Table 1.
Cell Proliferation Assays with 6-Substituted Pyrrolo[2,3-d]pyrimidine Analoguesa
| CHO (IC50, nM) | HeLa (IC50, nM) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| RFC | FRα | FRβ | PCFT | RFC | PCFT | FRα | ||||
| group | antifolate | 2′/3′-F | PC43–10 | R2 | RT16 | D4 | R2/PCFT4 | R1–11-RFC2 | R1–11-PCFT4 | R1–11-FR2 |
| − | 1 | − | 106(17) | 249(25) | 0.31(0.15) | 0.16(0.02) | 3.3(0.5) | 444(5) | 53.1(15.5) | 30.2(5.6) |
| a | 2 | − | >1000 | >1000 | 6.3(1.6) | 6.3(1.6) | 213(28) | >1000 | 5.21(1.33) | 11.6(2.7) |
| a | 8 | + | >1000 | >1000 | 0.58(0.12) | 1.6(0.44) | 23(2) | >1000 | 4.57(1.48) | 7.78(1.89) |
| b | 3 | − | 304(89) | >1000 | 4.1(1.6) | 5.6(1.2) | 23.0(3.3) | >1000 | 9.71(2.78) | 12.2(4.4) |
| b | 9 | + | 62(12)* | 140(27)* | 1.12(0.37)* | 3.87(0.14)* | 3.82(0.27)* | 507(76) | 5.54(3.35) | 5.22(1.32) |
| c | 4 | − | >1000 | >1000 | 1.3(0.1) | 0.51(0.09) | 30.4 (10.7) | 634(43.9) | 8.98(3.64) | 6.54(1.29) |
| c | 10 | + | 468(142) | >1000 | 0.69(0.39) | 0.44(0.13) | 67(25) | >1000 | 21.57(4.54)* | 21.7(3.9)* |
| d | 5 | − | >1000 | >1000 | 2.5(0.5) | 0.43(0.14) | 41.5(3.1) | >1000 | 72.6(18.1) | 35.3(6.2) |
| d | 11 | + | >1000 | >1000 | 0.36(0.13) | 0.75(0.42) | 6.00(0.94)* | 213(9) | 5.01(2.12)* | 2.84(0.67)* |
| e | 6 | − | 197(49) | 355(10) | 0.33(0.15) | 0.34(0.03) | 5.4(1.3) | 62.9(3.8) | 40.0(9.8) | 7.57(1.16) |
| e | 12 | + | 20.1(6.2)* | 20.5(1.6)* | 0.14(0.03)* | 0.19(0.02) | 1.5(0.4)* | 66.2(6.2) | 20.4(4.7)* | 3.11(0.69)* |
| − | PMX | − | 26.2(5.5) | 138(13) | 42(9) | 60(8) | 8.3(2.7) | 23.8(1.5) | 66.8(4.8) | 863(81) |
Growth inhibition assays were performed using CHO sublines derived from RFC-, PCFT-, and FR-null MTXRIIOuaR2–4 CHO cells (R2) engineered to express human RFC (PC43–10), PCFT (R2/hPCFT4), FRα (RT16), or FRβ (D4).25,43–46 Additional experiments were performed with isogenic HeLa sublines derived from RFC-, PCFT-, and FR-null R1–11 HeLa cells, expressing RFC (R1–11RFC2), PCFT (R1–11PCFT4), or FRα (R1–11FR2).28,47,48 For all experiments, folate-free RPMI 1640 with 10% dialyzed FBS and antibiotics was used including 2 nM LCV (RT16 and D4 CHO) or 25 nM LCV (R2, PC43–10, and R2/PCFT4 CHO; R1–11RFC2, R1–11PCFT4, R1–11FR2 HeLa). Results are shown as mean values from three to five experiments (± standard errors in parentheses) and are presented as calculated IC50 values representing the concentrations at which growth of 50% of cells was inhibited relative to untreated cells. IC50 values of fluorinated compounds that are statistically different from the corresponding non-fluorinated compounds within each cell line are marked with * (p < 0.05). Groups a, b, c, etc. designated paired structural homologs differing by virtue of the absence or presence of a 2′ or 3′ fluorine.