Evaluation of O^3α-, O²¹-Di-(N¹-methyloxycarbonyl-2, 4-dioxo-5-fluoropyrimidinyl)17α-hydroxy-5β-pregnan-20-one as a novel potential antiangiogenic codrug

Figures & data

Table I.  The angiostatic response between 5FU, THS, physical mixtures of THS and 5FU versus the THS-5FU codrug at varying dosages. Angiostatic effect is determined by the presence of an avascular zone. An avascular zone of ≥4 mm at the site of implantation indicates a positive response, otherwise a negative response is indicated.

Compound (molar ratio)	Dose	Positive response/Total number of embryos	% Positive response
5FU	100 μg	0/29	0%
	50 μg	1/29	3%
	25 μg	3/28	10%
THS	100 μg	0/28	0%
	50 μg	0/27	0%
	25 μg	0/30	0%
THS:5FU (1:1)	50 μg : 18 μg	10/27	37.0%
THS:5FU (1:2)	50 μg : 36 μg	15/33	45.5%
THS-BIS-5FU (LD)	25 μg : 18 μg	10/22	45.5%
THS-BIS-5FU (HD)	50 μg : 36 μg	12/17	70.6%

Figure 1 Synthesis of 3α-O, 21-O-(dichloroformyl)-5β-pregnane-17-ol-20-one (5) from Reichstein's Substance S (1).

Figure 2 Coupling of 3α-O, 21-O-(dichloroformyl)-5β-pregnane-17-ol-20-one (6) and N¹,N³-Bis-dihydroxymethyl 5-fluorouracil (8) to produce O³-(N¹-methyloxycarbonyl-5-fluorouracil)5-β-pregnane-17-ol-20-one (9),O²¹-(N¹-methyloxycarbonyl-5-fluorouracil)5-β-pregnane-17-ol-20-one (10), and O³-, O²¹-Di(N¹-methyloxycarbonyl-5-fluorouracil)5-β-pregnane-17-ol-20-one; (THS-BIS-5FU) (11).

Figure 3 Proposed hydrolysis of the model THS-BIS-5FU codrug (11). One molar equivalent of codrug should generate 1 molar equivalent of THS, 2 molar equivalents of 5FU, and 2 molar equivalents of both formaldehyde and carbon dioxide.

Figure 4 The appearance of 5FU (•) and THS (▴) in molar concentration from the degradation of THS-BIS-5FU codrug (▪) in phosphate buffer (0.1 M, pH 7.4).

Figure 5 The appearance of 5FU (•) and THS (▴) in molar concentration from the degradation of THS-BIS-5FU codrug (▪) in human serum.

Figure 6 The appearance of 5FU (•) and THS (▴) in molar concentration from the degradation of THS-BIS-5FU codrug (▪) in bovine vitreous humor.

Figure 7 Formation of a 17:20:21 cyclic intermediate via intramolecular cyclization.

Figure 8 Chemical structure of a proposed THS carbonate ester, the cyclic intermediate formed from the hydrolysis of the O^3α-, O²¹-di-(N¹-methyloxycarbonyl-2, 4-dioxo-5-fluoropyrimidinyl)17α- hydroxy-5β-pregnan-20-one codrug [THS-BIS-5FU] (11), and the degradation of the cyclic intermediate to form THS.

Figure 9 Proposed hydrolysis products, based upon ¹H-NMR spectrum of the isolated hydrolysis intermediate O^3α-, O²¹-di-(N¹-methyloxycarbonyl-2, 4-dioxo-5-fluoropyrimidinyl)17α- hydroxy-5β-pregnan-20-one codrug [THS-BIS-5FU] (11).