Development of substituted pyrido[3,2-d]pyrimidines as potent and selective dihydrofolate reductase inhibitors for pneumocystis pneumonia infection

Abstract

Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii (pj) can lead to serious health consequences in patients with an immunocompromised system. Trimethoprim (TMP), used as first-line therapy in combination with sulfamethoxazole, is a selective but only moderately potent pj dihydrofolate reductase (pjDHFR) inhibitor, whereas non-clinical pjDHFR inhibitors, such as, piritrexim and trimetrexate are potent but non-selective pjDHFR inhibitors. To meet the clinical needs for a potent and selective pjDHFR inhibitor for PCP treatment, fourteen 6-substituted pyrido[3,2-d]pyrimidines were developed. Comparison of the amino acid residues in the active site of pjDHFR and human DHFR (hDHFR) revealed prominent amino acid differences which could be exploited to structurally design potent and selective pjDHFR inhibitors. Molecular modeling followed by enzyme assays of the compounds revealed 15 as the best compound of the series with an IC₅₀ of 80 nM and 28-fold selectivity for inhibiting pjDHFR over hDHFR. Compound 15 serves as the lead analog for further structural variations to afford more potent and selective pjDHFR inhibitors.

Keywords: DHFR; PCP; Pneumocystis jirovecii; Pyrido[3,2-d]pyrimidines.

Figure 1.

Agents that have been used to treat PCP

Figure 2.

(A) Docked pose of 1 (magenta) in the homology model of pjDHFR and (B) Docked pose of 1 (magenta) in the crystal structure of hDHFR (PDB: 4QJC, 1.62 Å)[30]

Figure 3.

Proposed pyrido[3,2-d]pyrmidine compounds

Figure 4.

(A) Superimposition of docked pose of 1 (magenta) and 9 (cyan) in the homology model of pjDHFR and (B) Docked pose of 1 (magenta) and 9 (cyan) in the crystal structure of hDHFR (PDB: 4QJC, 1.62 Å)[30]

Figure 5.

(A) Superimposition of docked pose of 9 (cyan) and 18 (magenta) in the homology model of pjDHFR and (B) Docked pose of 9 (cyan) and 18 (magenta) in the crystal structure of hDHFR (PDB: 4QJC, 1.62 Å)[30]

Scheme 1.

Synthesis of intermediate 26 a) 0% HNO₃, H₂SO₄, reflux, 3 h; b) CuCN, 180 °C, 15 min; c) aniline, monoethyl glycol, pyridine, 140 °C, 12 h; d) aniline, LDA, THF, −78 °C, 12 h

Scheme 2.

Synthesis of target compounds 9–22 a) substituted aniline, isopropanol, 130 °C, 3–16 h; b) conc. HCl, Fe powder, reflux, 0.5–2 h; c) chlorformamidine HCl, dimethyl sulfone, 140 °C, 3–16 h