the 2'-oxo substituent had no reactivity toward such a reductive C(1')-N(1) bond dissociation. The decomposition of 2-oxo compounds in the radiolytic one-electron reduction was more enhanced, as the one-electron reduction potential measured by cyclic voltammetry in N,N-dimethylformamide became more positive. The efficiency of 5-fluorouracil release was strongly dependent on the structural flexibility of 2-oxo compounds. X-ray crystallographic studies of representative compounds revealed that the C(1')-N(1) bond possesses normal geometry and bond length in the ground state. MO calculations by the AM1 method demonstrated that the LUMO is primarily localized at the π* orbital of C(5)-C(6) double bond of the 5-fluorouracil moiety, and that the LUMO + 1 is delocalized between the π* orbital of 2'-oxo substituent and the σ* orbital of adjacent C(1')-N(1) bond. The one-electron reductive release of 5-fluorouracil 1 in anoxic aqueous solution was presumed to occur from the LUMO + 1 of radical anion intermediates possessing a partial mixing of the antibonding C(2')=O π* and C(1')-N(1) σ(*) MO's, that may be facilitated by a dynamic conformational change to achieve higher degree of (π + σ) MO mixing.

Full text of DOI:10.1021/jo000245u

J . Org. Chem. 2000, 65, 4641-4647
4641
Ster eoelectr on ic Effect on On e-Electr on Red u ctive Relea se of
-F lu or ou r a cil fr om 5-F lu or o-1-(2′-oxocycloa lk yl)u r a cils a s a New
Cla ss of Ra d ia tion -Activa ted An titu m or P r od r u gs
5
Mayuko Mori, Hiroshi Hatta, and Sei-ichi Nishimoto*
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University,
Sakyo-ku, Kyoto 606-8501, J apan
nishimot@scl.kyoto-u.ac.jp
Received February 22, 2000
A series of 5-fluoro-1-(2′-oxocycloalkyl)uracils (3-11) that are potentially novel radiation-activated
prodrugs for the radiotherapy of hypoxic tumor cells have been synthesized to evaluate a relationship
between the molecular structure and the reactivity of one-electron reductive release of antitumor
5
-fluorouracil (1) in anoxic aqueous solution. All the compounds 3-11 bearing the 2′-oxo group
-
were one-electron reduced by hydrated electrons (eaq ) and thereby underwent C(1′)-N(1) bond
dissociation to release 5-fluorouracil 1 in 47-96% yields upon radiolysis of anoxic aqueous solution,
while control compounds (12, 13) without the 2′-oxo substituent had no reactivity toward such a
reductive C(1′)-N(1) bond dissociation. The decomposition of 2-oxo compounds in the radiolytic
one-electron reduction was more enhanced, as the one-electron reduction potential measured by
cyclic voltammetry in N,N-dimethylformamide became more positive. The efficiency of 5-fluorouracil
release was strongly dependent on the structural flexibility of 2-oxo compounds. X-ray crystal-
lographic studies of representative compounds revealed that the C(1′)-N(1) bond possesses normal
geometry and bond length in the ground state. MO calculations by the AM1 method demonstrated
that the LUMO is primarily localized at the π* orbital of C(5)-C(6) double bond of the 5-fluorouracil
moiety, and that the LUMO + 1 is delocalized between the π* orbital of 2′-oxo substituent and the
σ* orbital of adjacent C(1′)-N(1) bond. The one-electron reductive release of 5-fluorouracil 1 in
anoxic aqueous solution was presumed to occur from the LUMO + 1 of radical anion intermediates
possessing a partial mixing of the antibonding C(2′)dO π* and C(1′)-N(1) σ* MO’s, that may be
facilitated by a dynamic conformational change to achieve higher degree of (π* + σ*) MO mixing.
In tr od u ction
Sch em e 1
We recently reported electrochemical synthesis of a
novel N(1)-C(5′)-linked dimer, 1-(5′-fluoro-6′-hydroxy-
5
′,6′-dihydrouracil-5′-yl)-5-fluorouracil (2), by anodic one-
electron oxidation of 5-fluorouracil (1) in Ar-purged
aqueous solution containing NaCl as a supporting elec-
1
trolyte (Scheme 1). The dimer 2 was also identified to
show a formally reverse reactivity, undergoing one-
electron reduction to release 5-fluorouracil 1 upon radi-
1
olysis in anoxic aqueous solution (Scheme 1). Among the
2
water radicals generated in the radiolysis, the reducing
-
species of hydrated electrons (eaq ) are responsible for the
activated prodrug of exhibiting selective toxicity toward
hypoxic tumor cells under radiation treatment. Actually,
1
4
reductive release of 1. Thus, the yield of 1 is significantly
1
diminished in the radiolysis of aerobic aqueous solution,
as demonstrated by a tumor growth-delay assay, 2 had
no antitumor effect in contrast to 1, but could potentiate
the radiotherapy to inhibit the tumor growth to consider-
able extent.1
In attempting the molecular design by reference to the
prototype compound 2 for a new class of radiation-
activated prodrugs that release more efficiently antitu-
mor 5-fluorouracil 1 under anoxic or hypoxic irradiation,
our attention has been directed to the presence of an
electron-accepting carbonyl group in the R position with
respect to the leaving 5-fluorouracil-1-yl group in the
compound 2. This aspect is associated with the previous
-
since the hydrated electrons eaq are readily scavenged
by dissolved oxygen to form less-active superoxide radical
-
. 2
ions (O
2
). In view of a well-specified antitumor action
3
of 1, the characteristic one-electron reduction reactivity
of releasing 1 may display a potential that the N(1)-
C(5′)-linked 5-fluorouracil dimer 2 can be a radiation-
*
351.
Corresponding author. Tel: +81(75)753-5688, Fax: +81(75)753-
3
(
1) Nishimoto, S.; Hatta, H.; Ueshima, H.; Kagiya, T. J . Med. Chem.
1
992, 35, 2711-2712.
(2) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor
&
Francis: London, 1987.
3) (a)Wilkinson, D. S.; Tlsty, T. D.; Hanas, R. J . Cancer Res. 1975,
(
3
1
6
5, 3014-3020. (b) Glazer, R. I.; Peale, A. L. Mol. Pharmacol. 1979,
6, 270-277. (c) Pinedo, H. M.; Peters, G. F. J . J . Clin. Oncol. 1988,
, 1653-1664.
(4) (a) Vaupel, P.; Kallinowski, F.; Okunieff, P. Cancer Res. 1989,
49, 6449-6465. (b) Denny, W. A. Cancer Chemotherapeutic Agents;
American Chemical Society: Washington, DC, 1995; pp 483-497.
1
0.1021/jo000245u CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/06/2000

4
642 J . Org. Chem., Vol. 65, No. 15, 2000
Mori et al.
Ch a r t 1
Ta ble 1. X-Ra d iolysis of 2-oxo Com p ou n d s 3-11 (1 m M)
in P h osp h a te Bu ffer Solu tion Con ta in in g
2
-Meth yl-2-p r op a n ol (1 M)
7
-1
G × 10 , mol J
substrate -substrate 5-fluorouracil % selectivitya Ered1/2, Vb
3
4
5
6
7
8
9
10
1
2.6
2.3
2.2
2.0
2.5
2.5
2.1
2.4
2.7
1.6
1.1
2.1
1.9
1.5
1.5
1.3
1.3
1.4
59
47
96
93
58
59
61
54
53
-0.48
-0.61
-0.63
-0.74
-0.55
-0.56
-0.72
-0.54
-0.71
reports by Rossi and co-workers5,6 that bridgehead chlo-
1
rides of 2-oxo-substituted bicyclic compounds are replaced
a
Defined as the ratio G(5FU)/G(-substrate). b One-electron
-
with diphenyl phosphide ions (Ph
2
P ) by a radical nu-
+
7
reduction potential vs Ag/Ag in DMF as measured by cyclic
voltammetry.
cleophilic substitution (SRN1) mechanism under photoir-
radiation in liquid ammonia, while analogues without the
2
-oxo substituent are unreactive under similar conditions.
5
2
-fluoro-2,4-bis(trimethylsiloxy)pyrimidine with 1-chloro-
Most commonly the SRN1 mechanism involves the forma-
tion of a radical anion intermediate of the substrates in
the initiation step.7 It has been postulated that the
antibonding π* MO of 2-oxo substituent as the LUMO of
substrates accepts an electron to form the CdO π* radical
anion, then transferring the electron intramolecularly to
the antibonding C-Cl σ* MO which results in an
-oxo compounds (for 5, 7, and 8) or 1-bromo-2-oxo
9
compounds (for 3, 4, 6, 9, and 10). trans-5-Fluoro-1-(5′-
tert-butyl-2′-oxocyclohexyl)uracil 11 was synthesized in
a similar manner, but was obtained only from cis-2-
bromo-4-tert-butylcyclohexanone. Control compounds 12
and 13 without the 2′-oxo group were prepared by the
reaction of 5-fluoro-2,4-bis(trimethylsiloxy)pyrimidine
electron-capturing dissociation of C-Cl bond to form the
1
0
carbon-centered radical and chloride ion.5,6 There is
with 1-chloropropane and 1-adamanthyl trifrate, re-
spectively.
another possibility that a mixing of the CdO π* MO and
the C-Cl σ* MO may be predominant and thereby the
Reduction potentials (Ered1/2 vs Ag/Ag ) of the 2-oxo
compounds 3-11 were measured by means of cyclic
voltammetry in Ar-purged N,N-dimethylformamide (DMF)
solution containing 0.1 M tetra-n-butylammonium bro-
mide (TBAB) as a supporting electrolyte. The cyclic
voltammograms were recorded upon scanning the work-
ing electrode potentials between -1.5 and 0 V vs Ag/Ag
at a rate of 500 mV s . In this potential range all the
2
+
radical anion intermediate favors spontaneous dissocia-
tion of the C-Cl bond.8
To get structural insight into the mechanism by which
radiation-activated prodrugs analogous to the N(1)-C(5′)-
linked dimer 2 undergo one-electron reduction to release
+
5
-fluorouracil 1, we synthesized 2-oxopropane (3) and a
series of 2-oxocycloalkanes (4-11) with a common leaving
-fluorouracil-1-yl group and evaluated their one-electron
-
1
-oxo compounds, except for 5 and 6, displayed a single
5
reversible redox process assigned to a redox couple of
each 2-oxo compound and its one-electron reduced anion
radical form, from which the corresponding one-electron
reduction potentials were evaluated as listed in Table 1.
In contrast to these 2-oxo compounds, 5-fluoro-1-(2′-
oxocyclopentyl)uracil 5 and 5-fluoro-1-(2′-oxocyclohexyl)-
uracil 6 showed irreversible cathodic reduction peak
reduction reactivity in comparison with those of control
compounds (12 and 13) bearing no 2′-oxo substituent (see
Chart 1). For better understanding of a possible overlap-
ping or mixing between the C(2′)dO π* and the C(1′)-
N(1) σ* MO’s in the radical anion intermediate, X-ray
crystallographic and computational studies of represen-
tative 2-oxo compounds were also performed. In this
paper, we disclose a definitive function of the 2′-oxo
substituent of 5-fluoro-1-(2′-oxocycloalkyl)uracils in the
radiation-activated reductive release of 5-fluorouracil 1
in anoxic aqueous solution, that may occur from the
LUMO + 1 of radical anion intermediates with a partial
mixing of the antibonding C(2′)dO π* and C(1′)-N(1) σ*
MO’s. The efficiency of 5-fluorouracil release is also
shown to be enhanced by a dynamic conformational
change to achieve higher degree of (π* + σ*) MO mixing.
1
/2
potentials (Ep
c
) at -0.74 V (Ered ) -0.63 V) and -0.86
1
/2
+
V (Ered ) -0.74 V) vs Ag/Ag , respectively (Table 1). A
common behavior of all compounds including 5 and 6 was
that an oxidation peak appeared at -0.38 V vs Ag/Ag
+
in the reverse anodic scan. This characteristic peak is
most likely attributable to the oxidation of N(1)-depro-
-
tonated 5-fluorouracil anion (1(-H) ) that could be a
common product derived from the one-electron reductive
C(1′)-N(1) bond dissociation of 2-oxo compounds 3-11
in an aprotic solvent of DMF. According to the above
electrochemical characterization, it is plausible that one-
electron reduction of the 2-oxo compounds 3-11 may
Resu lts a n d Discu ssion
Syn th esis a n d Red u ction Rea ctivity. 5-Fluoro-1-
-
release N(1)-deprotonated 5-fluorouracil anion 1(-H) ,
(2′-oxopropyl)uracil 3 and 5-fluoro-1-(2′-oxocycloalkyl)-
while producing rather stable radical anion intermediates
at least in aprotic solvents. In the latter regard, a
separate pulse radiolysis study11 demonstrated that the
N(1)-C(5′)-linked dimer 2 is one-electron reduced by
hydrated electrons in aqueous solution to form a consid-
uracils 4-10 were easily prepared by the reaction of
(
5) Santiago, A. N.; Takeuchi, K.; Ohga, Y.; Nishida, M.; Rossi, R.
A. J . Org. Chem. 1991, 56, 1581-1584.
6) Lukach, A. E.; Morris, D. G.; Santiago, A. N.; Rossi, R. A. J . Org.
Chem. 1995, 60, 1000-1004.
7) (a) Bunnet, J . F. Acc Chem Res. 1978, 11, 413-420. (b) Bowman,
W. R. Chem. Soc. Rev. 1988, 17, 283-316.
8) (a) Symons, M. C. R. Pure Appl. Chem. 1981, 53, 223-238. (b)
Andrieux, C. P.; Sav e´ ant, J - M.; Su, K. B. J . Phys. Chem. 1986, 90,
815-3823.
(
(
(9) Akashi, M.; Beppu, K.; Kikuchi, I.; Miyauchi, N. J . Macromol.
Sci., Chem. 1986, A23, 1233-1249.
(10) Takeuchi, K.; Moriyama, T.; Kinoshita, T.; Tachino, H.; Oka-
moto, K. Chem. Lett. 1980, 1395-1398.
(
3

Radiation-Activated Antitumor Prodrugs
J . Org. Chem., Vol. 65, No. 15, 2000 4643
erably stable electron adduct at the 5-fluorouracil moiety
with a lifetime of 8 ms.
Sch em e 2
For characterizing the one-electron reduction reactiv-
ity, X-radiolyses of 2-oxo compounds 3-11 (1 mM) in Ar-
purged phosphate buffer solutions containing excess
amount (1 M) of 2-methyl-2-propanol were carried out
at room temperature. In the radiolysis of a diluted
aqueous solution, water radicals such as oxidizing hy-
•
-
droxyl radicals ( OH), reducing hydrated electrons (eaq ),
•
and reducing hydrogen atoms (H ) are primarily gener-
1
2
•
-7
-1
ated with the G values of G( OH) ) 2.8 × 10 mol J
G(eaq ) ) 2.8 × 10 mol J , and G(H ) ) 0.6 × 10 mol
(reaction 1). Under the present conditions, the OH
,
-
-7
-1
•
-7
-
1
J
radicals are scavenged by excess 2-methyl-2-propanol into
substantially unreactive 2-methyl-2-propanol radicals
•
(
CH
2
(CH
3
)
2
COH) as in reaction 2. Thus, the hydrated
in Ar-purged methanol up to 90% decomposition resulted
in no free 5-fluorouracil 1. For comparison, representative
X-radiolysis of 6 in Ar-purged methanol was performed
to obtain the G values for the decomposition of 6 and the
formation of 1 as 1.2 and 0.78 (65% selectivity), respec-
tively. These results may be rationalized by a mechanism
involving one-electron reductive dissociation of the C(1′)-
N(1) bond of 3-11 into N(1)-deprotonated 5-fluorouracil
-
-
13
electrons eaq (E(nH
2
O/eaq ) ) -2.9 V vs NHE at pH
7
.0) are involved as the reductants for 3-11. Smaller
•
+
•
amount of the hydrogen atoms H (E(H /H ) ) -2.4 V vs
NHE13 at pH 7.0) would make relatively minor contribu-
tion to the reaction of 3-11, although they add to the
double bonds of pyrimidines.14
•
•
-
-
anion 1(-H) and 2-oxocycloalkyl radical (2-oxopropyl
H O f OH + H + e
(1)
2
aq
radical in the case of 3), as illustrated in Scheme 2.
Evidently, 2′-oxo group is an essential part of the radia-
tion-activated antitumor prodrugs that undergo one-
electron reductive C(1′)-N(1) bond dissociation to release
•
•
OH + (CH ) COH f H O + CH (CH ) COH (2)
3
3
2
2
3 2
In the case of water-insoluble compound 13, Ar-purged
5
-fluorouracil 1 in sufficient yield.
methanol solution was similarly X-irradiated. This reac-
tion system involves solvated electrons eMeOH and reduc-
ing radicals CH
concomitant primary radicals such as CH
and CH
ditionally the reducing radicals CH
Analytical HPLC of the X-irradiated solutions by
reference to authentic samples indicated that all the
X-r a y Cr ysta l Str u ctu r e a n d Ca lcu la ted Elec-
-
tr on ic Str u ctu r e. X-ray studies of single crystals were
carried out to elucidate the detailed structures of some
representative compounds 3-5 and 7-9. As illustrated
in Figure 1, the X-ray structures revealed that the C(1′)-
N(1) bonds of all compounds determined possess normal
geometry and bond lengths ranging from 1.41 to 1.49 Å.
The distortion angles of these 2-oxo compounds between
the C(1′)-N(1) bond and the C(2′)dO bond were -3.5°
•
2
OH for the reduction of 13, since the
•
•
•
3
O , H , OH,
•
3
react rapidly with methanol to produce ad-
•
15
2
OH.
2
-oxo compounds 3-11 release 5-fluorouracil 1 as a major
-
product upon reduction by hydrated electrons eaq . A few
fragments with higher polarity were also produced in
each reaction, as eluted faster in the HPLC. These
products may be derived from degradation such as
pyrimidine ring opening reaction, although they could not
be identified. The residual 2-oxoalkane moiety resulted
from the 5-fluorouracil release is also a possible product,
but could not be detected by NMR analysis of each
reaction mixture obtained after evaporation. Table 1 lists
the G values for the decomposition of 3-11 and the
formation of 5-fluorouracil 1 in the X-radiolytic reduction.
In a separate experiment we also confirmed that 5-fluoro-
(
(
3), -4.8° (4), 35.6° (5), -28.0° (7), 135.9° (8), and 3.8°
9), respectively. Thus, comparing these compounds with
each other, there would be no great difference in the
extent of overlapping between the C(2′)dO π MO and the
C(1′)-N(1) σ MO in the crystal. In fluid solution, how-
ever, conformational changes will be allowed for the 2-oxo
compounds under the influence of their molecular struc-
tures, causing various extents of overlapping of hypo-
thetical C(2′)dO π* radical anion with C(1′)-N(1) σ* MO.
Such an accessible conformational change in fluid solu-
tion may account for the observation that both the one-
electron reductive decomposition and the release of
1
-propyluracil 12, as a non-2-oxo analogue of 3, did not
5
-fluorouracil 1 varied depending on the molecular
release 1 upon X-irradiation up to 80% decomposition
under similar conditions. Furthermore, X-radiolysis of 13
structures of 2-oxo compounds (Table 1).
For better understanding of the electronic structural
characteristics of 2′-oxo substituent that is crucial for
promoting the one-electron reductive release of 5-fluo-
(11) (a) Mori, M.; Teshima, S.; Hatta, H.; Fujita, S.; Taniguchi, R.;
Nishimoto, S. Unpublished data. In the pulse radiolysis of 2 in Ar-
purged aqueous solution containing 2-methyl-2-propanol using pulsed
electron beam with a half-width duration 1 µs, we observed a transient
absorption spectrum with a maximum wavelength at 340 nm which
was almost identical with that of 5-fluorouracil radical anion (see also
ref 11b). (b) Rivera, E.; Schuler, R. H. J . Phys. Chem. 1983, 87, 3966-
1
6
rouracil 1, MO calculations by the AM1 method were
performed for the 2-oxo compounds 3-11. Figure 2 shows
representatively the LUMO and LUMO + 1 of compound
6. Each compound has similar LUMO that is primarily
localized at the π* orbital of C(5)-C(6) double bond of
the 5-fluorouracil moiety, as in Figure 2, therein accept-
ing hydrated electron to form the most stable radical
3
971.
12) The number of molecules produced or changed per 1 J of
radiation energy absorbed by the reaction system (see also ref 2).
(
(
13) Wardman, P. J . Phys. Chem. Ref. Data 1989, 18, 1637-1755.
(14) (a) Neta, P.; Schuler, R. H. Radiat. Res. 1971, 47, 612-627. (b)
Das, S.; Deeble, D. J . Z. Naturforsch. 1985, 40c, 292-294.
(15) Bensasson, R. V.; Land, E. J .; Truscott, T. G. Excited States
(16) Dewar, M. J . S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J . J . P.
J . Am. Chem. Soc. 1985, 107, 3902-3909. The AM1 calculations were
performed using the CAChe software.
and Free Radicals in Biology and Medicine; Oxford University Press:
Oxford, 1993.

4
644 J . Org. Chem., Vol. 65, No. 15, 2000
Mori et al.
F igu r e 1. ORTEP drawings of 3, 4, 5, 7, 8, and 9.
for R-halo or R-amino ketones, causing the UV spectral
perturbations and dominating the photochemical reactiv-
ity in the excited state.18 Since the non-2-oxo compounds
1
2 and 13 cannot release 5-fluorouracil 1 upon one-
electron reduction, the mixing of C(2′)dO π* orbital as a
possible electron-accepting site of secondary importance
with the C(1′)-N(1) σ* orbital at relatively higher energy
level may be responsible for the promoted one-electron
reductive C(1′)-N(1) bond dissociation of 2-oxo com-
pounds 3-11.
(17) A computational study on the electronic nature of radical anions
generated from various N1-substituted 5-fluorouracil derivatives and
their preferred fragmentation pathway has been recently reported
(Borosky, G. L.; Nishimoto, S.; Pierini, A. B. J . Mol. Struct. THEOCHEM
2000, 499, 151-160). This indicated that while the most stable radical
anion is of π-type for general N1-substituents, increase in the electron
affinity of the N1-substituents can enhance the spin density at the
C(1′)-N(1) bond favoring the formation of σ C(1′)-N(1) isomer of
radical anion intermediates. According to this result, it seems plausible
that the electron-adduct of 5-fluorouracil derivatives 3-11 bearing 2′-
oxoalkyl substituents at N(1) could have a (π* + σ*) SOMO energy
level closer to or even lower than the π* level.
F igu r e 2. LUMO and LUMO + 1 of the 2-oxo compound 6 as
calculated by AM1 method. The LUMO + 1 is delocalized by
mixing of the π* orbital of 2′-oxo substituent with the σ* orbital
of adjacent C(1′)-N(1) bond.
anion.17 In contrast, the LUMO + 1 is delocalized
between the π* orbital of 2′-oxo substituent and the σ*
orbital of adjacent C(1′)-N(1) bond (see Figure 2),
indicating a (π* + σ*) mixing of the antibonding orbitals.
Such a mixing between π* and σ* orbitals is well-known
(
18) (a) Allinger, N. L.; Tai, J . C.; Miller, M. A. J . Am. Chem. Soc.
966, 88, 4495-4499. (b) Levin, C. C.; Hoffmann, R.; Hehre, W. J .;
Hudec, J . J . Chem. Soc., Perkin Trans. 2 1973, 210-220.
1

Radiation-Activated Antitumor Prodrugs
J . Org. Chem., Vol. 65, No. 15, 2000 4645
an electronic structural feature of the 2-oxo compounds
3
-11 is expected to be evolved as a result of conforma-
tional changes that are allowed to occur more freely than
the 1-chloro-2-oxo-bicyclic compounds with more rigid
molecular structures. Although the 2′-oxo substituent
similarly plays a crucial function in the one-electron
reductive release of 5-fluorouracil 1, the reactivity of
3
1
-11 showed no correlation with the (π* + σ*) LUMO +
. This is not surprising in view of the molecular
structures that the 2-oxo compounds 3-11 studied may
be flexible enough to undergo conformational change
within the lifetimes of the (π* + σ*) LUMO + 1 radical
anion intermediates, distinct from the 1-chloro-2-oxo-
bicyclic compounds. In this context, the axial conformer
of R-chlorocyclohexanone dominates over the equatorial
1
9
conformer in the photolytic C-Cl bond cleavage, the
reactivity being correlated to the degree of mixing
between the C-Cl σ* and CdO π* orbitals in the LUMO
which becomes a maximum at the dihedral angle of Cl-
C-CdO ) 90°.20 It is thus reasonable to presume that
the observed reactivity of one-electron reductive 5-fluo-
rouracil release involves some contribution from a dy-
namic conformational change to achieve higher degree
of MO mixing in the (π* + σ*) LUMO + 1 between the
C(2′)dO π* and C(1′)-N(1) σ* orbitals.
F igu r e 3. Correlation between the G value for decomposition
of substrate (G(-substrate)) and the one-electron reduction
1
/2
+
potential (Ered
vs Ag/Ag ) in DMF.
On e-Electr on Red u ction Mech a n ism for Relea s-
in g 5-F lu or ou r a cil. Figure 3 shows that the reactivity
of all the 2-oxo compounds except 11 for radiolytic one-
electron reduction in phosphate buffer, as measured by
the G value for decomposition, increases as the reduction
potential Ered1 in DMF becomes more positive. Neither
the G value nor the selectivity of 5-fluorouracil release
has such a clear relationship with the Ered1/2 value. As
suggested by cyclic voltammetry and MO calculation, the
/2
Inspection of the data listed in Table 1 suggests strong
influences of not only the overlapping between the
C(2′)dO π* and C(1′)-N(1) σ* orbitals but also the
structural flexibility of 2-oxo compounds on the efficiency
of 5-fluorouracil release in the radiolytic one-electron
reduction. The four-membered ring compound 4 that has
probably the most rigid structure with rather small π*-
σ* MO overlapping showed the lowest selectivity (47%)
of 5-fluorouracil release among the tested 2-oxo com-
pounds. On the other hand, the most flexible acyclic
compound 3 or the relatively large ring compounds 7 and
8 which are unstrained and more flexible than the five-
and six-membered ring compounds 5 and 6 also resulted
in smaller selectivity (58-59%) of 5-fluorouracil release.
Therefore, extreme flexibility is also unfavorable for the
one-electron capturing C(1′)-N(1) bond dissociation,
probably because the π*- σ* MO overlapped state of
larger extent occurring with high frequency would have
too short lifetime to allow intramolecular electron trans-
fer from the C(2′)dO π* radical anion to the antibonding
C(1′)-N(1) σ* orbital. In contrast to these two extremes,
5-fluoro-1-(2′-oxocyclopentyl)uracil 5 and 5-fluoro-1-(2′-
oxocyclohexyl)uracil 6 released 5-fluorouracil 1 in nearly
quantitative yields (96 and 97%) upon radiolytic one-
electron reduction. These 2-oxo compounds may possess
moderately flexible structures with accessible overlap-
ping between the C(2′)dO π* and C(1′)-N(1) σ* orbitals.
The dependence of one-electron reductive 5-fluorouracil
release on the molecular flexibility was examined in fur-
ther details for a family of alkyl-substituted cyclohexa-
none derivatives. As indicated by comparing the reactiv-
ity among the 2-oxo compounds 6, 9-11 (Table 1), the
efficiency of C(1′)-N(1) bond dissociation to give 5-fluo-
rouracil 1 is reduced to more extent with increasing the
steric effect of 5′-alkyl substituent at cyclohexanone ring
on the restriction of conformational change. Diasteroiso-
meric 2-oxo compounds with a tert-butyl-substituent, cis-
1
/2
E
red
value listed in Table 1 is attributable to one-
electron reduction process involving the formation of π*
LUMO radical anion at the C(5)-C(6) double bond of
5
-fluorouracil moiety, which produces a few fragments
with higher polarity by pyrimidine ring opening reaction.
In view of the calculated result that the LUMO + 1
energy levels are higher than the corresponding LUMO
levels only by 0.32 to 0.58 eV, the 5-fluorouracil radical
anion thus formed could intramolecularly transfer elec-
tron from the antibonding π* orbital (LUMO) to the (π*
+
σ*) LUMO + 1 in competition with undergoing the
pyrimidine ring fragmentation, thereby making consider-
able contribution to the 5-fluorouracil release which
accounted for more than 47% of decomposed 3-11
(
Table 1).
Previously, Rossi and co-workers reported5,6 that the
LUMO belonging to the π* orbital of 2-oxo substituent
in 1-chlorobicyclic compounds with rigid molecular struc-
tures decreases the antibonding σ* MO with higher
energy of the C-Cl bond, thereby enhancing the reactiv-
ity of electron-capturing C-Cl bond dissociation. They
also suggested that the reactivity of 2-oxo substituted
1
-chlorobicyclic compounds correlates with the π* MO
order of CdO group but not with the σ* MO order of
C-Cl group, probably due to the most facilitated forma-
tion of CdO π* radical anions. This implies that stabi-
lization energy in the formation of π* radical anion at
2
-oxo substituent may determine the reactivity of C-Cl
bond dissociation, which occurs spontaneously via in-
tramolecular dissociative electron transfer from the
CdO π* without formation of the C-Cl σ* radical anion.
According to our calculations for the 2-oxo compounds
3
-11, there is a partial mixing of the C(2′)dO π* MO
and the relatively high energetic C(1′)-N(1) σ* MO to
generate lower energy (π* + σ*) LUMO + 1, while the
LUMO is the most likely π* MO of the C(5)-C(6) double
bond of leaving 5-fluorouracil moiety (see Figure 2). Such
(
19) Morrison, H.; de Cardenas, L. J . Org. Chem. 1987, 52 2590-
592.
20) Morrison, H.; Singh, T. V.; de Cardenas, L.; Severance, D. J .
Am. Chem. Soc. 1986, 108, 3862-3863.
2
(

4
646 J . Org. Chem., Vol. 65, No. 15, 2000
Mori et al.
Ch a r t 2
dissociation to release 5-fluorouracil 1 by the radiolytic
one-electron reduction of 2-oxo compounds 3-11 in anoxic
aqueous solution. The reactivity of one-electron reductive
releasing 5-fluorouracil 1 was strongly affected by the
molecular flexibility of 2-oxo compounds 3-11. In view
of the X-ray crystal data and the MO calculations, the
5
-fluorouracil release is presumed to occur from the
LUMO + 1, but not LUMO, of radical anion intermedi-
ates with a partial mixing of the antibonding C(2′)dO
π* and C(1′)-N(1) σ* MO’s, that may be facilitated by a
dynamic conformational change to achieve higher degree
of (π* + σ*) MO mixing. The structure-reactivity rela-
tionship reported herein may provide a guiding principle
of developing a family of radiation-activated prodrugs
that release antitumor 5-fluorouracil sufficiently in the
radiotherapy of hypoxic tumor cells. In addition, our
approach would be applicable to molecular design of
similar prodrugs21 consisting of various antitumor agents
with more potent toxicity than 5-fluorouracil.
5
-fluoro-1-(5′-tert-butyl-2′-oxocyclohexyl)uracil 10 and
trans-5-fluoro-1-(5′-tert-butyl-2′-oxocyclohexyl)uracil 11,
which possess the least flexible ring structures are of
particular interest. Since the one-electron reductive
C(1′)-N(1) bond dissociation should favor a conformation
in which dihedral angle between the C(2′)dO bond and
C(1′)-N(1) bond is orthogonal, the cis-isomer 10 in a
more stable conformation with 5-fluorouracil-1-yl and
tert-butyl groups in the equatorial positions needs flip-
ping for the efficient release of 5-fluorouracil 1. This
structural restriction may account for the reactivity that
the cis-isomer 10 had significantly smaller selectivity
Exp er im en ta l Section
Ma ter ia ls. 5-Fluorouracil (1) was used as purchased from
Tokyo Kasei Chemicals. N,N-Dimethylformamide (DMF) was
distilled under reduced pressure and acetonitrile was dried
2 5
over P O followed by distillation. Tetra-n-buthylammonium
bromide (TBAB, Nacalai Tesque) of reagent grade was recrys-
tallized from benzene-hexane and stored in a vacuum desic-
cator prior to use. All the other reagents were of the best
available grades and used as received.
(54%) of the C(1′)-N(1) bond dissociation than the
nonsubstituted cyclohexanone analogue 6 (93%) with less
strain energy for the conformational change. Thus, strain
energy of the 2′-oxocycloalkane moiety seems to be at
least one of the dominant factors in determining the
C(1′)-N(1) bond dissociation reactivity of 5-fluoro-1-(2′-
oxocycloalkyl)uracils. In contrast to the cis-isomer 10
isolated as the sole structure, there existed two distinct
conformers for the trans-isomer 11 arising from bulky
Gen er a l Meth od s. The high-resolution fast atom bombard-
ment mass spectrometry (FAB-HRMS) were performed using
glycerol matrix. Reaction products in the irradiated solution
(5 µL) were analyzed by high-performance liquid chromatog-
raphy (HPLC). Sample solutions were injected onto a reversed
phase column (YMC-Pack ODS-AM, φ 6.0 mm × 150 mm)
containing C18 chemically bonded silica gel (5 µm particle size).
The phosphate buffer solution (10 mM, pH 3.0) containing 5
vol % methanol or the 0.05 vol % trifluoroacetic acid buffer
solution containing 30 vol % acetonitrile was delivered as the
5
-fluorouracil-1-yl and tert-butyl groups, as confirmed by
-1
1
mobile phase at a flow rate of 0.6 mL min at 40 °C. A normal
phase column (5SL Nakarai Tesqu, φ 4.5 mm × 250 mm) was
used for HPLC analysis of substrate 13 and chloroform was
NMR. The H NMR study demonstrated that 11a with
axial 5-fluorouracl-1-yl group and 11b with axial tert-
butyl group are in dynamic equilibrium with a molar
-1
delivered as the mobile phase at a flow rate of 0.6 mL min
ratio of 1.3:1 at room temperature in CDCl
from doublet-doublet peak areas of the C(1′) protons at
.39 and 5.15 ppm, respectively (Chart 2). These con-
3
, as evaluated
at room temperature. The column eluents were monitored by
the UV absorbance at 210 nm.
5
Ra d iolytic Red u ction . Aqueous solutions were prepared
with water ion-exchanged using Corning Mega-Pure System
MP-190 (>16 MΩ cm). Typically, aqueous solutions (2 mL) of
the 2-oxo compounds 3-13 (1 mM) containing excess amount
formers could not be separately isolated. In light of the
overlapping between the C(2′)dO π* MO and the C(1′)-
N(1) σ* MO, the conformer 11a should undergo one-
electron reductive dissociation of the C(1′)-N(1) bond to
release 5-fluorouracil 1, in preference to the counterpart
(
1 M) of 2-methyl-2-propanol were buffered at pH 7.0 ( 0.1
with phosphate buffer (KH PO and Na HPO , 10 mM), purged
2
4
2
4
with Ar for 20 min and then irradiated in a sealed glass
ampule at room temperature with an X-ray source (8.0 Gy
1
1b. It is also noted that there is no substantial difference
in the selectivity of 5-fluorouracil release between the cis
isomer 10 (54%) and trans isomer 11 (53%), while the
latter showed somewhat higher reactivity for radiolytic
decomposition than the former. This observation implies
that the energy barrier for the conformational change
from the less reactive 11b to the more reactive 11a may
be kinetically similar to that of 10 involving dynamic
change from the conformer 10b (isolated as the sole
stable product) to a possible conformer 10a (not detect-
able in this work probably due to much smaller fraction)
-1
min ).
X-r a y Cr ysta llogr a p h y. The colorless needle-shaped crys-
tals of 3-5 and 7-9 were grown from acetone solutions (∼10
-1
mg mL ). The crystals were mounted directly on a glass fiber.
The data were collected at 23 °C with graphite-monochromated
Mo KR radiation (λ ) 1.54178 Å). Details of crystal data, data
collection, and structure refinement are summarized in the
Supporting Information. The intensities of three representative
reflections which were measured after every 150 reflections
remained constant throughout data collection, indicating
electronic stability of the sample crystals. The data were
corrected for Lorentz and polarization effects, but not for
absorption. The structures were solved by direct methods
(Chart 2).
Con clu sion s
The present study demonstrated that the 2′-oxo sub-
stituent is crucial for the efficient C(1′)-N(1) bond
(
21) (a) Silverman, R. B. Organic Chemistry of Drug Design and
Drug Action; Academic Press: San Diego, 1992; pp 352-401. (b)
Bundgaard, H. Design of Prodrugs; Elsevier: Amsterdam, 1985.

Radiation-Activated Antitumor Prodrugs
SHELXS-9722 for compounds 3, 7, and 8; SHELXS86 for
J . Org. Chem., Vol. 65, No. 15, 2000 4647
23
(
t r a n s-5-F lu o r o -1-(5′-t er t -b u t y l-2′-o x o c y c lo h e x y l)-
u r a cil (11). In a similar manner, compound 11 was prepared
from 5-fluoro-2,4-bis(trimethylsiloxy)pyrimidine (1.7 mL, 5.4
mmol) and cis-2-bromo-4-tert-butylcyclohexanone2 (1.26 g, 5.4
2
4
25
compounds 9; SAPI91 for compound 4; SIR88 for compound
) and refined anisotropically for non-hydrogen atoms. Hy-
5
6
drogen atoms were refined with isotropic temperature factors.
Tables of positional and thermal parameters, bond lengths,
bond angles, torsion angles, and nonbonding atom distances
are listed in the Supporting Information.
Cyclic Volta m m etr y. Cyclic voltammograms were re-
corded using a Pt working electrode, Pt wire for the common
electrode and aqueous Ag/AgCl as a reference in a five-necked
jacketed cell (20 mL). The redox potentials of 2′-oxo compounds
1
mmol) as yellow oil in 6% yield. As confirmed by H NMR the
resulting oil consisted of a mixture of two conformers, 11a with
axial 5-fluorouracil-1-yl group and 11b with axial tert-butyl
1
group, that could not be separately isolated: H NMR (400
MHz, CDCl
(
(
3
) δ 0.94 (s, 18H), 1.54-1.77 (m, 4H), 2.01-2.14
m, 4H), 2.25-2.42 (m, 4H), 2.64-2.74 (m, 2H), 5.15 (for 11b)
dd, 1H, J ) 6.4, 12.2 Hz), 5.39 (for 11a ) (dd, 1H, J ) 6.8, 11.7
(5 mM) were determined at room temperature in Ar-purged
Hz), 7.28 (d, 1H, J ) 4.5 Hz), 7.31 (d, 1H, J ) 4.4 Hz), 8.87 (br
s, 2H); 13C NMR (100 MHz, CDCl
) δ 25.2 (CH ), 25.5 (CH ),
7.5 (CH ), 29.9 (CH ), 30.3 (CH ), 32.6 (C), 40.0 (CH ), 40.1
CH ), 46.0 (CH), 46.1 (CH), 58.0 (CH), 59.8 (CH), 122.9 (d,
CH, J ) 32.2 Hz), 123.6 (d, CH, J ) 32.2 Hz), 140.2 (d, CF, J
233.5 Hz), 140.7 (d, CF, J ) 234.4 Hz), 149.9 (C), 151.3 (C),
DMF solution containing 0.1 M TBAB as a supporting elec-
trolyte.
3
2
2
2
3
2
2
2
5
-F lu or o-1-(2′-oxocycloa lk yl)u r a cil (4-10). Compounds
(
2
4
-10 were prepared following the method reported by Akashi
et al. Typically, to a stirred solution of 5-fluoro-2,4-bis-
trimethylsiloxy)pyrimidine (1.8 mL, 7.2 mmol) and 2-bro-
9
)
(
1
2
2
56.4 (d, C, J ) 25.7 Hz), 157.7 (d, C, J ) 25.7 Hz), 202.5 (C),
mocyclobutanone (1.07 g, 7.2 mmol) in dry MeCN (10 mL)
cooled in an ice bath for 15 min under nitrogen was added
+
03.3 (C); FAB-HRMS m/z: calcd for C14
83.1458, found 283.1458.
20 2 3
H N O F [(M + H) ]
0
4
.08 mL of SnCl . The solution was stirred for 3 h at room
temperature and for 15 h at 40 °C. After cooling to room
temperature and adding 1 mL of MeOH, the resulting reaction
mixture was stirred in an ice bath for 30 min and evaporated.
The residue was purified by medium-pressure column chro-
matography (Chloroform-MeOH 20:1) to afford 4 that was
recrystallized into yellow crystal from acetone in 45% yield:
Ack n ow led gm en t. This work was supported by the
Grant-in-Aids for Scientific Research (B) (No. 07455339
and No. 09557069) from the Ministry of Education,
Science, Sports, and Culture of J apan. We gratefully
acknowledge helpful and stimulating discussions with
Prof. Ken’ichi Takeuchi. We also thank Dr. T oˆ ru Ueno
for performing fast atom bombardment mass spectral
analysis.
1
H NMR (400 MHz, acetone-d
6
) δ 2.40-2.47 (m, 1H), 2.75 (d,
2
1
d
H, J ) 13.2 Hz), 2.88-3.05 (m, 1H), 5.33 (m, 1H), 7.74 (d,
1
3
H, J ) 6.8 Hz), 10.54 (br s, 1H); C NMR (100 MHz, acetone-
) δ 19.1 (CH ), 42.9 (CH ), 71.6 (CH), 129.7 (d, CH, J ) 34.0
6
2
2
Hz), 141.6 (d, CF, J ) 231.6 Hz), 150.3 (C), 158.1 (d, C, J )
2
5.7 Hz), 202.6 (C); FAB-HRMS m/z: calcd for C F [(M
8 8 2 3
H N O
+
Su p p or t in g In for m a t ion Ava ila b le: Full details of
X-ray crystallographic data for compounds 3, 4, 5, 7, 8,
and 9, procedures for the synthesis and NMR data of com-
pounds 5-10 is available free of charge via the Internet at
http://pubs.acs.org.
+
H) ] 199.0519, found 199.0514.
(
22) Scheldrick, G. M. Program for the Solution of Crystal Structures;
University of Goettingen: Germany, 1997.
23) Scheldrick, G. M. Crystallographic Computing 3; Oxford Uni-
versity Press: Oxford, 1985; pp 175-189.
24) Hai-Fu, F. Structure Analysis Programs with Intelligent Control;
Rigaku Corporation: Tokyo, J apan, 1991.
25) Burla, M. C.; Camalli, M.; Cascarano, G.; Giacovazzo, C.;
Polidori, G.; Spagna, R.; Viterbo, D. J . Appl. Cryst. 1989, 22, 389-
03.
(
(
J O000245U
(
(26) Hannaby, M.; Warren, S. J . Chem. Soc., Perkin Trans. 1 1992,
3007-3013.
4