Table 1. IC50s (in nM) for 6-substituted pyrrrolo[2,3-d]pyrimidine benzoyl antifolates with heteroatom replacements, and classical antifolates in RFC-, PCFT- and FR-expressing cell lines.
Proliferation assays were performed for CHO sublines engineered to express human RFC (PC43-10), FRa (RT16), FRβ (D4) or PCFT (R2/PCFT4), and transporter-null (R2) CHO cells,29, 46–48 and KB human tumor cells (express RFC, FRa, and PCFT). For the experiments measuring FR-mediated effects, assays were performed in the presence or absence of 200 nM folic acid (results are shown only for KB cells). Results are presented as IC50 values, corresponding to the concentrations that inhibit growth by 50% relative to cells incubated without drug. The data are mean values from 5–16 experiments (+/− standard errors in parentheses). Some of the data for 1, 2, 3, MTX, PDX, PMX and LMTX have been previously published.29–31, 34, 47, 49 Results are also summarized for KB cells for the protective effects of adenosine (60 μM), thymidine (10 μM), or 5-aminoimidazole-4-carboxamide (320 μM). For compounds 4, 5, 6, 7, 8, and 9, folic acid and nucleoside/AICA protection results are shown in Figure 10. Methods are summarized in the Experimental Section. Undefined abbreviations: Ade, adenosine; AICA, 5-aminoimidazole-4-carboxamide; FA, folic acid; ND, not determined; Thd, thymidine. *Significantly different from compound 4 with RT16 or KB cells, as appropriate (p<0.05); ϕθ¥, significantly different from RT16 with 4 (p=0.0006), 6 (p=0.0026), and 5 (p=0.0001)
| Antifolate | RFC | FRa | FRβ | PCFT | RFC/FRa/PCFT | ||||
|---|---|---|---|---|---|---|---|---|---|
|
| |||||||||
| PC43-10 | R2 | RT16 | D4 | R2/PCFT4 | R2 | KB | KB (+FA) | KB + Ade/Thd/AICA | |
|
| |||||||||
| 1 | 649(38) | >1000 | 4.1(1.6) | 5.6(1.2) | 23.0(3.3) | >1000 | 1.7(0.4) | >1000 | Ade/AICA |
| 2 | >1000 | >1000 | 6.3(1.6) | 10(2) | 213(28) | >1000 | 1.7(0.4) | >1000 | Ade/AICA |
| 3 | 101.0 (16.6) | 273.5 (49.1) | 0.31 (0.14) | 0.17 (0.03) | 3.34(0.26) | 288(12) | 0.26(0.03) | 101(7) | Ade/AICA |
| 4 | 510(90) | >1000 | 3.04 (0.71) | 0.62 (0.20)ϕ | 87.4(9.9) | >1000 | 0.32(0.05) | 666(46) | Ade/AICA |
| 5 | 642(239) | >1000 | 3.01 (0.57) | 0.53 (0.11)¥ | 88.5(13.4) | >1000 | 2.09(0.72)* | >1000 | Ade/AICA |
| 6 | 808(124) | >1000 | 4.87 (0.81) | 1.57 (0.53)θ | 81.7(91) | >1000 | 2.57(0.60)* | >1000 | Ade/AICA |
| 7 | 783(109) | >1000 | 1.08 (0.69) | 0.45 (0.11) | 109 (26) | >1000 | 0.56(0.05)* | 924(40) | Ade/AICA |
| 8 | 641(140) | 940(60) | 1.89 (0.85) | 2.59 (1.17) | 57.5(5.8) | >1000 | 0.34(0.05) | 704(73) | Ade/AICA |
| 9 | >1000 | >1000 | 0.51 (0.10)* | 0.34 (0.02) | 267(19) | >1000 | 1.66(0.26)* | >1000 | Ade/AICA |
| MTX | 12(1.1) | 114(31) | 114 (31) | 106 (11) | 121 (17) | >1000 | 6.00(0.60) | 20(2.4) | Thd/Ade |
| PMX | 138(13) | 42(9) | 42 (9) | 60 (8) | 13.2(2.4) | 974 (18) | 68(12) | 327(103) | Thd/Ade |
| RTX | 6.3(1.3) | 15(5) | 15 (5) | 22 (10) | 99.5(11.4) | >1000 | 5.90(2.20) | 22(5) | Thd |
| LMTX | 12(2.3) | 12(8) | 12 (8) | 2.6 (1.0) | 38.0(5.3) | >1000 | 1.20(0.60) | 31(7) | Ade/AICA |