Synthesis and antitrypanosomal activities of a series of 7-deaza-5'-noraristeromycin derivatives with variations in the cyclopentyl ring substituents

Previous work in our laboratories has suggested that (+)-5'-nor-7-deazaaristeromycin (compound 1) may represent a prototype structure for a series of compounds with significant antitrypanosomal activities. To test this possibility, a series of derivatives of compound 1 with changes in the cyclopentyl substituents (compounds 3 to 10) have been studied. Although some growth activity was obtained with the L-like compound 5, related compounds 3 and 7 had little or no activity below 100 microM. D-like compounds 4 and 6 showed some activity at or below 100 microM, but the most interesting finding was that both the D- and L-like compounds having a methyl substituent on the 4' position were most active.

Melting points were recorded on a Meltemp II melting point apparatus and are uncorrected. Combustion analyses were performed by M-H-W Laboratories, Phoenix, Ariz. 1 H and 13 C spectra were recorded on a Bruker AC 250 spectrometer (operated at 250 and 62.5 MHz, respectively), all referenced to internal TMS at 0.0 ppm. The spin multiplicities are indicated by the symbols s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). Optical rotations were measured on a JASCO DIP-370 polarimeter. Reactions were monitored by TLC with 0.25-mm E. Merck silica gel 60-F 254 -precoated silica gel plates, with visualization by irradiation with a Mineralight UVGL-25 lamp or exposure to iodine vapor. Column chromatography was performed on Aldrich silica gel (average particle size, 5 to 25 m; 60 Å) and elution with the indicated solvent system. Yields refer to chromatographically and spectroscopically ( 1 H and 13 C NMR) homogeneous materials. The structures of the compounds synthesized in this study are presented in Fig. 1. Note that the discrepancy in the [␣] D values between enantiomers 3 and 4 suggests that one or both of these compounds are not enantiomerically pure.
To a suspension of 4-chloropyrrolo[2,3-d]pyrimidine (4.31 g, 28.06 mmol) (6) and PPh 3 (7.40 g, 28.06 mmol) in dry THF (150 ml) was added DEAD (4.93 g, 28.06 mmol). After stirring this mixture vigorously under an argon atmosphere at room temperature for 5 min, a solution of (ϩ)-2,3-isopropylidenedioxy-4-cyclopenten-1-ol (4.0 g, 25.61 mmol) (14) in dry THF (100 ml) was added, and the reaction mixture was stirred at 55°C for 2 days (9,12). The solvent was removed and the residue was purified via column chromatography, eluting with hexane, followed by elution with hexane-EtOAc (9:1 and then 5:1). Fractions containing product were evaporated to dryness to give 3.81 g (42%) of compound 13 as a colorless syrup, which crystallized upon cooling (mp, 85 to 86°C); 1 10.60 mmol) in saturated methanolic NH 3 (75 ml) was sealed in a steel bomb, and the steel bomb was heated at 120°C for 2 days. The bomb was cooled, the solvent was removed, and the residue was refluxed with Dowex (50 ϫ 8) resin beads and 0.5 N HCl (150 ml) until no starting material remained by TLC (9:1, CH 2 Cl 2 -MeOH). The suspension was evaporated and loaded onto a Dowex (50 ϫ 8) resin column, and the product was eluted with concentrated NH 3 . Fractions containing product were combined and evaporated. The residue was purified via column chromatography, eluting with CH 2 Cl 2 -MeOH (9:1). Fractions containing product were combined and evaporated. The resultant tan solid was refluxed with decolorizing charcoal and was filtered hot. The filtrate was evaporated, and the resultant tan solid was triturated in MeOH to afford 1.9 g (77%) of compound 7 as a pale beige solid (

-[4-Methyl-2,3-(isopropylidenedioxy)-cyclopentan-1yl]-4-chloropyrrolo[2,3-d]pyrimidine (compound 17).
A slurry of copper (I) iodide (6.6 g, 34.7 mmol) in dry THF (75 ml) was cooled to Ϫ20°C, and methyllithium (1.4 M in Et 2 O, 49.8 ml, 69.72 mmol) was added over a period of 15 min under an argon atmosphere (18). After stirring for 5 min at Ϫ20°C, a solution of (4S,5S)-4,5-(isopropylidenedioxy)-2-cyclopentenone (3.0 g, 19.5 mmol) (14) in dry THF (50 ml) was added, and this mixture was stirred for 30 min, quenched (200 ml of saturated NH 4 Cl-concentrated NH 3 solution [1:1]), and stirred vigorously for an additional 15 min. The THF layer was separated, and the aqueous layer was extracted with Et 2 O (three times with 100 ml each time). The combined organic phases were dried (MgSO 4 ) and evaporated. The residual yellow liquid was purified via column chromatography, eluting with Et 2 O. The fractions containing product were combined and evaporated to yield 3.0 g of a yellow oil, which was dissolved in dry THF (50 ml). This solution was cooled to Ϫ20°C and treated with BH 3 ⅐ THF (41 ml, 1.0 M solution, 41 mmol). After stirring at room temperature under an argon atmosphere for 3 h, the solvent was evaporated and the residue was coevaporated with dry MeOH (three times with 50 ml each time) to afford 3.0 g (90%) of compound 15 as a yellow oil which was used directly in the next step without further purification.

RESULTS AND DISCUSSION
Target compounds 3 and 4 were obtained by catalytic hydrogenation of compounds 7 and 8, respectively. These latter precursors were prepared by a Mitsunobu coupling (12) of the enantiomeric allylic alcohols 11 and 12 with 4-chloropyrrolo [2,3-d]pyrimidine (6) to give compounds 13 and 14, respectively. Ammonolysis of compounds 13 and 14 followed by treatment with Dowex acidic resin for deblocking the 2Ј,3Јhydroxyl groups resulted in compounds 7 and 8, respectively. Catalytic hydrogenation of compounds 7 and 8 yielded target compounds 3 and 4, respectively.
The final new targets, compounds 9 and 10, were obtained by ammonolysis of chloro precursors 19 and 20, respectively, which were used previously as precursors to compounds 1 and 2, respectively (11). Table 1 details the results of growth inhibition studies with compounds 1 through 10. The data (11) from compound 1 are included as a reference. Of the other analogs, compound 6 showed activity, with IC 50 s of approximately 40 M for four test strains. The other compounds showing activity, although at reduced levels, were 4, which was 30 to 40% inhibitory to the growth of all strains; 5, which had modest activity against three of four isolates; 9, which had activity against the two isolates tested; and 10, with an IC 50 of 100 M for T. brucei brucei and 40% inhibition of T. brucei rhodesiense KETRI 243 at 100 M.
The results of this study clarify the structural components of compound 1 that are necessary for its antitrypanosomal activity, because none of the analogs showed the same potency as compound 1. Compound 3 indicated that the 4Ј-hydroxyl of compound 1 is necessary for the observed activity, while compound 9 demonstrated that the 2Ј-and 3Ј-hydroxyl substituents are needed. Methyl derivative 5 provided support that a lipophilic group at C-4Ј did not improve activity, as was expected for AdoHcy hydrolase inhibitors (19). Compound 7 was studied as an analog of the potent AdoHcy hydrolase inhibitor neplanocin A and was found to exhibit diminished activity. The D series of compounds, compounds 2, 4, 6, 8, and 10, were all less active than compound 1 with the L-like configuration. It is, thus, apparent from these results that any future compounds designed to improve upon the antitrypanosomal properties of a Trypanosomes were grown in modified IMDM plus 20% horse serum in 24-well microplates at 37°C (9). The wells were inoculated with 10 5 trypanosomes, and the compounds were solubilized in medium. The cells were diluted 1:1 daily with fresh medium and drug, and cell counts (Coulter counter) were made at 24 and 48 h. The final control count after 48 h was approximately 5 ϫ 10 6 /ml. IC 50 s were determined from semi-logarithmic plots by using duplicate determinations. compound 1 must posses the 2Ј-, 3Ј-, and 4Ј-hydroxyl groups and, preferably, be in the L-like configuration.