Bioorganic & Medicinal Chemistry Letters 10 (2000) 391±394
Mechanism-Based Inactivation of Thymidylate Synthase by
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5
-(3-Fluoropropyn-1-yl)-2 -deoxyuridine 5 -Phosphate
Thomas I. Kalman,* Zhe Nie and Ashwini Kamat
Department of Medicinal Chemistry, State University of New York at Bualo, Bualo, New York 14260, USA
Received 27 October 1999; accepted 17 December 1999
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AbstractÐ5-Fluoropropynyl-2 -deoxyuridine 5 -phosphate (3) was designed as a mechanism-based inactivator of thymidylate syn-
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thase (TS). The inhibitor was synthesized from 5-iodo-2 -deoxyuridine and propargyl alcohol by palladium-catalyzed coupling,
followed by ¯uorination and selective phosphorylation. Incubation of TS with 3, in the presence or absence of the CH H folate
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4
cofactor, caused rapid, irreversible inactivation of the enzyme. # 2000 Elsevier Science Ltd. All rights reserved.
Thymidylate (dTMP) synthase (EC 2.1.1.45) catalyzes
the conversion of deoxyuridylate (dUMP) and 5,10-
methylenetetrahydrofolate (CH H folate) to dTMP and
the presence of CH H folate; in addition, the activity
2 4
8±11
of the enzyme recovered very rapidly.
propynyl-dUMP (3), an improved derivative of 2,
5-Fluoro-
2
4
7
,8-dihydrofolate. This reaction is the sole de novo bio-
was designed, based on a mechanistic rationale, as a
prototype of a new generation of dTMP synthase
synthetic source of thymine in DNA. As a consequence,
inhibition of dTMP synthase, in the absence of pre-
formed thymidine, blocks DNA synthesis, and causes
``thymineless death'' of the cell. Due to its essential role
in cellular proliferation, this enzyme is recognized as a
inhibitors,1
2,13
operating by a novel strategy of enzyme
inactivation. In this communication, we describe the
synthesis of 3, and the initial results of our enzyme
inhibition studies. These results demonstrate that 3 can
cause rapid, irreversible inactivation of dTMP synthase,
in both the presence and the absence of CH H folate.
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2
±4
major target in cancer chemotherapy.
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The most important dTMP synthase inhibitors of clin-
ical utility are the 5-¯uoropyrimidines: 5-¯uorouracil (5-
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drug derivatives. The active form of these drugs is the
FU), 5-¯uoro-2 -deoxyuridine (FdUrd), and their pro-
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Results and Discussion
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5
-phosphate of FdUrd, FdUMP (1), which is a
Title compound 3 was synthesized as outlined in
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mechanism-based inhibitor of this enzyme. Its potent
inhibitory activity is due to the reversible formation
of an inactive, covalently bound ternary complex
composed of 1, the CH H folate cofactor, and the
Scheme 1. The sugar hydroxyl groups of 5-iodo-2 -
deoxyuridine (4) were protected by acetylation to yield
5, which was reacted with propargyl alcohol by Pd(0)-
catalyzed coupling14 to yield 6. Several methods of
¯uorination were tried for the conversion of 6 to 7. Tri-
2
4
2
,6
enzyme. The stability of this complex in vivo depends
on the intracellular levels of the polyglutamylated forms
of CH H folate, which are often inadequate for eective
¯ation of the alcohol followed by S 2 displacement with
N
¯uoride failed to yield the desired product. Fluorination
with Select¯uo (F-TEDA-BF ), in the presence of Me S,
was also unsuccessful. Direct ¯uorination of compound
2
4
inhibition of dTMP synthase. This prompted the search
for novel analogues that do not require the cofactor for
4
2
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potent inhibitory activity. 5-Ethynyl-2 -deoxyuridine 5 -
phosphate (2) was considered a promising candidate,
because it has the potential for irreversible interactions
via enzyme-mediated conversion of the acetylenic side-
6 could be carried out successfully with an excess of
ꢀ
15
DAST at � 78 C, in anhydrous methylene chloride.
At the end of the reaction, the excess reagent was
quenched with MeOH. After ¯ash column chromato-
7
16
chain to a chemically reactive allene. However, inacti-
vation of dTMP synthase by 2 was found also to require
graphy, 7 was obtained in 30±40% yield. Standard
deprotection conditions could not be used, due to the
reactivity of the ¯uoropropynyl side-chain. Dilute
methanolic ammonia converted 7 to the corresponding
amine, whereas the use of aqueous potassium carbonate
*
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Corresponding author.
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