WO2001014345A1

WO2001014345A1 - 5-propynylpyrimidine derivatives as cytotoxic enzyme inhibitors

Info

Publication number: WO2001014345A1
Application number: PCT/US2000/022908
Authority: WO
Inventors: Thomas I. Kalman; Zhe Nie
Original assignee: The Research Foundation Of State University Of New York
Priority date: 1999-08-20
Filing date: 2000-08-21
Publication date: 2001-03-01
Also published as: AU6792700A

Abstract

Disclosed are 5-propynylpyrimidine derivatives of formula (I) wherein X is a leaving group, R1 is selected from the group consisting of -OH,-NH2, and -SH; R2 is selected from the group consisting of -H, 2-deoxyribose and ester derivatives thereof. Also disclosed is a method for the synthesis of the 5-propynylpyrimidines. These compounds are useful as cytotoxic agents.

Description

5-PROPYNYLPYREVΠDINE DERΓVATΓVΈS AS CYTOTOXIC ENZYME

INHIBITORS

5

This invention was made with government support under grant R01 CA35212-12 awarded by the National Institutes of Health. The government has certain rights in the invention.

o FIELD OF INVENTION

This invention relates to novel cytotoxic pyrimidine derivatives. More particularly, this invention discloses 5-propynylpyrimidine derivatives that are useful as cytotoxic agents and a method of preparation of these compounds.

5 DESCRIPTION OF RELATED ART

Thymidylate synthase (dTMP synthase) is the sole de novo biosynthetic source of thymine in DNA. In the absence of preformed thymidine, inhibition of this enzyme stops DNA synthesis. This prevents cell division and the maintenance of the integrity of DNA. Cells entering the S-phase of the cell cycle under 0 conditions of inadequate supplies of thymine nucleotides will undergo "thymineless death" (Cohen, 1971, Ann. N.Y. Acad. Sci., 186:292), which may involve apoptosis in susceptible cells (Tillman et al., 1998, Proc. Am. Assoc. Cancer Res., 39: 177). Due to its essential role in cellular proliferation, dTMP synthase has become a target of clinically effective anticancer drugs. 5 One group of dTMP synthase inhibitors that is currently used in cancer chemotherapy is fluoropyrimidines, which include 5-fluorouracil and 5- fluorodeoxyuridine, as well as their prodrug derivatives. However, these compounds have many shortcomings which impose a limitation on their clinical application. For example, these drugs are associated with the development of resistance. A o common type of resistance involves increased cellular levels of the enzyme due to either gene amplification or post trans criptional events. Another type of resistance is due to inadequate levels of the folate cofactor required for inactive ternary complex formation. Thus, there is an ongoing need for novel enzyme inhibitors that can overcome these limitations and be used as effective cytotoxic agents in cancer therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation of the method of preparation of a representative nucleoside derivative, 5-(3-fluoropropyn-l-yl)-2'-deoxyuridine.

Figure 2 is a representation of the Η-NMR spectrum of 5-(3-fluoropropyn- 1 -yl)-2'-deoxyuridine. Figure 3 is a schematic representation of another method of preparation of a representative nucleoside derivative, 5-(3-fluoropropyn-l-yl)-2'-deoxyuridine and its 5'-phosphate derivative, 5-(3-fluoropropyn-l-yl)-dUMP.

Figure 4 is a representation of the inactivation of dTMP synthase by 5-(3- fluoropropyn-l-yl)-dUMP, the intracellularly active form of 5-(3-fluoropropyn-l- yl)-2'-deoxyuridine.

Figure 5 is a representation of the inactivation of dTMP synthase by 5-[3- (imidazole-l-yl)propyn-l-yl]-dUMP (triangles), and 5-[3-(methylimidazolium-l- yl)propyn-l-yl]-dUMP (circles).

Figure 6 is a representation of the time course of dTMP synthase activity during dialysis for enzyme inactivated by 5-(3-fluoropropyn-l-yl)-dUMP (O); and free enzyme control (Δ)

Figure 7 is a representation of the time course of dTMP synthase activity during dialysis for free enzyme control (O); and enzyme inactivated by 5-[3- (imidazole-l-yl)propyn-l-yl]-dUMP (D), and 5-[3-(methylimidazolium-l- yl)propyn- 1 -yl]-dUMP (Δ).

Figure 8 is a representation of the growth inhibitory effect of 5-(3- fluoropropyn-l-yl)-2'-deoxyuridine on human myelogenous leukemia cells K-562.

Figure 9 is a representation of the growth inhibitory effect of 5-(3- fluoropropyn-l-yl)-2'-deoxyuridine on mouse lymphocytic leukemia cells L-1210. SUMMARY OF THE INVENTION

The present invention provides novel 5-propynylpyrimidine derivatives that inhibit dTMP synthase. These compounds can generally be referred to as 5- propynyl-2'-deoxyuridines. These compounds cause rapid and irreversible inactivation of dTMP synthase. This inactivation does not require the presence of CH₂H₄ folate cofactor. These compounds are useful for inhibition of the growth of cells.

A method of making these compounds is also disclosed. The compounds are synthesized from 5-iodo-2'-deoxyuridine. The hydroxyl groups of the deoxyribose are protected and then the protected compound is reacted with a propargyl reagent in the presence of a palladium catalyst to introduce the desired side-chain at the 5- position. The synthesis is completed by deprotection of the sugar hydroxyl groups to obtain the desired 5-propynyl-2'-deoxyuridines. Ester derivatives of the above compounds can be obtained by reaction with phosphoric or carboxylic acid. Thus, an object of the present invention is to provide novel 5- propynylpyrimidine derivatives that are inhibitors of dTMP synthase.

Another object of the present invention is to provide a method of making 5- propynylpyrimidine derivatives that are inhibitors of dTMP synthase. A still further obj ect of the present invention is to provide a method for the use of 5-propynylpyrimidine derivatives in the inhibition of growth of cells such as in cancer chemotherapy.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The term "leaving group" as described herein for the purposes of specification and claims, means a group that is readily displaceable by conjugate elimination.

The compounds of the present invention can be represented by the following formula:

wherein X is a leaving group Suitable leaving groups include, but are not limited to, halides, heterocyclic groups, and other groups such as -SCN, -N₃, and - CN The heterocyclic leaving groups include, but are not limited to, lmidazole, N- methylimidazole, tetrazole and triazole Some examples of suitable X groups are as follows -OCOC₆H₄CH₃, -OC_fiH₄NO₂, -SC₆H₄NO₂, -F, -SCN, -OCOCH₃,

+

/=*N /=^NCH₃

-N and

Ri may be selected from the group consisting of -OH, -NH₂, and -SH Those skilled in the art will recognize that equivalent tautomeric forms exist, e g , the -OH group at Ri is akin to =O at Ri with the N at the 3 -position of the pyrimidine ring having an -H R₂ may be -H or it may be 2-deoxyribose or an ester derivative thereof, wherein the 3' and/or the 5' -hydroxyl groups are esterified by carboxylic or phosphoric acids When R₂ is 2-deoxyribose or an ester derivative thereof, the compounds of the present invention can be represented by the following formula

where R₃ and R4 independently can be -H, acyl, phosphoryl or silyl groups. Acyl or silyl groups, useful for the protection of the 3' and 5' - hydroxyl groups , are blocking groups known to those skilled in the art. Such groups include, but are not limited to, acetyl, benzoyl, toluoyl, mesyl, tosyl, trimethyl silyl and the like.

In a preferred embodiment, the compound of the present invention is 5-(3- fluoropropyn-l-yl)-2'-deoxyuridine and is represented by the following formula:

In another preferred embodiment, the compound of the present invention is 3' 5' _ diacetyl-5-(3-fluoropropyn-l-yl)-2'deoxyuridine and is represented by the following formula:

In another preferred embodiment, the compound of the present invention is 5-(3-fluoropropyn-l-yl)-2'-deoxyuridine 5'-phosphoric acid (or its salts) and is represented by the following formula

While not intending to be bound by any particular theory, it is considered that the compounds of the present invention act as follows. There is an initial nucleophilic attack by the cysteine residue of the active site of dTMP synthase at the 6-position of the pyrimidine ring, forming a covalent enolate intermediate, analogous to that in the normal enzyme catalyzed reaction. Then, expulsion of the leaving group X results in the formation of a covalently attached cumulene derivative.

The compounds of the present invention are synthesized from 5-iodo-2- deoxyuridine. The hydroxyl groups of the deoxyribose are protected and then the protected compound is reacted with a propargyl derivative in the presence of a palladium catalyst to introduce the desired side-chain at the 5-position. Identification of the appropriate propargyl derivative is well within the purview of one skilled in the art. In the case of the 5-fluoropropynyl side-chain, the corresponding propargyl alcohol derivative is made first, followed by fluorination of the alcohol with (diethylamino) sulfur trifluoride (DAST). The synthesis is completed by deprotection of the sugar hydroxyl groups to obtain the desired 5- propynyl-2'-deoxyuridines. Ester derivatives of the above compounds can be obtained by reaction with phosphoric or carboxylic acid. Thus, the steps in the synthesis of the compounds of the present invention may be summarized as follows: a) protection of the 3'- and 5'-hydroxy groups of 5-iodo-2'- deoxyuridine, such as by addition of blocking groups known to those skilled in the art including, but not limited to, acetyl, silyl, benzoyl, toluoyl, mesyl, tosyl, and the like; b) attachment of the side-chain to the 5- position of the pyrimidine ring via Pd(O)-catalyzed coupling of the appropriate propargyl reagent, such as propargylalcohol c) if desired, modification of the side-chain, such as by fluorination of the alcohol using DAST to yield the 3-fluoropropynyl side-chain; d) deprotection of the product by removal of the blocking groups from the 3' and 5 '-hydroxyl groups by standard methods; e) purification of the deprotected product, such as by chromatography

5' -Monophophate derivatives of the compounds of the present invention can be prepared by using phosphorus oxychloride, which is a standard phosphorylating agent. In general, 1-1.5 equivalent of phosphorus oxychloride reagent can be added to a solution of nucleoside in triethylphosphate at -10°C→0°C. Catalytic amounts of pyridine may be added to accelerate the reaction. This procedure was used to phosphorylate the compounds disclosed herein. The reaction progress was monitored by two solvent systems: (1) CH₂Cl₂/MeOH= 9: 1, used for observation of the consumption of the starting material, newly generated monophosphate does not move in the system; (2) nBuOH/HOAc/H₂O = 5:3:2 or 6:2:2, used for detection of the newly generated product. After the completion of the reaction, chlorophosphate intermediate may be hydrolyzed, such as by the addition of a few drops of water. The reaction times and yield for the synthesis of 5'-monophosphates of various X groups for the following compound are presented in Table 2, where Imd and Imd⁺- CH₃ designate imidazole and N-methylimidazole, respectively. X

The compounds of the present invention will be useful for treating conditions wherein inhibition of dTMP synthase contributes to achieving the desired effect. For example, the compounds of the present invention will be useful for inhibiting or controlling the unwanted growth of cells. One example wherein the compounds of the present invention are expected to be useful is in the treatment of cancer. The data presented herein demonstrates that these compounds are competitive inhibitors of dTMP synthase, causing time-dependent inactivation of dTMP synthase. This inactivation is demonstrated not to require the cofactor CH₂H₄ folate. The inactivation of dTMP synthase by the compounds of the present invention is irreversible and therefore particularly useful for inhibiting the growth of tumor cells. The compounds of the present invention or salts thereof, can be used for therapeutic purposes by administering a therapeutically effective dose in a pharmaceutically acceptable carrier. The term "therapeutically effective amount" as used herein means an amount which is required to cause a desired effect. As is know to those skilled in the art, a therapeutically effective amount of the compound can be administered by any standard means such as by injection, parenterally, orally, or topically. Thus the administration may take the form of tablets, capsules, liquids, suspensions, suppositories or the like. The determination of dosage and frequency of administration is well within the purview of one skilled in the art and will depend upon such factors as the severity of the illness and individual requirements. The dosage may be delivered as single precise dosage or as sustained or controlled release dosage forms.

The following examples are presented for illustrative purposes and are not to be construed as restrictive.

EXAMPLE 1

This embodiment discloses the synthesis of the compounds of the present invention. As an illustration, the method of synthesis of 5-fluoropropynyl-2'- deoxyuridine (4) is outlined in Figure 1. 5-Iodo-2'-deoxyuridine was first protected by acetylation, which was reacted with propargylalcohol in Pd(0)-catalyzed coupling conditions. Triflation of the alcohol followed by S_N2 displacement with 'naked' fluoride failed to yield the desired product. Direct fluorination with Selectfluor (F-TEDA-BF₄₎ in the presence of Me₂S was also unsuccessful. Fluorination of compound 2 was carried out successfully at -78 °C, in anhydrous methylene chloride, with excess DAST. MeOH was used in the end of the reaction to quench excess of DAST. After flash column chromatography, 3 was obtained in 30% - 40%' yield. The removal of the acetate protective groups was complicated by the presence of the fluorine. Dilute methanolic ammonia converted the fluoro compound to an amine and the use of potassium carbonate resulted in hydrolysis to the alcohol. Deprotection could be affected by heating 3 in MeOH at 50 °C for 2 days in the presence of a catalytic amount of HC1. The final product was isolated by HPLC in analytically pure form. The experimental procedures are outlined in details below. The 'H-NMR spectrum of 4 is shown in Figure 2.

3\5'-Di-O-acetyl-5-iodo-2'-deoxyuridine (lV To 2.12 g (6 mmol) of 5-iodo- 2'-deoxyuridine suspended in 20mL of acetic anhydride, 122 mg (1 mmol) of dimethylaminopyridine (DMAP) was added as catalyst. After stirring for 12 h at room temperature, the reaction was completed, and the mixture was then left at 4 °C overnight. Product 1 was obtained in quantitative yield as white crystals (2.6 g), mp 156-158°C. Η-NMR (CDC1₃, Gemini-300) δ 2.11-2.20 (2s + m, 7H, 2'-H + COCH₃), 2.52 (m, 1H, 2"-H), 4.29-4.42 (m, 3H, 4', 5' + 5"-H), 5.22 (d, J=6.6 Hz, 1H, 3'-H), 6.29 (t, J=6.9 Hz, 1H, l'-H), 7.96 (s, 1H, 6-H).

of 1 (2.5 mmol) in 5 mL of DMF, 145 mg (0.125 mmol) of Pd(PPh₃)₄and 49 mg

(0.25 mmol) of Cul were added, followed by slow addition of 582 μL (10 mmol) of propargyl alcohol and 1.4 mL of TEA. The reaction mixture was then stirred at room temperature overnight. The solvent was evaporated in vacuo, and the product was purified by flash column chromatography on silica gel with ethyl acetate/hexane as eluent. The ratio of the two solvents was gradually changed from 1 : 1, 2: 1, to finally 3:1. Slowly increasing the polarity of the elution system was found to be important for the separation of compound 2 from contaminating colored metal complexes. Fractions containing the product were pooled, concentrated, and recrystallized from MeOH. Repeated recrystallization yielded 510 mg (56%) of pure 2; mp. 170-172 °C. Η-NMR (CDC1₃, Gemini-300) δ 1.80 (br s, IH, OH), 2.11 (s, 3H, Me), 2.17 (s, 3H, Me), 2.20 (m, IH, 2'-H), 2.58 (m, 1H,2"-H), 4.30-4.43 (m, 3H, 4',5' + 5"-H), 5.23 (m, IH, 3'-H), 6.28 (t, J= 7.05 Hz, IH, l'-H), 7.84 (s, 1H,6-

H). 3'.5'-Di-O-acetyl-5-(3-fluoropropyn-l-yl -2'-deoxyuridine (3). To a suspension of 2 (366.2 mg, 1 mmol) in 10 mL of anhydrous dichloromethane at -78 °C, was added (diethylamino)sulfur trifluoride (DAST), 661 μL, (5 mmol). The reaction mixture was stirred at -78 °C, for one and a half hour, then gradually warmed up to room temperature with continued stirring for another half an hour. MeOH was added at 0 °C to quench excess DAST reagent. Flash column chromatography on silica gel (ethyl acetate/hexane = 1 : 1) afforded 145 mg (40%) 3. Η-NMR (CDCL₃, Inova-500) δ 2.12-2.25 (m + 2s, 7H, 2'-H + Me), 2.56 (m, IH, 2"-H), 4.31-4.41 (m, 3H 4',5' + 5"-H), 5.14 (d, J= 47.5 Hz, 2H, CH₂F), 5.24 (m, IH, 3'-H), 6.30 (t, J= 6.75 Hz, IH), 7.94 (s, IH, 6-H). Anal, calcd. for C₁₆H₁₇FN₂O₇: C, 52.18; H, 4.65; N, 7.61. Found: C, 52.33; H, 4.80; N/7.35.

5-(3-Fluoropropyn-l-ylV2'-deoxyuridine (4V For 36 h, 100 mg (0.7 mmol) of 3 was heated in MeOH (50 °C) in presence of catalytic amount of hydrochloric acid. Compound 4 was isolated by HPLC in an aqueous MeOH system. Η-NMR (D₂O, Inova-400) δ 2.17-2.27 (m, 2H, 2' + 2"-H), 3.59-3.66 (m, 2H, 5' + 5"-H), 3.87 (m, IH, 3^*-H), 4.27 (m, IH 4'-H), 5.04 (d, J= 47.6 Hz, 2H, CH₂F), 6.05 (t, J, = 6.6 Hz, IH, l'-H), 8.06 (s, IH, 6-H). Anal, calcd. for C₁₂H₁₃FN₂O₅-0.6H₂O: C, 48.85; H, 4.85; N, 9.49. Found: C, 48.86; H, 4.51; N, 9.45.

In another illustration of this embodiment, 5-(3-fluoropropyn-l-yl)-2'- deoxyuridine (4) was synthesized as outlined in Figure 3. 5-Iodo-2'-deoxyuridine was first protected by silyl protecting groups by reacting with l,3-dichloro-l,l,3,3-tetraisopropyldisiloxane, in the presence of imidazole as base, in anhydrous DMF. This reaction gave 5 in quantitative yield. This protecting reagent was chosen, because trimethylsilyl protection is incompatible with the fluorination conditions employed in step (c). Other bulky silyl protecting groups, such as tertiary-butyldiphenylsilyl, may also be used. Pd(0)- catalyzed coupling of 5 with propargylalcohol led to 6, which was purified by flash column chromatography, after extraction with 5% EDTA. Fluorination of 6 was carried out at 0 °C by stirring for 10 min in anhydrous CH₂C1₂, using (diethylamino)sulfur trifluoride (DAST) in slight excess. After quenching the excess reagent with MeOH, the resulting 7 was purified by column chromatography and deprotected, using KF, to yield 4. The 5'-monophosphate, 5-(3-fluoropropyn-l- yl)-dUMP (8), was synthesized by selective phosphorylation of 4, using POCl₃ in PO(OEt)₃, as described by Sowa et al. (1975, Bull. Chem. Soc. Japan, 48:2084- 2090.

A detailed description of the procedures are presented below. 3 ' .5 ' -O-(Tetraisoprop yldisiloxane- 1.3 -diylV 5-iodo-2 ' -deoxyuridine (5). To a solution of 5-iodo-2'-deoxyuridine (3.54 g, 10 mmol) in anhydrous DMF (10 mL), imidazole (2.72 g, 40 mmol) was added, followed by 1,3-dichloro-l, 1,3,3, - tetraisopropyldisiloxane (3.4 g, 10.7 mmol). The reaction mixture was stirred at room temperature under argon for 3 h. After the solvent was evaporated, the residue was dissolved in CH₂C1₂, washed by ice-cold water. The organic layer was dried over sodium sulfate, and evaporated to a foam-like solid, which was crystallized from ethanol to give 5 as a colorless solid, mp. 180-182 °C; Η-NMR (CDC1₃ Inova-400) δ 0.93-1.11 (m, 28H, iPr₄), 2.28 (m, IH, 2'-H), 2.48 (m, IH, 2"- H), 3.77 (m, IH, 4'-H), 4.02 (m, IH, 5'-H), 4.13 (m, IH, 5"-H), 4.45 (m, IH, 3'-H), 5.98 (dd, J= 1.6 Hz & 7.2 Hz, IH, l'-H), 8.01 (s, IH, 6-H), 8.24 (br s, IH, NH). Anal, calcd. for C₂₁H₃₇IN₂O₆Si₂: C, 42.28; H, 6.25; N, 4.70. Found: C, 42.10; H, 6.25; N, 4.70. 3',5'-O-(Tetraisopropyldisiloxane-l,3-diyl)-5-(3-hydroxypropyn-l-yl)-2'- deoxyuridine (6). To 596 mg of 5 (1 mmol) in 5 mL of freshly distilled triethylamine, 58 mg (0.05 mmol) of Pd(PPh₃)₄ and 19 mg (0.10 mmol) of Cul were added, followed by slow addition of 145 μL (2.5 mmol) of propargyl alcohol. The reaction mixture was stirred at room temperature for 2-10 hours. After the solvent was evaporated in vacuo, the residue was dissolved in CH₂C1₂, washed by 5% EDTA solution and water, and chromatographed on silica gel (ethyl acetate/hexane = 1 : 1) to give 360 mg (69%) of 6 as a white solid. Mp. 96-98 °C. Η-NMR (CDC1₃ Inova-500) δ 0.90-1.13 (m, 28H, iPr₄), 2.30 (m, IH, 2'-H), 2.53 (m, IH, 2"-H), 3.31 (br s, IH, OH), 3.79 (m, IH, 4'-H), 4.03 (m, IH, 5'-H), 4.17 (m, IH, 5"-H), 4.43- 4.48 (s & m, 3H, 3'-H + CH₂OH), 6.04 (d, J= 6 Hz, IH, l'-H), 7.95 (s, IH, 6-H), 9.83 (br s, IH, NH). Anal, calcd. for C₂₄H₄₀N₂O₇Si₂: C, 54.93; H7.68; N, 5.34. Found: C, 54.93; H, 7.73; N, 5.17.

3 ' .5 ' -O-(Tetraisopropyldisiloxane- 1.3 -diyl V 5-(3 -fluoropropyn- 1 -yl -2 ' - deoxyuridine (7). To 524 mg (1 mmol) of 6 in 20 mL of anhydrous CH₂C1₂ at 0°C, was added DAST (165 μL, 1.25 mmol). The reaction mixture was stirred for 10 min at 0°C, then MeOH was added at 0°C to quench the excess DAST. Flash column chromatography on silica gel (CH₂Cl₂:MeOH = 100: 1) afforded 116 mg (22%) of 7, which was used directly for next step. Η NMR (CDC1₃, Inova-500) δ 0.89-1.08 (m, 28H, iPr₄), 2.25 (m, IH, 2'-H), 2.50 (m, IH, 2"-H), 3.75 (m, IH, 4'- H), 3.99 (m, IH, 5'-H), 4.14 (m, IH, 5'-H), 4.42 (m, IH, 3'-H), 5.10 (d, J= 47.5 Hz, 2H, CH₂F), 5.98 (d, J= 7 Hz, IH, l'-H), 8.00 (s, IH, 6-H), 9.32 (br s, IH, NH).

5 -(3 -Fluoropropyn- 1 -yl )-2 ' -deoxyuridine (4) . The above obtained 116 mg of 7 in 5 mL of DMF was treated with 100 mg (2 mmol) of KF for 3 h. Chromatography on a silica gel column (CH₂Cl₂:MeOH = 20: 1) gave 45 mg (66%) of 4. Η NMR (DMSO- , Inova-500) δ 2.11 (m, 2H, 2' + 2"-H), 3.54-3.58 (m, 2H, 5' + 5"-H), 3.77 (m, IH, 4'-H), 4.20 (m, IH, 3'-H), 5.12 (t, J- 4.8 Hz, IH, 5'-OH), 5.23 (d, J= 4.4 Hz, IH, 3'-OH), 5.25 (d, J= 47.2 Hz, 2H, CH₂F), 6.07 (t, J= 6.4 Hz, IH, l'-H), 8.32 (s, IH, 6-H), 11.68 (s, IH, NH). ¹³C NMR (DMSO-- Inova- 500) δ 40.2, 60.8, 70.0, 71.9, 82.5, 84.9, 86.4, 87.7, 96.8, 145.2, 149.4, 161.4, UV λ^ 287 nm (ε 10,400, pH = 2; ε 10,800, pH = 7). Anal, calcd. for C₁₂H₁₃FN₂O₅ 0.6H₂O: C, 48.85; H, 4.85; N, 9.49. Found: C, 48.86; H, 4.51; N, 9.45.

5-(3-Fluoropropyn-l-yl -2'-deoxyuridine-5'-monophosphate (8). In 2 mL of newly distilled triethylphosphate, was added 28 mg (0.1 mmol) of 4, followed by the addition of 14 μL (0.15 mmol) of phosphorus oxychloride in two portions. Reaction mixture was stirred at -10 °C for 5 h. 1 mL of water was added to hydrolyze the intermediate. Product was purified by reverse-phase preparative HPLC (gradient 0-100% MeOH/H₂O in 30 min.) and lyophilized to give 14 mg of pure product: Η NMR (D₂O, Inova-400) δ 2.15-2.26 (m, 2H, 2' + 2"-H), 3.89-3.93 (m, 2H, 5' + 5"-H), 4.01 (m, IH, 4'-H), 4.36 (m, IH, 3'-H), 5.04 (d, J= 47.6 Hz, 2H, CH₂F), 6.08 (t, J= 6.4 Hz, IH, l '-H), 8.04 (s, IH, 6-H).

EXAMPLE 2

This embodiment illustrates the synthesis of the imidazole derivatives. The synthesis of the imidazole derivatives. The synthesis of the following derivative

involves the Pd(O) -coupling of 5-iodo-2'deoxyuridine with propargylimidazole, which in turn is prepared by reacting propargylbromide with imidazole as described (Broggini et al., 1990, j. Chem. Soc. Perkin Trans, 1 :533). The N-methylimizolium derivative

is made from the imidazole derivatives described above, by simple alkylation with methyliodide yielding the iodide salt as the product. Details of these procedures can be obtained from Zhe, 2000(Ph.D. thesis, SUNY at Buffalo).

EXAMPLE 3

This embodiment describes the effect of the compounds of the present invention on the activity of the enzyme dTMP synthase. For the illustration of this embodiment, dTMP synthase was incubated with 5-(3-fluoropropyn-l-yl)-dUMP at 30 °C. Following this incubation, enzyme activity was assayed by a standard spectrophotometric method.

As shown in Figure 4, incubation of 5 μM 5-(3-fluoropropyn-l-yl)-dUMP with the enzyme caused a rapid inactivation of the enzyme (O). Essentially all enzyme activity was lost after 30 minutes. The presence of a saturating concentration (20 mM) of the substrate, dUMP, provided protection against inactivation of the enzyme (Δ), demonstrating that the interaction is specific for the active site of the enzyme. It should be noted that inactivation did not require the cofactor, Q LI-Lfolate, as is the case for FdUMP, the active form of the 5- fluoropyrimi dines. In another illustration of this embodiment, the time course of inactivation was also investigated for ImdPdUMP (^Δ) —d Imd+PdUMP (O). As shown in Figure 5, essentially all the activity was lost by about 40 minutes. The presence of a saturating concentration (20 mM) of the substrate dUMP (—) f°^r ImdPdUMP and (•)for Imd+PdUMP demonstrates that the interaction is specific for the active site of the enzyme.

The inhibition of dTMP synthase by several compounds of the present invention was investigated. dTMP synthase activity was assayed by following the changes associated with the conversion of tetrehydrofolate to dihydrofolate by a previously described method (Wahba and Friedkin, 1961, J. Biol. Chem., 236:PC11). The increase in molecular extinction during this conversion is 6400 at 340 nm (ΔC₃₄₀ = 6400). The reaction was run at 30°C and monitored in a spectrophotometer. Initial velocities of the dTMP synthase catalyzed reaction were determined by monitoring the increase of absorbency at 340 nm over a period of 5 minutes. The results were analyzed by the double-reciprocal plot of velocity versus substrate concentrations. Ki -values were calculated using Km values from the same experiment. The results for the following substituents represented by Z at the 5- position for the following compound are presented in Table 3

Table 3

Abbreviation used Z (5-Substituent) Kj (M)^a

TolPdUMP C_≡C-CH₂OCOC₆H₄CH₃ 0.065 ± 0.007

HOPdUMP C=C-CH₂OH 1.8 ± 0.6

PhPdUMP C=C-CH₂OC₆H₄NO₂ 0.11 ± 0.00

SPhPdUMP C_≡C-CH₂SC₆H₄NO₂ 0.060 ± 0.010

FPdUMP C_≡C-CH₂F 0.015 ± 0.003

SCNPdUMP C≡C-CH₂SCN 0.44 ± 0.04

MeOPdUMP C_≡C-CH₂OCH₃ 0.74 ± 0.20

AcOPdUMP C=C-CH₂OCOCH₃ 1.5 ± 1.1

ImdPdUMP 0.54 + 0.08

C=CCH₂ -N I

Imd+PdUMP 0.50 + 0.22

/^NCH₃ C≡CCH₂— N^

^a A ^-value of 6.8 ± 2.7 M was obtained for the substrate, dUMP; Kj- values are the mean of three determinations ± SD.

Experiments were also carried out to determine if the inactivation of the enzyme was reversible. Enzyme inactivated by 5-fluoropropynyl-dUMP in the absence of CH-PLfolate, in 100 uL assay mixture, was dialyzed against 350 mL phosphate buffer (changed every 2 hours). Remaining activity was assayed as a function of the time period of dialysis. The results are shown in Figure 6, (Δ) representing the free enzyme control in the absence of the inhibitor. As shown in Figure 6, when dTMP synthase, inactivated in the absence of GHbFLfolate with 5- (3-fluoropropyn-l-yl)-dUMP, was dialyzed exhaustively, no enzyme activity could be recovered over a period of 24 hours (O). These results indicate that the inactivation process is irreversible. As shown in Figure 7, similar results were also obtained for ImdPdUMP and Imd+PdUMP.

EXAMPLE 4 This embodiment demonstrates that the compounds of the present invention inhibit the growth of cells. To illustrate this embodiment, the effect of some representative compounds of the present invention was investigated on human chronic myelogenous leukemia K-562 cells. These cells are widely used to study the efficacy of potential cytotoxic agents in the treatment of cancer. K562 was propagated as a suspension culture, and passaged at a density of 4 x 10-> (per T-25 flask) three days prior to use in the assay. Each cell line was used for a maximum of 20 passages after which time a new vial of frozen cells was revived for use in the assay. The cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. For the assay, the cell suspension from the tissue culture flasks was centrifuged and resuspended to give a final cell count of 4 x 10^ cells/mL, and seeded on 96-well plates. The plates were placed in the incubator at 37 °C for 24 hrs. After addition of a solution of compound in varying concentrations, the plates were incubated for 96 hours. Viable cells were counted by the sulforhodamine B colorimetric procedure, by measuring the absorbance at 570 nm using a Rainbow plate reader. As shown in Figure 8, 5-fluoropropynyl deoxyuridine (FPdUrd) caused an inhibition of the growth of the K-562 cells. A plot of percent inhibition versus concentration yielded an IC₅₀ value of about 13 nM.

In another illustration of this embodiment, the growth of mouse lymphocytic leukemia cells in culture was also investigated. As shown in Figure 9, the IC₅₀ for these cells was observed to be about 3 nM.

The foregoing description of the specific embodiments is for the purpose of illustration and is not to be construed as restrictive. From the teachings of the present invention, those skilled in the art will recognize that modifications may be made without departing from the spirit of the invention.

Claims

WHAT IS CLAIMED IS:

1. A compound of the formula:

wherein X is a leaving group selected from the group consisting of halides, hetetocyclic group, -SCN, -N₃, and -CN, R is selected from the group consisting of -OH, -NH₂, and -SH; R₂ is selected from the group consisting of -H, 2-deoxyribose and ester derivatives of 2-deoxyribose.

2. The compound of claim 1 , wherein the halide is -F.

3. The compound of claim 1, wherein the heterocyclic group is selected from the group consisting of imidazole, N-methylimidazole, tetrazole and triazole.

4. A compound of the formula:

wherein X is a leaving group selected from the group consisting of halides, hetetocyclic group, -SCN, -N₃, and -CN, and wherein R₃ is selected from the group consisting of -H, phosphate, phosphate ester, and derivatives of phosphates or phosphate esters. The compound of claim 5, wherein the compound has the following formula

X

Wherein X is selected from the group consisting of -OCOC_gH₄CH₃, -OC₆H₄NO₂, -SC₆H₄NO₂, -F, -SCN, -OCH₃, -OCOCH₃,

6. The compound of claim 5, wherein X is -OCOC₆H₄CH₃.

7. The compound of claim 5, wherein X is OC_fiH₄NO₂

8. The compound of claim 5, wherein X is -SC₆H₄NO₂

9. The compound of claim 5, wherein X is -F

10. The compound of claim 5, wherein X is -SCN

11. The compound of claim 5, wherein X is -OCOCH₃

12. The compound of claim 5, wherein X is

13. The compound of claim 5, wherein X is

+ /^NCH₃

14. A compound of the formula

wherein X is a leaving group, R is selected from the group consisting of -OH, - NH₂, and -SH, wherein R₃ and R4 are independently -H or a protecting group.

15. The compound of claim 16, wherein the protecting group is selected from the group consisting of acetyl, benzoyl and silyl.

16. A method for inhibiting the growth of cells comprising the steps of contacting the cells with a growth inhibiting amount of compound of claim 1.

17. A method for inhibiting the growth of cells comprising the steps of administering to an individual a therapeutically effective amount of the compound of claim 1.

18. A method for inhibiting the growth of cells comprising the steps of contacting the cells with a growth inhibiting amount of compound of claim 4.

19. A method for inhibiting the growth of cells comprising the steps of contacting the cells with a growth inhibiting amount of compound of claim 4.

20. A method for inhibiting the growth of cells comprising the steps of contacting the cells with a growth inhibiting amount of compound of claim 9.

21. A method for inhibiting the growth of cells comprising the steps of administering to an individual a therapeutically effective amount of the compound of claim 9.

22. A method for inhibiting the growth of cells comprising the steps of contacting the cells with a growth inhibiting amount of compound of claim 14.

23. A method for inhibiting the growth of cells comprising the steps of administering to an individual a therapeutically effective amount of the compound of claim 14.

24. A method of preparing the compound of claim 1 comprising the steps of a) protecting the 3 '- and 5'-hydroxy groups of 5-iodo-2'- deoxyuridine by a blocking group; b) attaching a side chain to the 5-position of the pyrimidine ring by a Pd(O)-catalyzed coupling of a propargyl derivative; c) deprotecting the product in b) by removal of the blocking groups from the 3' and 5 '-hydroxyl groups; d) purifying the deprotected product.

25. The method of claim 24, wherein the propargyl derivative is propargylalcohol and further comprising the step of fluorinating the propargylalcohol side chain following attachment to the 5-position of the pyrimidine ring.

26. The method of claim 24, wherein the blocking groups are selected from the group consisting of acetyl, silyl, benzoyl, toluoyl, mesyl, and tosyl.

27. The method of claim 24, wherein the propargyl derivative is propargylimidazole. 28. The method of claim 27 further comprising the step of methylating the propargylimidazole side chain following attachment to the 5-position of the pyrimidine ring.