Table 1.
IC50 Values (nM) for Inhibition of Proliferation of CHO and KB Sublinesa
| antifolate | RFC
|
FRα
|
FRβ
|
PCFT
|
FRα/PCFT/RFC
|
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| PC43-10 | R2 | RT16 | RT16 (+FA) | D4 | D4 (+FA) | R2/hPCFT4 | R2(VC) | KB | KB (+FA) | Ade/Thd/AICA | |
| 4 | 101.0(16.6) | 273.5(49.1) | 0.38(0.12) | 173(35) | 0.17(0.03) | 126(13) | 3.34(0.26) | 288(12) | 0.26(0.03) | 101(7) | Ade/AICA |
| 5 | 197(49) | 355(10) | 0.33(0.15) | 494(254 | 0.34(0.03) | 557(147) | 5.39(1.27) | >1000 | 0.17(0.05)* | 337(127) | Ade/AICA |
| 6 | 54.0(9.8) | >1000 | 154(50) | >1000 | 17.5(0.6) | >1000 | >1000 | >1000 | 20.7(4.9) | 275(25) | Ade/AICA |
| 7 | 189(51) | 290(8) | 0.61(0.11) | 506(118) | 0.10(0.01) | 654(169) | 6.51(1.30) | >1000 | 0.09(0.02)** | 105(3) | Ade/AICA |
| 8 | >1000 | >1000 | 148(28) | >1000 | 54.3(9.6) | >1000 | >1000 | >1000 | 70.3(13.7) | >1000 | Ade/AICA |
| MTX | 12(1.1) | 216(8.7) | 114(31) | 461(62) | 106(11) | 211(43) | 120.5(16.8) | >1000 | 6.0(0.6) | 20(2.4) | Ade/Thd |
| PDX | 0.69(0.07) | 819(94) | 43 (6) | 289 (33) | 2.5(0.1) | 951(29) | 57(12) | >1000 | 0.47(0.20) | 1.94(0.28) | Ade/Thd |
| PMX | 138(13) | 894(93) | 42(9) | 388(68) | 60(8) | 254(78) | 13.2(2.4) | 974.0(18.1) | 68(12) | 327(103) | Thd/Ade |
| LMTX | 12(2.3) | >1000 | 12(8) | 188(41) | 2.6(1.0) | 275(101) | 248.0(18.2) | >1000 | 1.2(0.6) | 31(7) | Ade/AICA |
Growth inhibition assays were performed with isogenic CHO sublines engineered to express human RFC (PC43-10), FRα (RT16), FRβ (D4), or PCFT (R2/PCFT4) from transporter-null (R2) CHO cells.24,36,37 R2(VC) were R2 cells transfected with empty PCDNA3. Results are also shown for the KB human tumor subline (expresses RFC, FRα, and PCFT).39 For the FRα experiments, growth inhibition assays were performed in the presence and absence of excess (200 nM) folic acid. Results shown are mean values from 3–10 experiments (± standard errors in parentheses) and are presented as IC50 values, representing the concentrations that inhibit growth by 50% relative to cells incubated without drug. Certain data for MTX, PDX, PMX, and LMTX were previously published. 24–27,29,37 Results are also summarized for the protective effects of adenosine (60 μM) or thymidine (10 μM), or 5-aminoimidazole-4-carboxamide (320 μM) with KB cells, as shown in Figure 9. The structures for compounds 4–8 are in Figure 3. Experimental details are provided in the Experimental Procedures. Undefined abbreviations: Ade, adenosine; AICA, 5-aminoimidazole-4-carboxamide; FA, folic acid; ND, not determined; Thd, thymidine. Statistics: *p < 0.005 and **p < 0.05 when compared to 4 in KB cells.