Identification, synthesis, and biological evaluation of metabolites of the experimental cancer treatment drugs indotecan (LMP400) and indimitecan (LMP776) and investigation of isomerically hydroxylated indenoisoquinoline analogues as topoisomerase I poisons

Abstract

Hydroxylated analogues of the anticancer topoisomerase I (Top1) inhibitors indotecan (LMP400) and indimitecan (LMP776) have been prepared because (1) a variety of potent Top1 poisons are known that contain strategically placed hydroxyl groups, which provides a clear rationale for incorporating them in the present case, and (2) the hydroxylated compounds could conceivably serve as synthetic standards for the identification of metabolites. Indeed, incubating LMP400 and LMP776 with human liver microsomes resulted in two major metabolites of each drug, which had HPLC retention times and mass fragmentation patterns identical to those of the synthetic standards. The hydroxylated indotecan and indimitecan metabolites and analogues were tested as Top1 poisons and for antiproliferative activity in a variety of human cancer cell cultures and in general were found to be very potent. Differences in activity resulting from the placement of the hydroxyl group are explained by molecular modeling analyses.

Figure 1

Representative Top1 Inhibitors.

Figure 2

Proposed metabolic pathways and potential metabolites of 6 and 7.

Figure 3

LC-MS retention times (a) and positive ion electrospray ion tandem mass spectra fragmentation patterns (b) for metabolites obtained upon incubation of 6 with human liver microsomes.

Figure 4

LC-MS retention times (a) and positive ion electrospray ion tandem mass spectra fragmentation patterns (b) for metabolites obtained upon incubation of 7 with human liver microsomes.

Figure 5

Top1-mediated DNA cleavage induced by indenoisoquinolines 22b, 14b, 35a, 44a, and 45a. Lane 1: DNA alone; lane 2: Top1 + DNA; lane 3: 1, 1 μM; lane 4: 5, 1 μM; lane 5–24: 22b, 14b, 35a, 44a, and 45a at 0.1, 1, 10 and 100 μM respectively from left to right. Numbers and arrows on the left indicate arbitrary cleavage site positions.

Figure 6

Minimized, top-ranked GOLD pose of compound 22b in ternary complex with DNA and Top1, constructed in SYBYL. The ligand is colored in purple, surrounding structures are colored by atom, water molecules are depicted as red spheres, and hydrogens have been omitted. All distances are measured from heavy-atom to heavy-atom. The diagram is programmed for wall-eyed (relaxed) viewing.

Figure 7

Minimized, top-ranked GOLD pose of compound 14b in ternary complex with DNA and Top1 (constructed in SYBYL). The ligand is colored in cyan, surrounding structures are colored by element, and hydrogens have been omitted. All distances are measured from heavy-atom to heavy-atom. The same water molecules from Figure 4 are shown as red spheres.

Figure 8

11-Hydroxycamptothecin.

Scheme 1^a

^aReagents and conditions: (a) BnBr, DMF, K₂CO₃, r.t.; (b) 3-bromopropylamine HBr; (c) CHCl₃, 10 °C - r.t.; (d) i. SOCl₂, r.t., ii. AlCl₃ (2 eq.), 1,2-dichloroethane, r.t.; (e) imidazole or morpholine, NaI, DMF, 70 °C.

Scheme 2^a

^aReagents and conditions: (a) BnBr, DMF, K ₂CO₃, r.t.; (b) 3-bromopropylamine HBr, Et₃N, Na₂SO₄, CHCl₃, r.t.; (c) 11, CHCl₃, 10 °C - r.t.; (d) SOCl₂, r.t.; (e) imidazole or morpholine, NaI, DMF, 70 ° C; (f), HBr, AcOH, H₂O, 55–70 °C.

Scheme 3^a

^aReagents and conditions: (a) PMB-Cl, DMF, K₂CO₃, 70 °C; (b) 3-bromopropylamine HBr, Et ₃N, Na ₂SO₄, CHCl ₃, r.t.; (c) 11, CHCl₃, 0 °C - r.t.; (d) SOCl₂, r.t.; (e) imidazole or morpholine, NaI, DMF, 70 °C or dioxane, reflux.

Scheme 4^a

^aReagents and conditions: (a) i. KOH, BnCl, EtOH, reflux, ii. KOH, H₂O, reflux; (b) H₂CO, H₂O, HCl, AcOH, 45 °C; (c) i. KOH, H₂O, r.t., ii. KMnO₄, H₂O, 0 °C-r.t., iii. EtOH, reflux; (d) AcCl, reflux; (e) 3-bromopropylamine HBr, Et ₃N, Na₂SO₄, CHCl₃, r.t.; (f) CHCl₃, 0 ° C - r.t.; (g) i. SOCl₂, r.t., ii. AlCl₃ (2 eq.), DCE, 0 °C; (h), imidazole or morpholine, NaI, DMF, 60 °C.

Scheme 5^a

^aReagents and conditions: (a) H₂CO, H₂O, HCl, AcOH, 120 °C-r.t.; (b) BnBr, K₂CO₃, acetone, 56 °C; (c) i. KOH, H₂O, r.t., ii. KMnO₄, H₂O, 0 °C-r.t., iii. EtOH, reflux; (d) AcCl, reflux; (e) 3-bromopropylamine HBr, Et ₃N, Na₂SO₄, CHCl₃, r.t.; (f) CHCl₃, 0 °C - r.t.; (g) SOCl₂, r.t.,(h) AlCl₃ (2 eq.), nitrobenzene, 90 °C; (i), imidazole or morpholine, NaI, DMF, 70 °C.

Scheme 6^a

^aReagents and conditions: (a) NaBH₄, MeOH, 0 °C.

Scheme 7^a

^aReagents and conditions: (a) BnBr, DMF, r.t.−70 °C; (b) 3-bromopropylamine HBr, Et₃N, Na₂SO₄, r.t.; (c) 11, CHCl₃, 0 °C - r.t.; (d) SOCl₂, −4 °C to r.t.; (e) imidazole or morpholine, NaI, dioxane, 65 °C; (f) 48% HBr-H₂O, 70 °C (for 51a); (g) H₂, Pd/C, MeOH-THF (for 51b).