Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood-brain barrier penetration

Abstract

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood-brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.

Figure 1

Primary amine prodrug analogues

Figure 2

Structure of nNOS inhibitor 5, and analogs 6-8, designed to test whether removing a charge and increasing lipophilicity leads to greater BBB penetration.

Scheme 1

i) nBuLi, THF, −78 °C to rt, 30 min, then 10, −78 °C to rt, 4h; ii) NaH, DMF, 0 °C, 30 min, then BnBr, rt, 16h; iii) PPh₃, DIAD, AcOH, THF, rt, 16h; iv) 1N NaOH (aq), MeOH, rt, 16h; v) NaH, DMF, 0 °C, 30 min, then allyl bromide; vi) O₃, MeOH, −78 °C, 1h, then NaBH₄, rt, 3h; vii) H₂, Pd / C, MeOH, 2d; viii) PPh₃, DIAD, DPPA, THF, rt, 16h; ix) 4N HCl, dioxanes, rt, 16h.

Scheme 2

i) Hg(tBuCOO)₂, pivalic acid, 80 °C; ii) BH₃-THF, then Boc₂O, p-dioxane, 10% NaHCO3; iii) H2, Pd/C, MeOH/H₂O/AcOH; iv) Fmoc-Arg(NO₂)-OH, TFFH, HOAt, CH2Cl2/aq. NaHCO3; v) BH3-THF, −10 °C; vi) 20% piperidine, DMF, then 21, DIEA, CH₃CN; vii) TFA, CH₂Cl₂.

Figure 3

Incubation of 1 and 3 in fresh mouse plasma. The area under the peak was integrated and the value was normalized based on the integration at 0 min. Two diastereoisomers of 3 are shown separately (3A and 3B).

Figure 4

Formation of compound 4 from prodrug 3 during incubation in fresh mouse plasma (not normalized)

Scheme 3

i) Ac₂O, MeOH, rt, 1h; ii) H₂, Pd(OH)₂ / C, MeOH, 60 °C, 1-2 d; iii) 4N HCl, dioxanes, rt 16h

Scheme 4

i) ClCOOMe, MeOH, rt, 4h; ii) 4N HCl, dioxanes, rt 16h; iii) ClCOOBn, MeOH, rt 4h.

Figure 5

Metabolic stability of 5 with microsomes at 37 °C. The experiment was performed in the presence (5 red) and absence (5 ox) of additional NADPH. Minaprine concentration at 20 min was >0 but was not quantifiable.

Figure 6

Concentration of 5 in plasma after an i.p. dose of 3.7 mg/kg. Error bars show standard error mean.

Figure 7

Comparison of the brain concentrations of 5 and 6 after administration of 3.7 mg/kg and 4.1 mg/kg respectively. Error bars show standard error mean.

Figure 8

Concentration of 6 in plasma after a dose of 4.1 mg/kg. Error bars show standard error mean.

Figure 9

Comparison of the brain concentrations of 5 and 7 after a dose of 3.7 and 4.3 mg/kg, respectively. Error bars show the standard error mean.

Figure 10

Comparison of the brain concentrations of 5 and 8 after a dose of 3.7 and 5.1 mg/kg, respectively. Error bars show the standard error mean.

Figure 11

Comparison of the plasma levels of 5, 6, 7, 8, 36 after i.p. injection.

Figure 12

Comparison of brain concentrations of 5, 6, 7, 8, 36 after i.p. injection.