Pyrrolo[2,3-d]pyrimidine thymidylate synthase inhibitors: Design and synthesis of one-carbon bridge derivatives

Article abstract of DOI:10.1248/cpb.49.1280

A series of novel pyrrolo[2,3-d]pyrimidine derivatives was designed and synthesized as thymidylate synthase (TS) inhibitors. Molecular design was performed on the human TS complex model built on the basis of the reported structure of TS-deoxyuridinemonoph

Full text of DOI:10.1248/cpb.49.1280

1280
Chem. Pharm. Bull. 49(10) 1280—1287 (2001)
Vol. 49, No. 10
Pyrrolo[2,3-d]pyrimidine Thymidylate Synthase Inhibitors:
Design and Synthesis of One-Carbon Bridge Derivatives
d
Kazuyoshi ASO, ,a Yumi IMAI,a Koichi YUKISHIGE,b Koichiro OOTSU,c and Hiroshi AKIMOTO
*
Medicinal Chemistry Research Laboratories,a Takeda RABICSc and Intellectual Property Department,d Takeda Chemical
Industries, Ltd., 2–17–85 Jusohonmachi, Yodogawa-ku, Osaka 532–8686, Japan and Pharmaceutical Business De-
velopment Department,b Takeda Chemical Industries, Ltd., 4–1–1 Doshomachi, Chuo-ku, Osaka 540–8645, Japan.
Received April 18, 2001; accepted July 23, 2001
A series of novel pyrrolo[2,3-d]pyrimidine derivatives was designed and synthesized as thymidylate synthase
(TS) inhibitors. Molecular design was performed on the human TS complex model built on the basis of the re-
ported structure of TS-deoxyuridinemonophosphate (dUMP)-CB3717 ternary complex. From a docking study,
we expected that a one-carbon bridge between pyrrolo[2,3-d]pyrimidine and an aromatic ring was suitable.
Moreover, we found that the bridge carbon could be replaced with an alkyl group to fill out the unoccupied
space. Based on this design, we synthesized five pyrrolo[2,3-d]pyrimidine derivatives with one-carbon bridge and
evaluated their TS inhibitory activities. All synthesized compounds inhibited TS more potently than compound 2
(LY231514), and the C8-ethyl analogue (7) showed a remarkable inhibitory activity against TS (IC50؍0.017 mM).
Key words pyrrolo[2,3-d]pyrimidine; thymidylate synthase (TS); molecular design; ternary complex
Thymidylate synthase (TS) catalyzes the conversion of 2Ј-
Design When we started our research, the 3D structure
deoxyuridine-5Ј-monophosphate (dUMP) to 2Ј-deoxythymi- of human TS was not available. Thus, the 3D model of
dine-5Ј-monophosphate (dTMP) and is essential for de novo human TS was built by comparative modeling techniques
DNA biosynthesis.1) Thus, TS has long been recognized as using the amino acid sequence of human TS and the crystal
an important target for cancer chemotherapy, and several TS structure of the reported E. coli TS–dUMP–CB3717 ternary
inhibitors have emerged as clinical candidates.2)
complex.16,18) After dUMP and CB3717 were docked manu-
We previously reported that N-[4-[3-(2,4-diamino-7H- ally according to the crystal structure, the energy of the com-
pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic plex model was minimized with moderate constraints on the
acid (1, TNP-351) exhibited potent antitumor activities backbone and dUMP.19) In human TS, the enzyme-ligand in-
against various solid tumors and methotrexate (MTX) resis- teractions might be altered by three replacements around the
tant mouse lymphocytic leukemia in vitro and in vivo.3—7) active site region [Glu82 (E. coli)→Ala (human), Trp83 (E.
After extensive chemical modification of pyrrolo[2,3-d]- coli)→Asn (human) and Val262 (E. coli)→Met (human)].
pyrimidine antifolates,8—12) the 4-oxo compounds (2, 3) were (The numbering of the human TS model corresponds to that
found to moderately inhibit TS (IC50ϭ4.7, 1.4 mM, respec- of E. coli TS.) The replacement of Trp83→Asn would intro-
tively).8) Taylor and co-workers independently reported the duce hydrogen-bonding sites, while the replacements of
synthesis and TS inhibitory activity of compound 2 Glu82→Ala and Val262→Met would alter hydrophobic in-
(LY231514) as a 5,10-dideaza-5,6,7,8-tetrahydrofolic acid teractions. After our research was completed, three crystal
(DDATHF)13) analogue.14) Clinical trials of LY231514 are structures of human TS complexed with its inhibitors have
underway.15)
become accessible from the Protein Data Bank.20) Compari-
The 3D (three-dimensional) structure of E. coli TS–dUMP– son of the crystal structures by superimposing the backbones
CB3717 ternary complex has been determined by X-ray demonstrated that the active site structure of our human TS
crystallography.16) The structural information of TS enabled model was consistent with that of the crystal structures and
us to design potent TS inhibitors of pyrrolo[2,3-d]pyrimidine suitable for structure-based drug design.21)
using molecular modeling techniques. Agouron’s group re-
We started the drug design by studying the binding mode
ported the drug design and synthesis of a new class of TS in- of CB3717 in the human TS model (Fig. 3a). Its binding fac-
hibitors by an iterative crystallographic analysis of an E. coli tors were speculated to include three hydrogen-bonding inter-
TS–dUMP–inhibitor ternary complex.17) Although their iter- actions between 2-NH2 and Ala263, 3-NH and Asp169, and
ative process is a useful strategy for ligand design, it usually 4-O and Gly173. The p-aminobenzoyl and propargyl moi-
takes considerable time to isolate and to solve the crystal- eties contributed to the hydrophobic interactions with Ile79,
lized structure of the complex. In addition, their design was Leu172, Pro175 and Phe176. Aromatic ring stacking interac-
carried out on TS from E. coli. The sequence of E. coli TS at tions were also present between the quinazolinone and the
the active site has high homology (75% identity) to the pyrimidine of dUMP. In addition, a hydrophobic cavity sur-
human sequence (Fig. 2), but we thought it would be advan- rounded by Glu58, Trp80 and the propargyl moiety was ob-
tageous for an accurate design to use a human TS model. served (Fig. 3b).
Thus, to explore novel TS inhibitors we built a human
We then focused our attention on the design of novel
TS–dUMP–inhibitor complex model on the basis of the re- pyrrolo[2,3-d]pyrimidine derivatives as TS inhibitors, based
ported E. coli TS–dUMP–CB3717 ternary complex.16) Here on the information from the human TS–dUMP–CB3717
we report our design and the synthesis of unique pyrrolo[2,3- ternary complex model. A docking study of compound 2
d]pyrimidine derivatives with one-carbon bridge and their (LY231514) with the human TS model suggested that the hy-
potent inhibitory activities against TS.
drogen-bond between 4-O and Gly173 was absent while the
To whom correspondence should be addressed. e-mail: Aso_Kazuyoshi@takeda.co.jp
© 2001 Pharmaceutical Society of Japan

October 2001
1281
hydrophobic interactions of the benzoyl moiety were pre- expected to exhibit potent inhibitory activities against TS.
served. These results led us to speculate that the pyrrolo[2,3- We further examined the ternary complex model of com-
d]pyrimidine ring moved from the position of the quinazoline pound 4, and found that there could be a hydrophobic space
of CB3717, because the benzoyl moiety was accommodated surrounded by Glu58, Trp80 and the bridge chain (Fig. 7).
into the hydrophobic pocket (Fig. 4). This could be caused by This space existed in the complex model of CB3717 and was
the replacement of the quinazoline ring with the pyrrolo[2,3- not occupied by the propargyl moiety. Filling the space by a
d]pyrimidine ring and the deletion of the propargyl moiety. hydrophobic moiety was expected to stabilize the binding en-
Using this information, we replaced the two-carbon bridge of ergy, thus we introduced a hydrophobic substituent at the 8-
compound 2 with a one-carbon bridge (compound 4) to re- position of compound 4 [6 (Rϭmethyl), 7 (Rϭethyl), 8
store the important binding factors, that is, three hydrogen- (Rϭvinyl)]. Each of the enantiomers of 6—8 were incorpo-
bonds (between 2-NH2 and Ala263, 3-NH and Asp169, and rated in the human TS complex model and examined. These
4-O and Gly173) and the hydrophobic interactions of the models demonstrated that the length between each sub-
benzoyl moiety with the enzyme. Actually, when the binding stituent at C8 and Glu58 (or Trp80) was shortened and the
mode of 4 in the human TS model was examined, three hy- substituent filled the hydrophobic space efficiently, regardless
drogen-bonds of the pyrrolo[2,3-d]pyrimidine system, hy- of the absolute configuration of C8 (Figs. 7, 8). In addition,
drophobic interactions of the benzene moiety, and ring stack- three hydrogen-bonds between the pyrrolo[2,3-d]pyrimidine
ing between the pyrimidine of dUMP and pyrrolo[2,3- system and TS were well preserved for 6—8 and the lengths
d]pyrimidine, were observed similar to those for CB3717 of the hydrogen-bonds were equivalent to those of 4 (Fig. 8).
(Fig. 6). The substitution of the thiophene ring for a benzene These results suggested that both the enantiomers of com-
ring (5) led to similar interactions, but the hydrogen-bond pounds 6—8 could inhibit TS more potently than compound
length (3.46 Å) between 4-O and Gly173 was a little longer 4.
than that of 4 (3.04 Å). The hydrophobic interactions of the
Recently, a pyrrolo[2,3-d]pyrimidine antifolate with a sul-
bridge chain moiety of compound 5 seemed enhanced, be- fur bridge and potent inhibitory effect on TS were re-
cause the length between C8 and Glu58 was shortened (Fig. ported.22) When this compound was docked into the human
6). Based on these considerations compounds 4 and 5 were TS model, similar interactions were observed such as three
hydrogen bonds and the ring stacking with dUMP. However,
the sulfur bridge would not be important except for the role
as a spacer. Our independently designed compounds are
characterized by one carbon bridge, and the fact that they can
adopt a hydrophobic substituent on the bridge carbon.
Chemistry
The syntheses of compounds 4—8 are summarized in
Charts 1 and 2. The key reaction was the synthesis of the
pyrrolo[2,3-d]pyrimidine nucleus by the coupling of 2,6-di-
amino-4-hydroxypyrimidine with an acyclic precursor a-bro-
moaldehyde. This reaction was performed using the condi-
tions reported by Secrist III,23) with some modifications.
Fig. 1. Pyrrolo[2,3-d]pyrimidine Antifolates
The key intermediates 10a and 10b for the syntheses of
Fig. 2. Sequence Alignments of Human, Mouse and E. coli TS
The conserved residues are indicated by shaded boxes. The active site is defined as the region within a radius of 5 Å from the ligand in the crystal structure of E. coli TS ternary
complex, and indicated with bold characters. The numbering corresponds to E. coli TS.

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Vol. 49, No. 10
Fig. 3a. A Schematic Model for the Binding Mode of CB3717
Fig. 3b. A Hydrophobic Space Surrounded by Glu58 and Trp80
with Human TS
The models viewed from two directions are shown. dUMP is shown by brown CPK model and
CB3713 by white. The hydrophobic residues around CB3717 are also shown by CPK model.
Glu58 and Trp80 are shown by ball-and-stick model.
CB3717 is shown by white wire model and dUMP by brown.
Fig. 4. A Schematic Model for Binding Mode of 2 with
Human TS
Fig. 7. Interaction of the C8 Substituents of 4, 6—8 with the Hydrophobic Space Sur-
rounded by Glu58 and Trp80
Compound 2 is shown by white wire model and dUMP by brown.
dUMP is shown by brown CPK model and inhibitors by white CPK model. The hydrophobic residues
around the inhibitors and Glu58 are also shown by CPK model. Trp80 is shown by ball-and-stick
model.
compounds 4 and 5 were prepared from the bromination of Chart 2. These three compounds 6—8 were synthesized as
propionaldehydes 9a and 9b8) by 5,5-dibromobarbituric acid racemates because they should bind to TS efficiently, regard-
(Chart 1). Condensation of 2,6-diamino-4-hydroxypyrimi- less of the absolute configuration of C8. Wittig reaction of
dine (11) and the a-bromoaldehydes 10a and 10b in DMSO tert-butyl 4-formylbenzoate 14 with methyl (triphenylphos-
containing K2CO3, gave pyrrolo[2,3-d]pyrimidines 12a and phoranyliden)acetate gave the cinnamate 15. Treatment of
12b. After acidic deprotection of the tert-butyl ester of 12a the a,b-unsaturated ester 15 with lithium dimethylcuprate in
and 12b using trifluoroacetic acid (TFA), the resulting ben- the presence of trimethylchlorosilane24) in anhydrous ether
zoic acids were coupled with diethyl L-glutamate using di- afforded the 1,4-addition product 16a. The 1,4-adducts of
ethyl cyanophosphonate (DEPC). Basic hydrolysis of 13a ethyl 16b or vinyl 16c were synthesized by the conjugate ad-
and 13b with NaOH in H2O–THF followed by an acid treat- dition using mixed cuprates [PhSCu(RMgBr)n (Rϭethyl or
ment gave the desired TS inhibitors 4 and 5.
The synthetic pathway of compounds 6—8 is shown in nard reagent. After selective reduction of methyl esters of
vinyl)]25) prepared from cuprous thiophenoxide and the Grig-

October 2001
1283
zyme has high homology (91% identity) to the human TS se-
quence and the active site region is exactly conserved (Fig.
2). Thus, it can be assumed that the activity of mouse TS is
not significantly different from that of human TS.
Compound 4, with the simple one-carbon bridge, showed
moderate activity against TS, and its potency was almost 5-
fold higher than that of compound 2 (LY231514) with the
two-carbon bridge. Replacement of the benzene ring of 4
with its bioisoster thiophene (5) gave a 12-fold increase of
TS inhibitory potency, which may be due to a favorable hy-
drophobic interaction of the bridge chain moiety caused by
the replacement. The introduction of a hydrophobic sub-
stituent at C8 of 4 resulted in a 30—60 times improvement
(6—8). The C8-ethyl analogue 7, the most potent compound
(IC50ϭ0.017 mM), possessed a 276-fold higher activity than 2
(LY231514). These results indicated that the substituent at
C8 established a hydrophobic interaction and filled the unoc-
cupied space created by Glu58, Trp80 and the bridge chain.
The docking study to the human TS model was shown to be
suitable for the design of potent TS inhibitors.
Fig. 5. Pyrrolo[2,3-d]pyrimidine TS Inhibitors
In conclusion, we designed the unique pyrrolo[2,3-
d]pyrimidine TS inhibitors with one-carbon bridge based on
the examination of the binding mode of compound 2 to the
human TS model. During the course of these studies, five
pyrrolo[2,3-d]pyrimidine derivatives (4—8) were synthesized
and evaluated for their inhibitory activities against TS. They
exhibited more potent TS inhibitory activities than com-
pound 2 (LY231514). These results suggested that our mod-
eling method was effective for designing novel and potent TS
inhibitors.
Experimental
Melting points were determined on a Yanagimoto micro melting point ap-
paratus and are uncorrected. 1H-NMR spectra were recorded on a Varian
Gemini-200 (200 MHz) instrument with tetramethylsilane as an internal
standard. IR spectra were obtained on a JASCO IR-810 IR spectrophotome-
ter. Secondary ionization mass spectra (SIMS) were measured on a Hitachi
M-80A mass spectrometer. Elemental analyses were carried out by Takeda
Analytical Research Laboratories, Ltd. Flash chromatography was per-
formed on Merck silica gel 60 (230—400 mesh).
tert-Butyl 4-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
5-yl)methyl]benzoate (12a) A mixture of tert-butyl 4-(3-oxopropyl)ben-
zoate (11.52 g) and 5,5-dibromobarbituric acid (7.03 g) in Et2O (225 ml) was
stirred for 64 h at room temperature. The resulting precipitate was removed
by filtration. The filtrate was washed with aqueous Na2CO3 and brine, dried
over MgSO4 and concentrated under vacuum to afford 10a (12.51 g, 65%).
To a solution of 2,4-diamino-6-hydroxypyrimidine (11, 1.45 g) in Me2SO (6
ml) was added K2CO3 (60 mg), followed by addition of 10a (3.00 g) in
Me2SO (5 ml). The mixture was stirred for 4 h at room temperature and then
poured into water. The resulting precipitate was collected by filtration and
purified by flash chromatography (CHCl3/MeOH, 97 : 3) to give 12a (1.43 g;
41%) as a white solid. IR (KBr): 3380, 3200, 3150, 2980, 1705, 1670, 1640,
Fig. 6. Interaction Mode for TS-Inhibitor Complex
Hydrogen bonds are denoted by dashed lines with their distances (Å). The distances
(Å) between Glu58 and the bridge carbon are indicated with arrows.
1
1605, 1525 cmϪ1. H-NMR (Me2SO-d6) d: 1.53 (9H, s), 3.99 (2H, s), 6.00
16a—c using lithium borohydride, the resulting alcohols
17a—c were converted to the corresponding aldehydes
18a—c by Swern’s oxidation. The key intermediates 19a—c
were prepared by bromination using 5,5-dibromobarbituric
acid. The remaining procedures used in the synthesis of the
TS inhibitors 6—8 were similar to those for the syntheses of
compounds 4 and 5 described above.
(2H, s), 6.32 (1H, s), 7.38 (2H, d, Jϭ8.0 Hz), 7.77 (2H, d, Jϭ8.0 Hz), 10.12
(1H, s), 10.72 (1H, s).
tert-Butyl 5-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
5-yl)methyl]thiophene-2-carboxylate (12b) Compound 12b (923 mg, 31%)
as a white solid was synthesized from 9b (2.42 g) by the same method as
that described for 12a. IR (KBr): 3420, 3320, 3200, 2980, 2920, 1680, 1600,
1
1605, 1540 cmϪ1. H-NMR (Me2SO-d6) d: 1.48 (9H, s), 4.14 (2H, s), 6.05
(2H, s), 6.48 (1H, s), 6.95 (1H, d, Jϭ3.8 Hz), 7.50 (2H, d, Jϭ3.8 Hz), 10.19
(1H, s), 10.83 (1H, s).
Diethyl N-{4-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
5-yl)methyl]benzoyl}-L-glutamate (13a) Trifluoroacetic acid (0.6 ml) was
added to a solution of 12a (1.39 g) in CH2Cl2 (40 ml). The mixture was
stirred for 4 h at room temperature and concentrated under vacuum. Triethyl-
Biological Results and Discussion
Compounds 4 to 8 were evaluated for their inhibitory ac-
tivities against TS purified from mouse fibrosarcoma Meth-A
cells (Table 1). The amino acid sequence of the mouse en- amine (1.62 g) was added dropwise to a solution of the resulting free acid, L-

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Vol. 49, No. 10
Fig. 8. Interaction Mode for TS–Inhibitor Complex
Hydrogen bonds are denoted by dashed lines with their distances (Å). The distances (Å) between Glu58 (Trp80) and C8 substituent are indicated with arrows.
Chart 1. Synthesis of Pyrrolo[2,3-d]pyrimidine TS Inhibitors (4, 5)
1735, 1710, 1675, 1650, 1620, 1580, 1540 cmϪ1 1H-NMR (Me2SO-d6) d
.
glutamic acid diethyl ester hydrochloride (1.20 g) and diethyl cyanophospho-
nate (DEPC, 0.72 g) in DMF (5 ml) with ice cooling. The mixture was
stirred at room temperature for 18 h and diluted with AcOEt. The AcOEt so-
lution was successively washed with 5% aqueous HCl solution, H2O and
brine, dried over MgSO4 and concentrated under vacuum. The residue was
purified by flash chromatography (CHCl3/10% NH3 in EtOH, 9 : 1) to give
13a (1.70 g, 100%) as a white solid. IR (KBr): 3430, 3280, 3150, 2980,
1.16 (3H, t, Jϭ7.0 Hz), 1.20 (3H, t, Jϭ7.0 Hz), 1.95—2.15 (2H, m), 2.40
(2H, t, Jϭ7.0 Hz), 3.99 (2H, s), 4.04 (2H, q, Jϭ7.0 Hz), 4.10 (2H, q, Jϭ7.0
Hz), 4.37—4.47 (1H, m), 5.99 (2H, s), 6.32 (1H, s), 7.37 (2H, d, Jϭ8.0 Hz),
7.74 (2H, d, Jϭ8.0 Hz), 8.85 (1H, d, Jϭ7.0 Hz), 10.11 (1H, s), 10.71 (1H, s).
Diethyl N-({5-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
5-yl)methyl]thien-2-yl}carbonyl)-L-glutamate (13b) Compound 13b

October 2001
1285
Chart 2. Synthesis of Pyrrolo[2,3-d]pyrimidine TS Inhibitors (6—8)
Table 1. Inhibition of Thymidylate Synthase
and dried over P2O5 under vacuum to give 4 (1.40 g, 78%). mp 186—189 °C.
IR (KBr): 3320, 2930, 1700, 1650, 1650, 1535, 1500 cmϪ1 1H-NMR
.
(Me2SO-d6) d: 1.85—2.20 (2H, m), 2.34 (2H, t, Jϭ7.0 Hz), 3.98 (2H, s),
4.34—4.47 (1H, m), 5.99 (2H, s), 6.32 (1H, s), 7.37 (2H, d, Jϭ8.0 Hz), 7.75
(2H, d, Jϭ8.0 Hz), 8.46 (1H, d, Jϭ8.0 Hz), 10.13 (1H, s), 10.71 (1H, s). Sec-
ondary ion mass spectrometry (SIMS) m/z 414 (MHϩ). Anal. Calcd for
C19H19N5O6·0.7H2O: C, 53.57; H, 4.83; N, 16.44. Found: C, 53.53; H, 4.96;
N, 16.31.
IC50 (mM)
Compound
R
H
A
TS
N-({5-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)-
methyl]thien-2-yl}carbonyl)-L-glutamic Acid (5) Compound 5 (416 mg,
43%) as a white crystalline solid was synthesized from 13b (1.10 g) by the
same method as that described for 13a. mp 179—182 °C. IR (KBr): 3350,
4
1.0
3240, 2930, 1700—1640, 1540 cmϪ1 1H-NMR (Me2SO-d6) d: 1.81—2.19
.
5
H
0.083
(2H, m), 2.22—2.47 (2H, m), 4.14 (2H, s), 4.24—4.41 (1H, m), 6.05 (2H,
s), 6.45 (1H, s), 6.91 (1H, d, Jϭ2.8 Hz), 7.65 (1H, d, Jϭ2.8 Hz), 8.45 (1H, d,
Jϭ7.0 Hz), 10.18 (1H, s), 10.81 (1H, s). SIMS m/z 420 (MHϩ). Anal. Calcd
for C17H17N5O6S·0.5H2O: C, 47.66; H, 4.23; N, 16.35. Found: C, 47.60; H,
4.35; N, 16.21.
tert-Butyl 4-[(1E)-3-Methoxy-3-oxo-1-propenyl]benzoate (15) A mix-
ture of tert-butyl 4-formylbenzoate (14, 75 g) and methyl (triph-
enylphoranyliden)acetate (122 g) in toluene (1 l) was refluxed for 2 h. The
insoluble material was removed by filtration. The filtrate was concentrated
under vacuum. The residue was triturated with MeOH. The resulting white
precipitate was collected by filtration, washed with MeOH and ether and
dried under vacuum to give 15 (74.5 g, 78%). IR (KBr): 2950, 1710, 1640,
1600, 1560 cmϪ1. 1H-NMR (CDCl3) d: 1.69 (9H, s), 3.82 (3H, s), 6.50 (1H,
d, Jϭ16.0 Hz), 7.55 (2H, d, Jϭ8.4 Hz), 7.70 (1H, d, Jϭ16.0 Hz), 7.99 (2H, d,
Jϭ8.4 Hz).
6
CH3
0.039
0.017
0.037
4.7
7
CH2CH3
CHϭCH2
H
8
2 (LY231514)
(1.01 g, 91%) as a white solid was synthesized from 12b (2.42 g) by the
same method as that described for 13a. IR (KBr): 3300, 3210, 2980, 2940,
tert-Butyl 4-(3-Methoxy-1-methyl-3-oxopropyl)benzoate (16a) Under
an argon atmosphere methyllithium (1.5 M solution in Et2O, 64 ml) was
added dropwise to a suspension of copper(I) iodide (9.14 g) in anhydrous
Et2O at Ϫ40 °C. To the solution of Me2CuLi were added successively
trimethylchlorosilane (6.7 ml) and 15 (10.49 g) in anhydrous Et2O (300 ml)
at Ϫ78 °C. The stirred mixture was allowed to warm slowly up to room tem-
perature (1 h). Stirring was continued at room temperature for 2 h, then hy-
drolyzed with aqueous NH4Cl. The aqueous layer was extracted with Et2O.
The combined ether layer was washed with brine, dried over MgSO4 and
concentrated under vacuum. The residue was purified by flash chromatogra-
phy (n-hexane/AcOEt, 95 : 5) to afford 16a (4.36 g, 39%) as a colorless oil.
1735, 1710—1580 cmϪ1 1H-NMR (Me2SO-d6) d: 1.16 (3H, t, Jϭ7.0 Hz),
.
1.18 (3H, t, Jϭ7.0 Hz), 1.91—2.20 (2H, m), 2.42 (2H, t, Jϭ7.0 Hz), 4.13
(2H, s), 4.04 (2H, q, Jϭ7.0 Hz), 4.10 (2H, q, Jϭ7.0 Hz), 4.32—4.45 (1H,
m), 6.10 (2H, s), 6.45 (1H, s), 6.91 (1H, d, Jϭ4.0 Hz), 7.65 (1H, d, Jϭ4.0
Hz), 8.58 (1H, d, Jϭ7.0 Hz), 10.22 (1H, s), 10.82 (1H, s).
N-{4-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)-
methyl]benzoyl}-L-glutamic Acid (4) A solution of 13a (1.70 g) in H2O–
THF (1 : 1, 36 ml) was treated with 1 N aqueous NaOH solution (14.5 ml) at
room temperature for 4 h. THF was evaporated under vacuum and 1 N HCl
solution was added dropwise to the residue. The white crystalline precipitate
was collected by filtration, washed successively with water, MeOH and ether
IR (KBr): 2980, 2940, 1710, 1720, 1605 cmϪ1 1H-NMR (CDCl3) d: 1.30
.

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Vol. 49, No. 10
(3H, d, Jϭ7.0 Hz), 1.58 (9H, s), 2.55 (1H, dd, Jϭ7.6, 15.5 Hz), 2.64 (1H, dd,
Jϭ17.0, 10.6, 6.8 Hz), 7.26 (2H, d, Jϭ8.0 Hz), 7.94 (2H, d, Jϭ8.0 Hz), 9.72
Jϭ7.6, 15.5 Hz), 3.23—3.43 (1H, m), 3.61 (3H, s), 7.26 (2H, d, Jϭ8.6 Hz), (1H, t, Jϭ1.8 Hz).
7.92 (2H, d, Jϭ8.6 Hz).
tert-Butyl 4-[1-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimi-
tert-Butyl 4-(1-Ethyl-3-Methoxy-3-oxopropyl)benzoate (16b) Under din-5-yl)ethyl]benzoate (20a) Compound 20a (916 mg, 24%) as a white
an argon atmosphere ethylmagnesium bromide (1.0 M solution in THF, 85
ml) was added dropwise to a suspension of phenylthiocopper (5.79 g) in an-
hydrous THF at Ϫ40 °C. The mixture was allowed to warm up to Ϫ15 °C.
After a solution of 15 (7.50 g) in anhydrous THF (50 ml) was added drop-
solid was synthesized from 18a (3.03 g) by the same method as that de-
scribed for 12a. IR (KBr): 3340, 3200, 3150, 2980, 2940, 1720, 1640, 1600,
1
1540 cmϪ1. H-NMR (Me2SO-d6) d: 1.52 (9H, s), 1.54 (3H, d, Jϭ8.0 Hz),
4.34—4.48 (1H, m), 5.92 (2H, s), 6.34 (1H, d, Jϭ1.6 Hz), 7.40 (2H, d,
wise, the mixture was stirred at 0 °C for 1 h and poured into aqueous NH4Cl. Jϭ8.2 Hz), 7.77 (2H, d, Jϭ8.2 Hz), 10.05 (1H, s), 10.73 (1H, d, Jϭ1.6 Hz).
The precipitate was removed by filtration. The filtrate was extracted with tert-Butyl 4-[1-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
Et2O. The combined ether layer was washed with brine, dried over MgSO4 5-yl)propyl]benzoate (20b) Compound 20b (510 mg, 45%) as a white
and concentrated under vacuum. The residue was purified by flash chro- solid was synthesized from 18b (911 mg) by the same method as that de-
1H-
matography (n-hexane/AcOEt, 95 : 5) to afford 16b (2.68 g, 32%) as a color- scribed for 12a. IR (KBr): 3330, 3200, 1710, 1665, 1600, 1520 cmϪ1
.
less oil. IR (KBr): 2960, 2930, 1740, 1705, 1605 cmϪ1. 1H-NMR (CDCl3) d: NMR (Me2SO-d6) d: 0.80 (3H, t, Jϭ7.2 Hz), 1.52 (9H, s), 1.83—1.98 (1H,
0.78 (3H, t, Jϭ7.4 Hz), 1.59 (9H, s), 1.53—1.78 (2H, m), 2.57 (1H, dd, m), 2.08—2.23 (1H, m), 4.15 (1H, t, Jϭ8.2 Hz), 5.98 (2H, s), 6.40 (1H, d,
Jϭ8.4, 15.0 Hz), 2.68 (1H, dd, Jϭ6.5, 15.0 Hz), 2.93—3.17 (1H, m), 3.57 Jϭ1.8 Hz), 7.43 (2H, d, Jϭ8.2 Hz), 7.77 (2H, d, Jϭ8.2 Hz), 10.06 (1H, s),
(3H, s), 7.23 (2H, d, Jϭ8.2 Hz), 7.92 (2H, d, Jϭ8.2 Hz).
10.74 (1H, d, Jϭ1.8 Hz).
tert-Butyl 4-[1-(2-Methoxy-2-oxoethyl)-2-propenyl]benzoate (16c)
Compound 16c (2.39 g, 31%) as a colorless oil was synthesized from 15
(7.00 g) by the same method as that described for 16b. IR (KBr): 2980,
tert-Butyl 4-[1-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
5-yl)-2-propenyl]benzoate (20c) Compound 20c (171 mg, 24%) as a
white solid was synthesized from 18c (622 mg) by the same method as that
1
1740, 1640, 1605 cmϪ1. H-NMR (CDCl3) d: 1.58 (9H, s), 1.53—1.78 (2H,
described for 12a. IR (KBr): 3340, 3200, 2980, 1705, 1670, 1635, 1600,
m), 2.70 (1H, dd, Jϭ7.8, 15.4 Hz), 2.80 (1H, dd, Jϭ7.8, 15.4 Hz), 3.61 (3H, 1540 cmϪ1. H-NMR (Me2SO-d6) d: 1.52 (9H, s), 4.99—5.05 (1H, m), 5.00
s), 3.86—3.89 (1H, m), 5.06 (1H, dt, Jϭ16.8, 1.2 Hz), 5.10 (1H, dt, Jϭ10.6,
1
(1H, d, Jϭ17.0 Hz), 5.06 (1H, d, Jϭ10.0 Hz), 6.01 (2H, s), 6.43 (1H, ddd,
1.2 Hz), 5.96 (1H, ddd, Jϭ16.8, 10.6, 6.8 Hz), 7.26 (2H, d, Jϭ8.4 Hz), 7.93 Jϭ17.0, 10.0, 8.0 Hz), 6.45 (1H, s), 7.33 (2H, d, Jϭ8.2 Hz), 7.78 (2H, d,
(2H, d, Jϭ8.4 Hz).
Jϭ8.2 Hz), 10.07 (1H, s), 10.83 (1H, d, Jϭ2.2 Hz).
tert-Butyl 4-(3-Hydroxy-1-methylpropyl)benzoate (17a) Lithium boro-
hydride (348 mg) was added to a solution of 16a (4.24 g) in anhydrous Et2O
Diethyl N-{4-[1-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrim-
idin-5-yl)ethyl]benzoyl}-L-glutamate (21a) Compound 21a (974 mg,
(30 ml). After the mixture was stirred at room temperature for 13 h, the reac- 89%) as a white solid was synthesized from 20a (800 mg) by the same
tion was quenched with 1 N aqueous KHSO4 solution. The aqueous layer was
extracted with Et2O. The combined ether layer was washed with brine, dried
over MgSO4 and concentrated under vacuum. The residue was purified by
method as that described for 13a. IR (KBr): 3350, 2970, 2920, 1735, 1680—
1580, 1500 cmϪ1. 1H-NMR (Me2SO-d6) d: 1.16 (3H, t, Jϭ7.2 Hz), 1.18 (3H,
t, Jϭ7.2 Hz), 1.55 (3H, d, Jϭ7.4 Hz), 1.94—2.18 (2H, m), 2.42 (2H, t,
flash chromatography (n-hexane/AcOEt, 4 : 1) to afford 17a (3.56 g, 96%) as Jϭ7.4 Hz), 4.05 (2H, q, Jϭ7.2 Hz), 4.10 (2H, q, Jϭ7.2 Hz), 4.30—4.49 (2H,
a colorless oil. IR (KBr): 3400, 2980, 2940, 2880, 1720, 1610 cmϪ1
.
1H- m), 5.98 (2H, s), 6.34 (1H, d, Jϭ1.6 Hz), 7.39 (2H, d, Jϭ8.4 Hz), 7.75 (2H,
d, Jϭ8.4 Hz), 8.58 (1H, d, Jϭ7.6 Hz), 10.05 (1H, s), 10.71 (1H, t, Jϭ1.6
NMR (CDCl3) d: 1.28 (3H, d, Jϭ7.0 Hz), 1.39 (1H, m), 1.59 (9H, s), 1.80—
1.91 (2H, m), 2.86—3.06 (1H, m), 3.50 (1H, dd, Jϭ6.6, 10.6 Hz), 3.61 (1H, Hz).
dd, Jϭ6.6, 10.6 Hz), 7.25 (2H, d, Jϭ8.2 Hz), 7.93 (2H, d, Jϭ8.2 Hz).
Diethyl N-{4-[1-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrim-
idin-5-yl)propyl]benzoyl}-L-glutamate (21b) Compound 21b (528 mg,
(1.07 g, 88%) as a colorless oil was synthesized from 16b (1.34 g) by the 78%) as a white solid was synthesized from 20b (500 mg) by the same
same method as that described for 17a. IR (KBr): 3400, 2970, 2930, 2860, method as that described for 13a. IR (KBr): 3300, 3240, 3150, 2960, 2925,
tert-Butyl 4-(3-Hydroxy-1-ethylpropyl)benzoate (17b) Compound 17b
1
1710, 1605 cmϪ1. H-NMR (CDCl3) d: 0.77 (3H, t, Jϭ7.4 Hz), 1.34—1.51 1730, 1680, 1650, 1625, 1600 cmϪ1 1H-NMR (Me2SO-d6) d: 0.81 (3H, t,
.
(1H, m), 1.59 (9H, s), 1.61—1.99 (4H, m), 2.60—2.76 (1H, m), 3.37—3.60
(2H, m), 7.21 (2H, d, Jϭ8.2 Hz), 7.92 (2H, d, Jϭ8.2 Hz).
Jϭ7.0 Hz), 1.16 (3H, t, Jϭ7.0 Hz), 1.18 (3H, t, Jϭ7.0 Hz), 1.84—2.23 (4H,
m), 2.42 (2H, t, Jϭ7.4 Hz), 4.04 (2H, q, Jϭ7.0 Hz), 4.10 (2H, q, Jϭ7.0 Hz),
tert-Butyl 4-[1-(2-Hydroxyethyl)-2-propenyl)benzoate (17c) Com- 4.11—4.19 (1H, m), 4.30—4.49 (1H, m), 5.98 (2H, s), 6.40 (1H, d, Jϭ1.4
pound 17c (791 mg, 35%) as a colorless oil was synthesized from 16c (2.38 Hz), 7.41 (2H, d, Jϭ8.4 Hz), 7.74 (2H, d, Jϭ8.4 Hz), 8.58 (1H, d, Jϭ7.8
g) by the same method as that described for 17a. IR (KBr): 3400, 2980, Hz), 10.06 (1H, s), 10.72 (1H, t, Jϭ1.4 Hz).
2940, 1715, 1640, 1605 cmϪ1
1.59 (9H, s), 1.87—2.09 (2H, m), 3.47—3.68 (3H, m), 5.08 (1H, dt, Jϭ10.0,
.
1H-NMR (CDCl3) d: 1.28—1.38 (1H, m),
Diethyl N-{4-[1-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrim-
idin-5-yl)-2-propenyl]benzoyl}-L-glutamate (21c) Compound 21c (171
1.4 Hz), 5.08 (1H, dt, Jϭ17.0, 1.4 Hz), 5.96 (1H, ddd, Jϭ17.0, 10.0, 7.8 Hz), mg, 77%) as a white solid was synthesized from 20c (165 mg) by the same
7.25 (2H, d, Jϭ8.2 Hz), 7.93 (2H, d, Jϭ8.2 Hz). method as that described for 13a. IR (KBr): 3350, 3215, 2980, 1740, 1680—
tert-Butyl 4-(1-Methyl-3-oxopropyl)benzoate (18a) Under an argon at- 1600, 1540, 1500 cmϪ1 1H-NMR (Me2SO-d6) d: 1.16 (3H, t, Jϭ7.0 Hz),
mosphere a solution of DMSO (2.57 g) in CH2Cl2 (35 ml) was added to a so- 1.18 (3H, t, Jϭ7.0 Hz), 1.92—2.16 (2H, m), 2.42 (2H, t, Jϭ7.4 Hz), 4.04
.
lution of oxalyl chloride (2.09 g) in CH2Cl2 (70 ml) at Ϫ60 °C. After 2 min, a (2H, q, Jϭ7.0 Hz), 4.09 (2H, q, Jϭ7.0 Hz), 4.33—4.44 (1H, m), 4.99 (1H, d,
solution of 17a (3.43 g) in CH2Cl2 (30 ml) was added. Stirring was contin- Jϭ16.4 Hz), 5.04 (1H, d, Jϭ10.0 Hz), 5.01—5.07 (1H, m), 6.00 (2H, s),
ued at Ϫ60 °C for 15 min, and then triethylamine (6.93 g) was added. The 6.43 (1H, d, Jϭ1.6 Hz), 6.44 (1H, ddd, Jϭ16.4, 10.0, 8.2 Hz), 7.31 (2H, d,
mixture was allowed to warm to 0 °C in 30 min, and poured into water (350
Jϭ8.4 Hz), 7.74 (2H, d, Jϭ8.4 Hz), 8.58 (1H, d, Jϭ7.6 Hz), 10.07 (1H, s),
ml). The aqueous phase was extracted with CH2Cl2. The combined organic 10.81 (1H, t, Jϭ1.6 Hz).
phase was dried over MgSO4 and concentrated under vacuum. Purification
by flash chromatography (n-hexane/AcOEt, 5 : 1) gave 18a (3.16 g, 93%) as
N-{4-[1-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-
yl)ethyl]benzoyl}-L-glutamic Acid (6) Compound 6 (674 mg, 83%) as a
a colorless oil. IR (KBr): 2980, 2840, 1735, 1715, 1610 cmϪ1
(CDCl3) d: 1.32 (3H, d, Jϭ6.8 Hz), 1.59 (9H, s), 2.68 (1H, ddd, Jϭ16.8, 6.8,
1.8 Hz), 2.78 (1H, ddd, Jϭ16.8, 6.8, 1.8 Hz), 3.32—3.52 (1H, m), 7.27 (2H,
d, Jϭ8.4 Hz), 7.94 (2H, d, Jϭ8.4 Hz), 9.72 (1H, t, Jϭ1.8 Hz).
.
1H-NMR white crystalline solid was synthesized from 21a (920 mg) by the same
method as that described for 4. mp 187—191 °C. IR (KBr): 3350, 3200,
2960, 1700, 1660 cmϪ1
.
1H-NMR (Me2SO-d6) d: 1.55 (3H, d, Jϭ7.2 Hz),
1.88—2.18 (2H, m), 2.34 (2H, t, Jϭ7.4 Hz ), 4.31—4.47 (2H, m), 5.98 (2H,
s), 6.32 (1H, s), 7.38 (2H, d, Jϭ8.2 Hz), 7.75 (2H, d, Jϭ8.2 Hz), 8.45 (1H, d,
tert-Butyl 4-(1-Ethyl-3-oxopropyl)benzoate (18b) Compound 18b (955
mg, 91%) as a colorless oil was synthesized from 17b (1.06 g) by the same Jϭ8.0 Hz), 10.04 (1H, s), 10.70 (1H, s). SIMS m/z 428 (MHϩ). Anal. Calcd
method as that described for 18a. IR (KBr): 2970, 2930, 1700, 1605 cmϪ1
.
for C20H21N5O6·0.5H2O: C, 55.04; H, 5.08; N, 16.05. Found: C, 54.92; H,
1H-NMR (CDCl3) d: 0.80 (3H, t, Jϭ7.2 Hz), 1.59 (9H, s), 1.61—1.78 (2H, 5.29; N, 16.04.
m), 2.74 (1H, dd, Jϭ7.4, 1.8 Hz), 3.07—3.23 (1H, m), 7.24 (2H, d, Jϭ8.4
Hz), 7.93 (2H, d, Jϭ8.2 Hz), 9.67 (1H, t, Jϭ1.8 Hz).
N-{4-[1-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-
yl)propyl]benzoyl}-L-glutamic Acid (7) Compound 7 (292 mg, 64%) as a
white solid was synthesized from 21b (500 mg) by the same method as that
tert-Butyl 4-[1-(2-Oxoethyl)-2-propenyl)benzoate (18c) Compound
18c (635 mg, 84%) as a colorless oil was synthesized from 17c (784 mg) by described for 4. mp 116—119 °C. IR (KBr): 3350, 2960, 2920, 1700—1600,
the same method as that described for 18a. IR (KBr): 2980, 1725, 1715, 1530 cmϪ1 1H-NMR (Me2SO-d6) d: 0.80 (3H, t, Jϭ7.4 Hz), 1.81—2.21
.
1640, 1605 cmϪ1. 1H-NMR (CDCl3) d: 1.58 (9H, s), 2.81 (1H, ddd, Jϭ17.0, (4H, m), 2.34 (2H, t, Jϭ7.4 Hz), 4.14 (2H, q, Jϭ7.4 Hz), 4.31—4.45 (1H,
7.5, 1.8 Hz), 2.91 (1H, ddd, Jϭ17.0, 7.5, 1.8 Hz), 3.95—4.07 (1H, m), 5.08 m), 5.97 (2H, s), 6.39 (1H, d, Jϭ1.4 Hz), 7.40 (2H, d, Jϭ8.2 Hz), 7.74 (2H,
(1H, dt, Jϭ17.0, 1.0 Hz), 5.14 (1H, dt, Jϭ10.6, 1.0 Hz), 5.97 (1H, ddd, d, Jϭ8.2 Hz), 8.45 (1H, d, Jϭ8.2 Hz), 10.05 (1H, s), 10.71 (1H, s). SIMS

October 2001
1287
m/z 442 (MHϩ). Anal. Calcd for C21H23N5O6·0.5H2O: C, 56.00; H, 5.37; N,
15.55. Found: C, 56.02; H, 5.55; N, 15.62.
6) Itoh F., Russello O., Akimoto H., Beardsley G. P., Cancer Chemother.
Pharmacol., 43, 230—235 (1994).
7) Yukishige K., Wajima M., Kusanagi T., Aso K., Akimoto H., Ootsu,
K., J. Takeda Res. Lab., 54, 97—107 (1995).
8) Miwa T., Hitaka T., Akimoto H., J. Org. Chem., 58, 1696—1701
(1993).
9) Aso K., Hitaka T., Yukishige K., Ootsu K., Akimoto H., Chem.
Pharm. Bull., 43, 256—261 (1995).
10) Itoh F., Yukishige K., Wajima M., Ootsu K., Akimoto H., Chem.
Pharm. Bull., 43, 230—235 (1995).
11) Itoh F., Yoshioka K., Yukishige K., Yoshida M., Wajima M., Ootsu K.,
Akimoto H., Chem. Pharm. Bull., 44, 1498—1509 (1996).
12) Itoh F., Yoshioka K., Yukishige K., Yoshida M., Ootsu K., Akimoto H.,
Chem. Pharm. Bull., 48, 1271—1281 (2000).
13) Taylor E. C., Harrington P. J., Fletcher S. R., Beardsley G. P., Moran,
R. G., J. Med. Chem., 28, 914—921 (1985).
14) Taylor E. C., Kuhnt D., Shih C., Rinzel S. M., Grindey G. B., Barredo
J., Jannatipour M., Moran R. G., J. Med. Chem., 35, 4450—4454
(1992).
15) John W., Picus J., Blanke C. D., Clark J. W., Schulman L. N., Rowin-
sky E. K., Thornton D. E., Loehrer P. J., Cancer, 88, 1807—1813
(2000).
N-{4-[1-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-
yl)-2-propenyl]benzoyl}-L-glutamic Acid (8) Compound 8 (82 mg, 77%)
as a white solid was synthesized from 21c (146 mg) by the same method as
that described for 4. mp 180—183 °C. IR (KBr): 3350, 2940, 1700—1600,
1540 cmϪ1. 1H-NMR (Me2SO-d6) d: 1.87—2.18 (2H, m), 2.34 (2H, t, Jϭ7.2
Hz), 4.34—4.45 (1H, m), 4.95—5.12 (3H, m), 6.00 (2H, s), 6.34—6.53 (1H,
m), 6.44 (1H, s), 7.31 (2H, d, Jϭ8.0 Hz), 7.75 (2H, d, Jϭ8.0 Hz), 8.46 (1H,
d, Jϭ7.8 Hz), 10.07 (1H, s), 10.81 (1H, s). SIMS m/z 440 (MHϩ). Anal.
Calcd for C21H21N5O6·0.7H2O: C, 55.79; H, 4.99; N, 15.49. Found: C,
55.98; H, 5.25; N, 15.50.
Preparation of Crude TS A crude fraction of TS was prepared from
Meth-A fibrosarcoma cells. Meth-A cells (2ϫ104 ml), in 2.0 l of Eagle’s
minimum essential medium (MEM) supplemented with 10% fetal bovine
serum (FBS), were divided into ten culture flasks and incubated for 72 h at
37 °C in 5% CO2. Cells were washed twice in phosphate-buffered saline and
resuspended in 5 ml of 250 mM sucrose, 10 mM Tris–HCl buffer (ph 7.5), and
then disrupted ultrasonically. Cell lysate was centrifuged at 100000ϫg for 1
h at 4 °C. The cytosol fraction (crude TS fraction) was diluted with 250 mM
sucrose and 10 mM Tris–HCl buffer (pH 7.5) to adjust the protein concentra-
tion to 10 mg/ml and then it was stored at Ϫ20 °C.
16) Montfort W. R., Perry K. M., Fauman E. B., Finer-Moore J. S., Maley
G. F., Hardy L., Maley F., Stroud R. M., Biochemistry, 29, 6964—
6977 (1990).
TS Inhibition Assay TS activity was measured by the method of
Roberts26) with some modification. In brief, 10 ml of crude TS fraction was
added to 40 ml of reaction mixture in a flat-bottom 96 well plate, in tripli-
cate. The final concentrations of the constituents of the reaction mixture
were as follows; 800 mM tetrahydrofolic acid, 18 mM formaldehyde, 80 mM
dUMP, 0.51 mM [3H]dUMP, 10.2 mM 2-mercaptoethanol, 100 mM sucrose, 68
mM NaF, 174 mM Tris–HCl (pH 7.5), 6.24 mg/ml bovine serum albumin and
various concentrations of test compounds. The reaction mixture was incu-
bated on a water bath at 37 °C for 1 h and then chilled on ice. Ice cold
26.65% trichloroacetic acid (20 ml), 3.33 mg/ml cold dUMP (10 ml) and 114
mg/ml charcoal (220 ml) were added to the reaction mixture, which was then
transferred to a centrifuge tube. After centrifugation, the amount of [3H]H2O
released from [3H]dUMP in the supernatant of the reaction mixture was
measured using a liquid scintillation counter.
17) Appelt K., Bacquet R. J., Bartlett C. A., Booth C. L. J., Freer S. T.,
Fuhry M. A. M., Gehring M. R., Herrmann S. M., Howland E. F., Jan-
son C. A., Jones T. R., Kan C.-C., Kathardekar V., Lewis K. K., Mar-
zoni G. P., Matthews D. A., Mohr C., Moomaw E. W., Morse C. A.,
Oatley S. J., Ogden R. C., Reddy M. R., Reich S. H., Schoettlin W. S.,
Smith W. W., Varney M. D., Villafanca J. E., Ward R. W., Webber S.,
Webber S. E., Welsh K. M., White J., J. Med. Chem., 34, 1925—1934
(1991).
18) Protein Data Bank ID code: 2TSC.
19) The modeling studies were performed on a Silicon Graphics IRIS
4D/220GTX workstation (Silicon Graphics Inc., Mountain View, CA,
U.S.A.) with the Insight II/Discover program package (Accelrys Inc.,
Princeton, NJ, U.S.A.).
20) Protein Data Bank ID codes: 1HVY, 1HW3 and 1HW4.
21) The average pairwise root mean square deviation (RMSD) for the
backbone was 2.06 Å. The RMSD for the backbone at the active site
was 0.65 Å.
Acknowledgements We are grateful to Drs. T. Aono and T. Miwa for
their encouragement and valuable advice. We thank Mr. T. Hitaka for his ex-
cellent technical support.
References and Notes
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4495—4502 (1995).
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(1986).
2) Brandt D. S., Chu E., Oncology Research, 9, 403—410 (1997).
3) Miwa T., Hitaka T., Akimoto H., Nomura H., J. Med. Chem., 34,
555—560 (1991).
25) Behforouz M., Curran T. T., Bolan J. L., Tetrahedron Lett., 27, 3107—
3110 (1986).
4) Akimoto H., Hitaka T., Miwa T., Yukishige K., Kusanagi T., Ootsu K.,
Pro. Am. Cancer Res., 32, 327 (1991).
26) Roberts D., Biochemistry, 5, 3546—3548 (1966).
5) Yukishige K., Kusanagi T., Fujita T., Aso K., Akimoto H., Ootsu K.,
Proc. Jpn. Cancer Assoc., 1991, 385.