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. 2018 May 15;26(9):2437-2451.
doi: 10.1016/j.bmc.2018.04.010. Epub 2018 Apr 4.

Structure based drug design and in vitro metabolism study: Discovery of N-(4-methylthiophenyl)-N,2-dimethyl-cyclopenta[d]pyrimidine as a potent microtubule targeting agent

Weiguo Xiang  1 , Shruti Choudhary  1 , Ernest Hamel  2 , Susan L Mooberry  3 , Aleem Gangjee  4
Affiliations

Structure based drug design and in vitro metabolism study: Discovery of N-(4-methylthiophenyl)-N,2-dimethyl-cyclopenta[d]pyrimidine as a potent microtubule targeting agent

Weiguo Xiang et al. Bioorg Med Chem. .

Abstract

We report a series of tubulin targeting agents, some of which demonstrate potent antiproliferative activities. These analogs were designed to optimize the antiproliferative activity of 1 by varying the heteroatom substituent at the 4'-position, the basicity of the 4-position amino moiety, and conformational restriction. The potential metabolites of the active compounds were also synthesized. Some compounds demonstrated single digit nanomolar IC50 values for antiproliferative effects in MDA-MB-435 melanoma cells. Particularly, the S-methyl analog 3 was more potent than 1 in MDA-MB-435 cells (IC50 = 4.6 nM). Incubation of 3 with human liver microsomes showed that the primary metabolite of the S-methyl moiety of 3 was the methyl sulfinyl group, as in analog 5. This metabolite was equipotent with the lead compound 1 in MDA-MB-435 cells (IC50 = 7.9 nM). Molecular modeling and electrostatic surface area were determined to explain the activities of the analogs. Most of the potent compounds overcome multiple mechanisms of drug resistance and compound 3 emerged as the lead compound for further SAR and preclinical development.

Keywords: Cyclopenta[d]pyrimidine; Metabolism; Microtubule targeting agent.

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Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Structures of microtubule targeting agents.
Figure 2
Figure 2
A. Superimposition of the docked poses of 1 (green), 3 (magenta) and colchicine (pink) in the colchicine site of tubulin (PDB ID: 4O2B). A dash line represented the border of α-tubulin and β-tubulin. B. Superimposition of the docked poses of 1 (green), 5 (orange) and colchicine (pink) in the colchicine site of tubulin (PDB ID: 4O2B).
Figure 3
Figure 3
A. Electrostatic surface of the colchicine site binding pocket in tubulin. B. Electrostatic surface of colchicine. C. Electrostatic surfaces of compounds 1–8. Red surface indicates electron rich surface, blue surface indicates electron deficient surface, and white surface represents hydrophobic surface. Residues Val238, Cys241, Leu248 and Asp251 belong to the β-chain of tubulin.
Figure 3
Figure 3
A. Electrostatic surface of the colchicine site binding pocket in tubulin. B. Electrostatic surface of colchicine. C. Electrostatic surfaces of compounds 1–8. Red surface indicates electron rich surface, blue surface indicates electron deficient surface, and white surface represents hydrophobic surface. Residues Val238, Cys241, Leu248 and Asp251 belong to the β-chain of tubulin.
Figure 4
Figure 4
Identification the metabolites of 3 by LC/MS/MS. HPLC spectra of 3 incubated with human liver microsomes at 0 min and 60 min. Metabolites M1–M5 and parent 3 were illustrated on the spectra. Some peaks (retention times larger than compound 3) in 60 min sample have different absorbance compared to that in 0 min sample, which may due to metabolic changes of the microsomes initiated by compound 3.
Figure 5
Figure 5
1H NMR of N4-desmethyl compound 3a and compound 3. In 3a, the 5-position hydrogens have a chemical shift 2.76 ppm. In compound 3, the 5-position hydrogens have a chemical shift 1.84 ppm. This is the result of the shielding effect of the aniline moiety. (400 MHz, solvent: DMSO-d6).
Scheme 1
Scheme 1
Synthesis of intermediate 4-chloro-2-methylcyclopenta[d]pyrimidine, 17. Reagents and conditions: (a) Acetamidine hydrochloride, KOt-Bu, DMF, 120 °C, 79%; (b) POCl3, 100 °C, 4 h, 73%.
Scheme 2
Scheme 2
Synthesis of anilines and methylthio derivatives. Reagents and conditions: (a) NaH, THF, CH3I, 0 °C, 65–75%; (b) mCPBA (1.1 eq.), ACN, 0 °C, 1 h, 67%; (c) mCPBA (1.1 eq.), ACN, 0 °C, 1 h, 59%.
Scheme 3
Scheme 3
Synthesis of anilines, derivatives and related intermediates. Reagents and conditions: (a) HCOOH, Ac2O, DCM, rt, 4–6 h; (b) THF, LiAlH4, 60 °C, 53–72%; (c) CBzCl, ACN, TEA, 0 °C, 90%; (d) NaH, THF, CH3I, 0 °C, 68%; (e) MeOH, Pd/C, H2 (55 psi), 2 h, 92%.
Scheme 4
Scheme 4
General synthesis of target compounds 3–7, 9–14. Reagents and conditions: compounds 19–21, 23, 25, 28a–b, 32 and 35a–c, dioxane, HCl (2 N in dioxane, 1 drop), μW, 120–160 °C, 3–6 h, 61–93%.
Scheme 5
Scheme 5
Synthesis of target compound 2. Reagents and conditions: HBr (48% aq.), 80 °C, 3–6 h, 72%.
Scheme 6
Scheme 6
Synthesis of target compound 8. Reagents and conditions: (a) N-methyl-4-nitroaniline, dioxane, HCl (2 N in dioxane, 1 drop), μW, 130 °C, 3 h, 79%; (b) MeOH, Pd/C, H2 (55 psi), rt, 69%;
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