Synthesis, StructureâActivity Relationships and Brain Uptake of a

Article pubs.acs.org/jmc

Synthesis, Structure−Activity Relationships and Brain Uptake of a Novel Series of Benzopyran Inhibitors of Insulin-Regulated Aminopeptidase Simon J. Mountford,† Anthony L. Albiston,‡ William N. Charman,§ Leelee Ng,∥ Jessica K. Holien,⊥ Michael W. Parker,⊥,# Joseph A. Nicolazzo,○ Philip E. Thompson,*,† and Siew Yeen Chai*,∥ †

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052, Australia College of Health and Biomedicine, Victoria University, St. Albans Campus, St. Albans, Victoria 8001, Australia § Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia ∥ Department of Physiology, Monash University, Clayton, Victoria 3800, Australia ⊥ ACRF Rational Drug Discovery Centre and Biota Structural Biology Laboratory, St. Vincent’s Institute of Medical Research, Fitzroy, Victoria 3065, Australia # Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia ○ Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia ‡

ABSTRACT: Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) enhance fear avoidance and spatial memory and accelerate spatial learning in a number of memory paradigms. Using a virtual screening approach, a series of benzopyran compounds was identified that inhibited the catalytic activity of IRAP, ultimately resulting in the identification of potent and specific inhibitors. The present study describes the medicinal chemistry campaign that led to the development of the lead candidate, 3, highlighting the key structural features considered as critical for binding. Furthermore, the in vivo pharmacokinetics and brain uptake of compounds (1 and 3) were assessed in rats and were complemented with in vitro human and rat microsomal stability studies. Following intravenous administration to rodents, 3 exhibits brain exposure, albeit it is rapidly converted to 1, a compound which also exhibits potent inhibition of IRAP.

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sequence.13−18 These angiotensin IV mimetics are proposed to be metabolically more stable than the native peptide, but optimal blood−brain barrier (BBB) permeability is still an issue for the development of these compounds as therapeutically useful cognitive enhancers. We previously reported on small molecule inhibitors of IRAP identified by elaboration from a virtual screening campaign.19 The inhibitors are benzopyran-based compounds that are specific, competitive inhibitors of IRAP (Figure 1). The inhibitors were efficacious in a hippocampal glucose uptake assay and were demonstrated to enhance the performance of rats tested in two distinct memory paradigms, the novel object recognition and spontaneous alternation tasks.19 The current study describes the medicinal chemistry pathway from in silico screening to the current candidate compounds, the structure− activity relationships governing their potency in inhibiting IRAP

INTRODUCTION Cognitive and memory impairments afflict one-quarter of the population over 65 years of age and result from a wide range of clinical conditions including Alzheimer’s disease, brain trauma, and stroke. Currently, most Food and Drug Administration approved treatments belong to the class of cholinesterase inhibitors and have limited efficacy. The M1 aminopeptidase, insulin regulated aminopeptidase (IRAP EC 3.4.11.3), is a relatively new target for the development of therapies for memory impairments.1 Peptide inhibitors of IRAP (angiotensin IV or LVV-hemorphin 7) have been demonstrated to enhance performance in both fear avoidance2−4 and spatial memory5,6 tasks. More importantly, these peptides reverse performance deficits induced by global ischemia,7 bilateral perforant pathway lesion,5 perturbations of central cholinergic systems,8−11 or chronic alcohol exposure.12 Peptidomimetic inhibitors of IRAP have been designed based on β-homo amino acid substitutions, disulfide cyclizations, and aromatic scaffold replacement of the angiotensin IV © XXXX American Chemical Society

Received: October 24, 2013

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dx.doi.org/10.1021/jm401540f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Structures of the benzopyran based IRAP inhibitors.

Figure 2. Benzopyran based IRAP inhibitors identified by virtual screening.

Scheme 1. Synthetic Pathway for the Production of 4H-Benzopyran Derivatives

Scheme 2. Synthesis of Compounds Derived from the Precursor Amine

develop more potent inhibitors based on modification of this template. Highly functionalized 4H-benzopyrans in general have shown a range of biological activities, and various methods for their synthesis have been described. Cai and co-workers have previously prepared a number of 4H-benzopyran derivatives based on the general synthetic pathway described in Scheme 1 for use as apoptosis inducers,20 including the use of catalysts such as DABCO21 and TBAB.22 As well as compounds synthesized in-house as described, compounds 1−4, 13, 15a−f, 16a,f,g,i−n,s,t, 17a−c,e−h, and 18a−f were obtained from commercial sources or custom synthesis (Epichem Pty Ltd.).

activity, and the evaluation of pharmacokinetic properties of this new class.

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RESULTS AND DISCUSSION

Chemistry. Our starting point for the structure−activity studies was a range of compounds identified by virtual screening against a homology model of IRAP followed by searching of the databases for similar core chemical structure compounds, as previously reported.19 Compound 1 (HFI142),19 along with compounds 5−7 (Figure 2), was identified and had common elements such as the benzopyran nucleus with 2-amino-4-aryl substitution and demonstration of high micromolar range affinity. We therefore pursued a campaign to B



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Typically, the 4H-benzopyran derivatives are obtained in two steps, with aromatic aldehydes 8 and nitriles 9 subject to Knoevenagel condensation in the presence of piperidine to give the corresponding arylidene-nitrile intermediate 10, followed by reaction with a substituted phenol 11 to give the desired products 12 (Scheme 1). Alternatively, a number of the 4Hbenzopyran derivatives obtained were prepared by a one-pot reaction with the reaction proceeding via the intermediate 10 in situ. It is worth noting that, while straightforward, the yields of these syntheses depended heavily on the choice of reaction conditions, with neither the stepwise nor one-pot approach offering a generally preferred method. In terms of reliability, it is suggested that the two-step pathway was preferred to reduce the complexity of reaction mixtures and ease of isolation of products. For example, compound 17d was prepared in a one-pot reaction from 3-pyridinecarboxaldehyde, t-butyl cyanoacetate, and resorcinol in 37% yield. In general however, the one-pot conditions gave yields below 20%. The two-step procedure was particularly useful when diversifying at the level of the phenol. The efficient isolation of intermediates such as ethyl 2-cyano-3(pyridin-3-yl)acrylate, prepared in 90% yield by the reaction of 3-pyridinecarboxaldehyde and ethyl cyanoacetate in refluxing toluene, made the subsequent condensation reaction cleaner and as seen in the case of 1- and 2-naphthol could be condensed in ethanol at ambient temperature to give 18g and 18h in 65% and 74% yield, respectively. Finally, for the synthesis of compounds 13 and 3 (HFI419)19 derived from the precursor amine, treatment of 1 with acetic anhydride selectively yielded the acetate, but extended treatment with a large excess allowed triacetylation to occur. Subsequent hydrolysis yielded the monoacetamide (Scheme 2). The same approach was applied to the synthesis of 4 (HFI437).19 Further attempts to modify the amino group of compound 1, including reductive alkylation, failed to yield any products. Structure−Activity Relationships. This investigation of the SAR has been categorized into specific molecular classes. The results relating to derivatives of 2-amino-4H-chromen-7-ol with various substituents at the 3- and 4-positions are shown in Table 1. Of the 3-cyano compounds, the first identified compound 15c with a 3,4-dimethoxyphenyl substituent was the only compound in the series to elicit inhibitory activity albeit at a modest Ki value of 49.7 μM. The Ki values for monomethoxy substitutions at either the 4- or 3-position (15a,b) could not be determined under the assay conditions used, likewise with the 3,5-dimethoxy and 3,4,5-trimethoxy analogues (15d,e). Where an ethyl ester substituent was included in the 3position, the inhibitory potencies were much stronger, and many of the compounds prepared in this series had Ki values of less than 20 μM. Notably, with the dimethoxyphenyl substituted compound (15c), the change to an ethyl ester (16r) yielded an 8-fold increase in potency. The unsubstituted phenyl derivative (16a) had a Ki value of >50 μM; however, the nature and placement of substituents on the ring greatly altered potency. The 3-cyano derivative (16g) had a Ki of 3.2 μM, while the 4-cyano derivative (16l) had a Ki of 11.4 μM, and the 2-cyano phenyl derivative (16b) was inactive. This trend in potency was also observed for the 3-chloro derivative (16f) over the inactive 4-chloro derivative (16k), and likewise, the 4nitro derivative (16o) was 5-fold more potent than the 2-nitro derivative (16e). Compounds that failed to produce a Ki value in the assay range were the unsubstituted phenyl derivative

Table 1. Substitutions at the 3- and 4-Position of the 4HBenzopyrans

a

compd

R

Ar

Ki (μM)a

1 2 15a 15b 15c 15d 15e 15f 15g 16a 16b 16c 16d 16e 16f 16g 16h 16i 16j 16k 16l 16m 16n 16o 16p 16q 16r 16s 16t

CO2Et CO2Et CN CN CN CN CN CN CN CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et CO2Et

pyridin-3-yl quinolin-3-yl 4-methoxyphenyl 3-methoxyphenyl 3,4-dimethoxyphenyl 3,5-dimethoxyphenyl 3,4,5-trimethoxyphenyl pyridin-3-yl 4-N,N-dimethylaminophenyl phenyl 2-cyanophenyl pyridin-2-yl quinolin-2-yl 2-nitrophenyl 3-chlorophenyl 3-cyanophenyl 2,4-dichloropyridine-3-yl 4-methylphenyl 4-bromophenyl 4-chlorophenyl 4-cyanophenyl pyridin-4-yl quinolin-4-yl 4-nitrophenyl 4-(pyridin-2-yl)phenyl 4-N,N-dimethylaminophenyl 3,4-dimethoxyphenyl 2-thiophene 2-thiazole

1.8 0.36 >100 >100 50 >100 >100 >100 >100 >100 >100 2.9 3.0 42 35 3.2 14 >100 >100 >100 11 3.7 0.9 7.7 >100 5.3 6.2 (50) (50)

The numbers in parentheses represent % inhibition at 100 μM.

(16a), and the mono substituted phenyl derivatives 2-cyano (16b), 4-methyl (16i), 4-bromo (16j), and 4-chloro (16k) having Ki values >50 μM. The greatest gains in potency derived from heteroaryl substitution at the 4-position of the 4H-benzopyrans were with either pyridine or quinoline groups. The quinoline derivatives, in general, were more potent than the pyridine derivatives. The 3-quinoline derivative (2) (HFI-435)19 was the most potent of the series (Ki 0.36 μM) followed by the 4-quinoline derivative (16n) (Ki 0.9 μM). The most active pyridyl derivative, again like the quinoline, had the ring nitrogen in the 3-position (1); however, it was 5-fold less potent than 2. The 2-pyridyl and 2quinoline derivatives (16c and 16d, respectively) had very similar activity (Ki 2.9 and 3.0 μM, respectively) while the 4pyridyl derivative (16m) was the least potent of the unsubstituted heteroaryls examined. The incorporation of the 2,4-dichloro substitution on the 3-pyridyl ring (16h) suppressed activity and was almost 8-fold less potent than 1. The collected data for compounds 16, give no obvious clue to the structural basis for activity, with electron-rich and -deficient substituents in para- or meta-positions capable of exhibiting inhibitory potency. The series of compounds 16 may also be able to adopt more than one binding pose to block enzyme activity,23 and it should be noted that these are all C



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racemates. We have previously attempted to rationalize the binding of compounds 1 (HFI142) and 2 (HFI435) by molecular docking.23 Overall, the docking suggests the Senantiomer will be more active than the R-enantiomer, which is unable to dock in a reasonable orientation for any active compound. The S-enantiomer of compound 1 was predicted to have a direct ring−stack interaction between the benzopyran rings and Phe544 of IRAP, while the benzopyran oxygen interacts with the Zn2+ ion. This was supported by mutagenesis studies that showed sensitivity to mutation at Phe544.23 In that model, the ester at the 3-position protrudes out of the binding pocket such that its polar groups are orientated toward the solvent. This may explain its significant potency advantages over the cyano compounds (15a−g). The poorly active cyano compound (15f) was unable to dock in the same pose as 1. The quinolone derivative 2 cannot be accommodated in this pose either, but the S-enantiomer readily adopts a flipped pose where the quinolone nitrogen interacts with the Zn2+ ion. This demands different interactions with Phe544, but the inhibitor is still sensitive to mutation. Given the activity of compound 1, we then examined variation to the ester functionality (Table 2), while retaining the

Table 3. Substitutions at the 7-Position of the 4HBenzopyrans

a

a

R

Ki (μM)a

17a 17b 17c 17d 17e 17f 17g 17h

methoxy propoxy n-butoxy t-butoxy 2-methoxyethoxy cyclohexylmethoxy benzyloxy phenyl

4.9 1.6 2.6 11.9 4.0 (50) 1.7 (50)

R

Ki (μM)a

1 13 18a 18b 18c 18d 18e 18f 18g 18h

7-OH 7-OAc 7-NH2 7-NMe2 7-OMe 6-Cl, 7-OH 6-Br, 7-OH 8-OH 7−8, fused phenyl 5−6, fused phenyl

1.8 (25) (50) (25) (25) 5.6 (50) 9.8 >100 >100


the 6-chloro for a 6-bromo (18e) caused complete loss of activity. Interestingly, shifting the hydroxyl group from the 7position to the 8-position (18f) resulted in just a 5-fold loss of potency. In our model of 1 described above, the 7-OH moiety is predicted to make a hydrogen bond with Glu295. Substitutions to this group (13, 18a−c,f−h) results in a significant loss of potency. The docked solution of 18f suggests that it retains a hydrogen bond to Glu295, the core shifting slightly in the binding pocket relative to 1. The distance of the benzopyran oxygen to the Zn ion is increased which may explain the modest loss in potency (Figure 3). The requirement for a phenolic substituent, in contrast to the amino group of 18a or the ether 18c, suggests that Glu295 has quite specific requirements for binding ligands. Consistent with that, 18c is unable to dock into the IRAP binding pocket. Finally, variations at the 2-amino group were limited to acetylation, and this proved to be effective for inhibitory potency with both the 3-pyridinyl (3) and 3-quinolinyl (4) substituted compounds (Table 4). The two acetamide compounds (3 and 4) dock into the IRAP model structure analogously to their parent compounds 1 and 2, resepctively. As previously described, the acetyl group allows for additional binding contacts in the binding site, albeit in different positions. In compound 3, the acetamide makes a second contact with the catalytic zinc ion and in 4 with aromatic residues Phe550 and Tyr495. Mutagenesis studies give support to the hypothesis that the pyridinyl and quinolinyl inhibitors bind in distinct poses. While strikingly different, the poses predicted by docking seem plausible based upon the collected SAR data (Figure 4). The solution of cocrystal structures with IRAP remains an important goal to progress the development of IRAP inhibitors. Biopharmaceutical Properties. Four compounds were selected for further study with respect to selectivity and biopharmaceutical properties, including solubility, and octanol−water partition coefficients. Although the substitution of the pyridinyl moiety for a quinolinyl at position 4 of the benzopyran resulted in an approximate order of magnitude increase in affinity for IRAP (i.e., 2 and 4), the aqueous solubility of the inhibitors was

Table 2. Variation of the Ester at the 3-Position of the 4HBenzopyrans

compd

compd


2-amino, 4-(3-pyridyl), and 7-hydroxyl groups. The resultant compounds showed a reasonable tolerance to changes in this position, with the methyl ester (17a), propyl ester (17b), nbutyl ester (17c), methoxyethyl ester (17e), and benzyl ester (17g) only marginally altering potency in comparison to that of 1. The introduction of more sterically demanding side chains, such as the t-butyl ester (17d), did cause a decrease in potency (Ki 11.9 μM), and the cyclohexyl derivative (17f) was found to be inactive. Replacement of the ester altogether by phenyl ketone resulted in the inactive derivative (17h). Modification of the 7-position of the 4H-benzopyrans was examined, while the 2-amino, 3-ethyl ester and 4-(3-pyridyl) groups of 1 were retained (Table 3). It was found that the phenolic group had no effective counterparts. Replacing the 7hydroxyl group with 7-amino (18a), 7-dimethylamino (18b), 7methoxy (18c), 7-acetyloxy (13), and a benzo ring fused at the 7−8-positions (18g) and the 5−6-positions (18h) all yielded inactive analogues. Addition of a chloro group at the 6-position of the 4H-benzopyran ring while retaining the 7-hydroxyl group (18d) caused a 3-fold decrease in potency, while substituting D



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Figure 3. Overlay of docking solutions for 1 (cyan carbons) and compound 18f (purple carbons) in the IRAP homology model (white carbons). The movement of the hydroxyl group around the ring to the 8-position causes the compound to shift relative to 1. However, the ability to retain a hydrogen bond to Glu295 may explain the small 5-fold drop in potency. The figure is presented in cross-eyed stereo with the zinc highlighted as a sphere and residues Phe544 and Glu295 highlighted as sticks. Potential hydrogen bonds are shown as yellow dashed lines.

both intravenous and intraperitoneal injections suggesting that urinary excretion is not a major pathway for elimination of this compound. Given the rapid in vivo clearance of 3, in vitro hepatic microsomal incubations were undertaken to identify potential pathways involved in the metabolism of both 3 and 1. In addition to the formation of glucuronide metabolites, P+16 metabolites (suggestive of oxygenated products of 3 and 1) were detected in vitro. Furthermore, in microsomal matrices lacking NADPH and UDPGA cofactors, 3 was converted to 1, suggesting that nonenzymatic (such as amide hydrolysis) processes were contributing to the rapid in vitro (and likely in vivo) disappearance of 3. The human and rat microsomepredicted hepatic extraction ratios for 1 were 0.71 and 0.86, respectively, and for 3, the corresponding human and rat microsome-predicted hepatic extraction ratios were 0.71 and 0.61. These values are suggestive of intermediate-to-high extraction ratios, which would be expected to result in a relatively high clearance in vivo, concordant with that observed for 3 in rat pharmacokinetic studies. In separate studies assessing BBB penetration in rats, plasma and brain concentrations of 3 were detected at 5 and 30 min following an intravenous dose (2 mg/kg) but not at 240 min postdose in line with a rapid systemic elimination of 3. The concentrations of 3 in the brain ranged between a mean value of 87.6 ng/g (5 min postdose) to 10.9 ng/g (30 min postdose), resulting in B/P ratios varying between 0.23 and 0.35. Similarly, 10 min following intraperitoneal administration of 3 (10 mg/ kg), a B/P ratio of 0.20 was detected (Figure 6). These values are similar to those obtained following intravenous administration of 3 to mice, where a 5 min postdose B/P ratio of 0.39 was reported.19 Following intravenous administration of 3, plasma concentrations of 1 were higher than those obtained for 3 (as observed in the pharmacokinetic studies), and these were detectable for up to 240 min postdose. Similarly, 1 was detected in the brain at all time points, resulting in mean B/P ratios ranging between 0.13 and 0.38. It is unknown whether the 1 detected within the brain was a product of 3 amide hydrolysis within the brain or whether systemically formed 1 penetrated the BBB. Given that the physicochemical properties of 1 are

Table 4. Acetylation of the 2-Amino Group of the 4HBenzopyrans

compd

Ar

Ki (μM)

3 4

pyridine-3-yl quinolin-3-yl

0.48 0.03

compromised. Although all inhibitors demonstrated good aqueous solubility under acidic conditions, at pH 6.5, 1 and 3 displayed only moderate solubility values, ranging from 25 to 50 μg/mL and 50−100 μg/mL, respectively, and solubility values for 2 and 4 were approximately 10-fold lower.19 The partition coefficient, effective LogD7.4 (eLogD7.4) values, for all four compounds exhibited acceptable values for good oral absorption (Table 5): however, based on lipophilicity alone, 1 and 3 appear better candidates for BBB penetration, as a logD7.4 of 1−3 is recommended for optimum BBB penetration.24 Pharmacokinetic Properties of 3. Given that 3 exhibited the most appropriate biochemical activity and pharmaceutical properties (Table 5),19 the pharmacokinetics and brain uptake of this analogue were determined following intravenous administration to rats. Concentrations of 3 were only detectable in plasma for up to 65 min following intravenous administration of 2 mg/kg, with this analogue exhibiting a short apparent elimination half-life (11 min), a high plasma clearance (172.8 mL/min/kg), and a large volume of distribution (2.8 L/ kg). This rapid disappearance of 3 was associated with the immediate appearance of the active metabolite 1 in the plasma, indicative of a rapid in vivo hydrolytic process (Figure 5). While 3 plasma concentrations declined rapidly following intravenous administration, the plasma concentrations of 1 were detected at all postdose time points. Following intraperitoneal injection of 10 mg/kg 3, the apparent half-life was extended to 4.6 h, although there was still rapid deacetylation to 1. A very small proportion of 3 was recovered in urine (less than 1%) with E



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Figure 4. Surface representation of the IRAP homology model (gray) displaying the alternate binding positions of 3 (magenta sticks) and 4 (cyan sticks). Theses alternate modes are supported in part by the SAR surrounding the 3 series and by mutagenesis studies of Phe544 (yellow sticks), which is predicted to ring-stack with the benzopyran of 3 but alternatively be an edge-face hydrophobic packing point with the benzopyran of 4. Also displayed is the Zn ion (purple sphere, positioned behind the benzopyran of 4) and Gln295 (yellow sticks, positioned behind the pyridine ring of 3).

Table 5. Summary of the Pharmacological/Physicochemical Properties of 4 Key IRAP Inhibitors compd

Ki (μM)

molecular mass (Da)

eLogD7.4a

aqueous solubility (μg/mL) at pH 6.5

1 2 3 4

1.8 0.36 0.48 0.02

312.33 362.39 354.37 404.43

2.99 3.88 3.16 4.05

25−50 3−6 50−100 6−12

a

For the acetylated analogues, 3 and 4, 0.17 units were added to the partition coefficient values of the base molecules resulting in calculated LogD7.4 (cLogD7.4) values of 3.16 and 4.05, respectively. eLogD7.4 is the experimentally determined effective LogD7.4

favorable for BBB penetration, it is likely that a substantial amount of the 1 detected within the brain was derived from the plasma, in line with that observed in mice.19

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Figure 5. Plasma concentrations of 3 (●) and 1 (○) following intravenous administration of 3 (2 mg/kg) to male Sprague−Dawley rats. Data are presented as the mean of n = 2 replicates.

CONCLUSIONS We have identified and developed the first generation small molecular weight inhibitors of IRAP based on the structure of 1 identified through virtual screening on a homology model of the catalytic site of IRAP. This study presents the structure− activity relationship of analogues of 1 and the physicochemical and pharmacokinetic properties of 4 of the most active analogues of this series. These small molecule IRAP inhibitors will be useful pharmacological tools to probe the physiological roles of IRAP.

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LTA4H25 and is described elsewhere.19,23 The model of IRAP was then used for structure-based drug design. An initial in-house virtual screen was conducted and is described elsewhere.19 Further docking analysis has been conducted on the synthesized analogues. Compounds were drawn and minimized under the Tripos forcefield26 in Sybylx1.2. (Tripos Inc., St Louis, MO USA). The docking of the synthetic analogues into the IRAP model was performed using Fred V.2.2.5,27 ensuring that the Zn2+ ion was included into the docking site. Then, 4 Å was added to the docking box, and the top 20 poses were retained for further analysis in Vida (OpenEye). Docking solutions as described were all present in a large cluster of solutions and/or were highly ranked using the G-score scoring function. All

EXPERIMENTAL SECTION

Computer Modeling. A molecular model of the catalytic site of IRAP was generated based on the structure of the homologous enzyme F



Article

Ethyl 2-Amino-4-(2-cyanophenyl)-7-hydroxy-4H-chromene-3carboxylate (16b). Piperidine (46 μL, 0.46 mmol) was added to a solution of ethyl cyanoacetate (100 mg, 0.89 mmol) and 2cyanobenzaldehyde (119 mg, 0.91 mmol) in EtOH (5 mL), and the mixture stirred at ambient temperature for 20 h. The resulting precipitate was collected and washed with cold EtOH to afford ethyl 2cyano-3-(2-cyanophenyl)acrylate as a yellow solid (70 mg, 35%). 1H NMR (300 MHz, CDCl3) δ 13.08 (s, 1H), 8.66 (dd, J = 5.8, 3.1 Hz, 2H), 7.80 (dd, J = 5.9, 3.1 Hz, 2H), 4.45 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H). Piperidine (60 μL, 0.6 mmol) was added to a solution of ethyl 2cyano-3-(2-cyanophenyl)acrylate (70 mg, 0.3 mmol) and resorcinol (34 mg, 0.3 mmol) in EtOH (5 mL) and the mixture stirred at reflux for 12 h. Removal of the solvent in vacuo followed by purification on silica (CH2Cl2/Et2O, 9:1) afforded 16b as a cream solid (30 mg, 30%).1H NMR (300 MHz, CDCl3) δ 9.75 (s, 1H), 8.52 (d, J = 7.8 Hz, 1H), 7.69−7.46 (m, J = 23.2, 7.4 Hz, 4H), 7.26 (s, 1H), 6.12 (d, J = 8.9 Hz, 1H), 5.76 (d, J = 9.1 Hz, 1H), 4.34−4.19 (m, 2H), 1.33 (td, J = 7.0, 1.4 Hz, 3H). MS (ESI+) m/z: 337.5 (M + H)+ (20%); 169.3 (M + 2H)2+ (100%). Ethyl 2-Amino-7-hydroxy-4-pyridin-2-yl-4H-chromene-3-carboxylate (16c). Piperidine (93 μL, 0.94 mmol) was added to a solution of ethyl cyanoacetate (0.25 g, 2.21 mmol), resorcinol (0.25 g, 2.25 mmol), and 2-pyridine carboxaldehyde (0.24 g, 2.25 mmol) in EtOH (10 mL) and stirred at ambient temperature for 20 h. The resulting precipitate was removed by filtration and the filtrate purified on silica (EtOAc/CH2Cl2, 1:1). Recrystallization from EtOH gave 16c as an off-white solid (42 mg, 6%). HRMS (ESI+): found m/z 313.1197 (M + H)+; C17H17N2O4 requires m/z 313.1188. 1H NMR (300 MHz, DMSO) δ 9.56 (s, 1H), 8.37 (ddd, J = 4.7, 1.7, 0.8 Hz, 1H), 7.67−7.60 (m, 1H), 7.23 (dd, J = 4.8, 3.9 Hz, 1H), 7.09 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.02 (d, J = 8.5 Hz, 1H), 6.44 (dd, J = 8.3, 2.4 Hz, 1H), 6.39 (d, J = 2.4 Hz, 1H), 4.91 (s, 1H), 3.99−3.79 (m, 2H), 0.95 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 313.2 (M + H)+ (100%). Ethyl 2-Amino-7-hydroxy-4-quinolin-2-yl-4H-chromene-3-carboxylate (16d). Piperidine (46 μL, 0.46 mmol) was added to a solution of ethyl cyanoacetate (100 mg, 0.88 mmol) and 2quinolinecarboxaldehyde (139 mg, 0.88 mmol) in EtOH (5 mL) and the mixture stirred at ambient temperature for 24 h. The resulting precipitate was collected and washed with cold EtOH to afford the intermediate ethyl 2-cyano-3-(quinolin-2-yl)acrylate as a yellow solid (83 mg, 37%). 1H NMR (300 MHz, CDCl3) δ 8.49 (s, 1H), 8.31 (d, J = 8.6 Hz, 1H), 8.22 (d, J = 8.6 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 7.70−7.62 (m, 1H), 4.44 (q, J = 7.1 Hz, 1H), 1.43 (t, J = 7.1 Hz, 1H). Piperidine (16 μL, 0.16 mmol) was added to a solution of ethyl 2cyano-3-(quinolin-2-yl)acrylate (80 mg, 0.32 mmol) and resorcinol (35 mg, 0.32 mmol) in EtOH (5 mL) and the mixture stirred at ambient temperature for 24 h. Removal of the solvent in vacuo followed by purification on silica (EtOAc/hexane, 3:2) and trituration with CH2Cl2 afforded afforded 16d as a white solid (14 mg, 12%). HRMS (ESI+): found m/z 363.1349 (M + H)+; C21H19N2O4 requires m/z 363.1345. 1H NMR (300 MHz, DMSO) δ 9.63 (s, 1H), 8.21 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.79−7.64 (m, 3H), 7.50 (t, J = 6.9 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 9.3 Hz, 1H), 6.47−6.39 (m, 2H), 5.12 (s, 1H), 3.84 (q, J = 7.0 Hz, 2H), 0.82 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 363.6 (M + H)+ (100%). Ethyl 2-Amino-7-hydroxy-4-(2-nitrophenyl)-4H-chromene-3-carboxylate (16e). Piperidine (184 μL, 1.84 mmol) was added to a solution of ethyl cyanoacetate (100 mg, 0.89 mmol), resorcinol (100 mg, 0.91 mmol), and 2-nitrobenzaldehyde (138 mg, 0.91 mmol) in EtOH (5 mL) and the mixture stirred at reflux for 18 h. Removal of the solvent in vacuo followed by purification on silica (CH2Cl2/EtOAc, 9:1) and trituration with CH2Cl2 afforded 16e as a yellow solid (102 mg, 32%). HRMS (ESI+): found m/z 357.1090 (M + H)+; C18H17N2O6 requires m/z 357.1087. 1H NMR (300 MHz, CDCl3) δ 7.64 (d, J = 7.9 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.29−7.11 (m, 3H), 6.57−6.49 (m, J = 11.1, 2.6 Hz, 2H), 5.57 (s, 1H), 4.01−3.87 (m, 2H), 0.99 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 357.3 (M + H)+ (100%).

Figure 6. B:P ratio of 3 (■) and 1 (□) following intravenous administration (2 mg/kg) of 3 to male Sprague−Dawley rats. Data are presented as the mean ± SD (n = 3). ND − 3 was not detected in the plasma or brain homogenate. figures were constructed using PyMOL (The PyMOL Molecular Graphics System, version 1.3, Schrödinger, LLC). Chemical Synthesis. All reagents and solvents were used as received. Proton nuclear magnetic resonance (1H NMR) spectra were recorded at 300 MHz with a Bruker Advance DPX-300. The 1H NMR spectra refer to solutions in deuterated solvents as indicated. The residual solvent peaks have been used as an internal reference, with each resonance assigned according to the following convention: chemical shift (δ) measured in parts per million (ppm) relative to the residual solvent peak. High resolution mass spectometry (HRMS) analyses were collected on a Bruker Apex II Fourier transform ion cyclotron resonance mass spectometer fitted with an electrospray ion source (ESI). Low resolution mass spectrometry analyses were performed using a Micromass Platform II single quadropole mass spectrometer equipped with an atmospheric pressure (ESI/APCI) ion source. Analytical HPLC was performed on a Waters 2690 HPLC system incorporating a diode array detector (254 nm), employing a Phenomenex column (Luna C8(2), 100 × 4.5 mm ID) eluting with a gradient of 16−80% acetonitrile in 0.1% aqueous trifluoroacetic acid, over 10 min at a flow rate of 1 mL/min. Analytical thin layer chromatography (TLC) was performed on Merck aluminum sheets coated in silica gel 60 F254 and visualization accomplished with a UV lamp. Column chromatography was carried out using silica gel 60 (Merck). The purity of compounds (≥95%) was established by reverse phase HPLC. Compounds were also custom synthesized by Epichem Pty Ltd. as indicated. 2-Amino-4-[4-(dimethylamino)phenyl]-7-hydroxy-4H-chromene3-carbonitrile (15g). Piperidine (2 drops) was added to a solution of malononitrile (122 mg, 1.84 mmol) and 4-dimethylaminobenzaldehyde (250 mg, 1.68 mmol) in EtOH (5 mL) and stirred at ambient temperature for 4 h. The resulting precipitate was collected and washed with cold EtOH to afford the intermediate 2-(4(dimethylamino)benzylidene)malononitrile as a bright orange solid (274 mg, 83%). 1H NMR (300 MHz, CDCl3) δ 7.82 (d, J = 9.1 Hz, 2H), 7.47 (s, 1H), 6.69 (d, J = 9.2 Hz, 2H), 3.14 (s, 6H). Piperidine (3 drops) was added to a solution of 2-(4(dimethylamino)benzylidene) malononitrile (50 mg, 0.25 mmol) and resorcinol (28 mg, 0.25 mmol) in EtOH (3 mL) and the mixture stirred at reflux for 24 h. The resulting precipitate was collected and washed with cold EtOH. Purification on silica (CH2Cl2 followed by EtOAc/hexane, 1:1) and trituration with cold CH2Cl2 gave 15g as a beige solid (20 mg, 26%). HRMS (ESI+): found m/z 308.1405 (M + H)+; C18H18N3O2 requires m/z 308.1399. 1H NMR (300 MHz, DMSO) δ 9.62 (s, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.80−6.69 (m, 3H), 6.64 (d, J = 8.6 Hz, 2H), 6.46 (dd, J = 8.4, 2.2 Hz, 1H), 6.37 (d, J = 2.1 Hz, 1H), 4.46 (s, 1H), 2.84 (s, 6H). MS (ESI+) m/z: 308.4 (M + H)+ (100%). G



Article

tert-Butyl 2-amino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-carboxylate (17d). Piperidine (3 drops) was added to a solution of tbutyl cyanoacetate (128 mg, 0.91 mmol), resorcinol (100 mg, 0.91 mmol), and 3-pyridinecaroxaldehyde (96 mg, 0.90 mmol) in EtOH (5 mL) and the mixture stirred at ambient temperature for 20 h. The solvent was removed in vacuo and the residue treated with Et2O. The resulting precipitate was collected and washed with Et2O. Purification on silica (Et2O) afforded 17d as a white solid (114 mg, 37%). 1H NMR (300 MHz, CDCl3) δ 9.65 (s, 1H), 8.44 (d, J = 1.7 Hz, 1H), 8.32 (dd, J = 4.7, 1.6 Hz, 1H), 7.59 (s, 2H), 7.47−7.41 (m, 1H), 7.24 (dd, J = 7.8, 4.7 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.46 (dd, J = 8.3, 2.4 Hz, 1H), 6.42 (d, J = 2.4 Hz, 1H), 4.76 (s, 1H), 1.24 (s, 9H). MS (ESI+) m/z: 341.5 (M + H)+ (100%). Ethyl 2-Amino-4-pyridin-3-yl-4H-benzo[h]chromene-3-carboxylate (18g). Acetic acid (100 μL) and piperidine (40 μL) were added to a stirred solution of ethyl cyanoacetate (2.3 g, 20.3 mmol) and 3pyridinecarboxaldehyde (2.2 g, 20.5 mmol) in toluene (20 mL). The reaction mixture was heated to reflux for 6 h. The resulting precipitate was collected and washed with toluene to afford the intermediate ethyl 2-cyano-3-(pyridin-3-yl)acrylate as a beige solid (3.69 g, 90%). 1H NMR (300 MHz, CDCl3) δ 8.92 (d, J = 2.3 Hz, 1H), 8.76 (dd, J = 4.8, 1.6 Hz, 1H), 8.58 (dt, J = 8.1, 1.8 Hz, 1H), 8.26 (s, 1H), 7.48 (dd, J = 8.2, 4.8 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 203.3 (M + H)+ (100%). Piperidine (2 drops) was added to a solution of ethyl 2-cyano-3(pyridin-3-yl)acrylate (100 mg, 0.49 mmol) and 1-naphthol (71 mg, 0.49 mmol) in EtOH (3 mL) and the mixture stirred at ambient temperature for 24 h. The resulting precipitate was collected and washed with cold EtOH to give 18g as a beige solid (110 mg, 65%). HRMS (ESI+): found m/z 347.1396 (M + H)+; C21H19N2O3 requires m/z 347.1396. 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 8.38 (d, J = 4.5 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.62−7.44 (m, 4H), 7.17−7.05 (m, J = 9.6 Hz, 2H), 5.10 (s, 1H), 4.10 (q, J = 7.2 Hz, 2H), 1.19 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 374.4 (M + H)+ (100%). Ethyl 3-Amino-1-pyridin-3-yl-1H-benzo[f ]chromene-2-carboxylate (18h). Piperidine (2 drops) was added to a solution of ethyl 2cyano-3-(pyridin-3-yl)acrylate (100 mg, 0.49 mmol) and 2-naphthol (71 mg, 0.49 mmol) in EtOH (3 mL) and the mixture stirred at ambient temperature for 24 h. The resulting precipitate was collected and washed with cold EtOH to give 18h as a white solid (126 mg, 74%). HRMS (ESI+): found m/z 347.1398 (M + H)+; C21H19N2O3 requires m/z 347.1396. 1H NMR (300 MHz, CDCl3) δ 8.70 (s, 1H), 8.31 (d, J = 4.7 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.78 (t, J = 7.5 Hz, 2H), 7.54−7.33 (m, 3H), 7.27 (d, J = 8.8 Hz, 1H), 7.13−7.03 (m, 1H), 6.37 (s, 2H), 5.60 (s, 1H), 4.22 (q, J = 7.0 Hz, 2H), 1.36 (t, J = 7.0 Hz, 3H). MS (ESI+) m/z: 374.4 (M + H)+ (100%). Enzyme Assay. Solubilized, crude membranes isolated from HEKT cells, transfected with pCI-IRAP, were used as a source of the enzyme to analyze the efficacy of the candidate compounds to inhibit its enzymatic activity. As a negative control, membranes from vector-only transfected cells were used. The enzymatic activity of IRAP was determined by the hydrolysis of a synthetic aminopeptidase substrate L-leucine-4-methyl-7-coumarinylamide (Leu-MCA) (Sigma Aldrich, St Louis, MO, USA) monitored by the release of a fluorogenic product at excitation and emission wavelengths of 380 and 430 nm, respectively. Assays were performed in 96-well plates; each well containing 1 μg of solubilized membrane protein and 25 μM substrate in a final volume of 100 μL 50 mM Tris-HCl buffer (pH 7.4). Reactions proceeded at 37 °C for 30 min in a Wallac Victor3 V Multilabel counter (Perkin-Elmer, Waltham, MA, USA). Inhibitory constants (Ki) for each of the inhibitors were determined over a range of concentrations (at 0.01 to 100 μM) and were calculated from the relationship IC50 = Ki (1 + [S]/KM) with GraphPad Prism 3 software (GraphPad Software Inc. San Diego, CA, USA). Physicochemical Characterization. Octanol−water partition coefficients or effective LogD (ElogD) values for 1 and 2 were measured using a chromatographic method employing a SUPELCOSIL LC-ABZ column with an octanol saturated mobile phase at pH 7.4.28 The values obtained for 1 and 2 were used to provide trained

Ethyl 2-Amino-4-(2,6-dichloropyridin-3-yl)-7-hydroxy-4H-chromene-3-carboxylate (16h). Piperidine (184 μL, 1.84 mmol) was added to a solution of ethyl cyanoacetate (100 mg, 0.89 mmol), resorcinol (100 mg, 0.91 mmol), and 2,6-dichloropyridine-3carboxaldehyde (160 mg, 0.91 mmol) in EtOH (5 mL) and the mixture stirred at reflux for 10 h. Removal of the solvent in vacuo followed by purification on silica (CH2Cl2/Et2O, 9:1) and trituration with CH2Cl2 afforded 16h as a yellow solid (61 mg, 18%). HRMS (ESI+): found m/z 381.0413 (M + H)+; C17H1535Cl2N2O4 requires m/ z 381.0409; found m/z 383.0396 (M + H)+; C17H1535Cl37ClN2O4 requires m/z 383.0379. 1H NMR (300 MHz, CDCl3) δ 8.68 (s, 2H), 7.44 (d, J = 8.1 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.54−6.45 (m, 2H), 5.41 (s, 1H), 4.06−3.91 (m, 3H), 1.10 (t, J = 7.1 Hz, 2H). MS (ESI+) m/z: 381.3 (M (35Cl) + H)+ (100%), 382.9 (M (35Cl)(37Cl) + H)+ (40%). Ethyl 2-Amino-7-hydroxy-4-(4-nitrophenyl)-4H-chromene-3-carboxylate (16o). Piperidine (184 μL, 1.84 mmol) was added to a solution of ethyl cyanoacetate (100 mg, 0.89 mmol), resorcinol (100 mg, 0.91 mmol), and 4-nitrobenzaldehyde (138 mg, 0.91 mmol) in EtOH (5 mL) and the mixture stirred at reflux for 18 h. Removal of the solvent in vacuo followed by purification on silica (CH2Cl2/Et2O, 9:1) and trituration with CH2Cl2 afforded 16o as a yellow solid (15 mg, 5%). HRMS (ESI+): found m/z 357.1077 (M + H)+; C18H17N2O6 requires m/z 357.1087. 1H NMR (300 MHz, CDCl3) δ 8.76 (s, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 9.0 Hz, 1H), 6.50 (d, J = 6.7 Hz, 1H), 4.93 (s, 1H), 4.00 (q, J = 7.1 Hz, 2H), 1.09 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 357.2 (M + H)+ (100%). Ethyl 2-Amino-7-hydroxy-4-(4-pyridin-2-ylphenyl)-4H-chromene3-carboxylate (16p). Piperidine (5 drops) was added to a solution of ethyl cyanoacetate (154 mg, 1.36 mmol) and 4-(2-pyridyl)benzaldehyde (250 mg, 1.36 mmol) in EtOH (5 mL) and stirred at ambient temperature for 20 h. The resulting precipitate was collected and washed with cold EtOH to afford the intermediate ethyl 2-cyano3-(4-(pyridin-2-yl)phenyl)acrylate as a pale yellow solid (278 mg, 74%). 1H NMR (300 MHz, CDCl3) δ 8.74 (d, J = 4.7 Hz, 1H), 8.29 (s, 1H), 8.19−8.07 (m, 4H), 7.84−7.77 (m, 2H), 7.31 (dd, J = 8.8, 4.5 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). Piperidine (3 drops) was added to a solution of ethyl 2-cyano-3-(4(pyridin-2-yl)phenyl)acrylate (50 mg, 0.18 mmol) and resorcinol (20 mg, 0.18 mmol) in EtOH (3 mL) and the mixture stirred at reflux for 24 h. Removal of the solvent in vacuo followed by purification on silica (CH2Cl2/EtOAc, 3:1) and recrystallization from EtOH gave 16p as a cream solid (30 mg, 43%). HRMS (ESI+): found m/z 389.1512 (M + H)+; C23H21N2O4 requires m/z 3389.1501. 1H NMR (300 MHz, DMSO) δ 9.64 (s, 1H), 8.65−8.57 (m, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.89−7.79 (m, 2H), 7.63 (s, 2H), 7.34−7.20 (m, 3H), 7.01 (d, J = 8.3 Hz, 1H), 6.52−6.41 (m, 2H), 4.85 (s, 1H), 3.95 (q, J = 7.1 Hz, 2H), 1.06 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 389.3 (M + H)+ (100%). Ethyl 2-Amino-4-[4-(dimethylamino)phenyl]-7-hydroxy-4H-chromene-3-carboxylate (16q). Piperidine (5 drops) was added to a solution of ethyl cyanoacetate (190 mg, 1.68 mmol) and 4dimethylaminobenzaldehyde (250 mg, 1.68 mmol) in EtOH (5 mL) and stirred at ambient temperature for 6 h. The resulting precipitate was collected and washed with cold EtOH to afford the intermediate ethyl 2-cyano-3-(4-(dimethylamino)phenyl)acrylate as a bright yellow solid (367 mg, 89%). 1H NMR (300 MHz, CDCl3) δ 8.07 (s, 1H), 7.94 (d, J = 9.1 Hz, 2H), 6.69 (d, J = 9.1 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.11 (s, 6H), 1.37 (t, J = 7.1 Hz, 3H). Piperidine (3 drops) was added to a solution of ethyl 2-cyano-3-(4(dimethylamino)phenyl)acrylate (100 mg, 0.41 mmol) and resorcinol (68 mg, 0.61 mmol) in EtOH (5 mL) and the mixture stirred at reflux for 24 h. The precipitate obtained on cooling was removed by filtration and the filtrate reduced in vacuo. Purification on silica (Et2O/hexane, 1:1) gave 16q as a beige solid (10 mg, 7%). HRMS (ESI+): found m/z 355.1666 (M + H)+; C20H23N2O4 requires m/z 355.1658. 1H NMR (300 MHz, DMSO) δ 9.54 (s, 1H), 7.50 (s, 2H), 6.97−6.87 (m, J = 8.6 Hz, 3H), 6.56 (d, J = 8.7 Hz, 2H), 6.45 (dd, J = 8.3, 2.4 Hz, 1H), 6.40 (d, J = 2.3 Hz, 1H), 4.65 (s, 1H), 4.01−3.88 (m, 2H), 2.79 (s, 6H), 1.09 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 355.6 (M + H)+ (100%). H



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partition coefficient values for the corresponding acetylated derivatives, 3 and 4, using the ACD LogD (version 9.0) of software programs. Pharmacokinetics and Brain Uptake of 3. All animal studies were performed in accordance with the Australian National Health and Medical Research Council code of practice for the care and use of animals for scientific purposes and were approved by the Monash University (Victorian College of Pharmacy) Animal Ethics committee. Male Sprague−Dawley rats (7−9 weeks) were intravenously administered (via an indwelling jugular vein cannula) a 1.0 mL solution of 3 (2 mg/kg in an aqueous solution containing 30% w/v hydroxypropyl-β-cyclodextrin and 1% v/v DMSO) over a 5 min period. In additional studies, rats were intraperitoneally administered a 0.5 mL solution of 3 (10 mg/kg in in 10% DMSO (v/v)) and 30% hydroxypropyl-β-cyclodextrin (w/v). Samples of arterial blood were collected (from an indwelling carotid artery cannula) over 24 h using a Culex automated blood sampler (BASi Instruments, West LaFayette, IN, USA). Blood samples were collected directly into heparinized borosilicate vials (at 4 °C) containing Complete protease inhibitor cocktail (80 mg/mL, Roche, Mannheim, Germany), EDTA (0.1 M), and potassium fluoride (4 mg/mL) (stabilization mixture). Blood samples were immediately centrifuged and the plasma supernatant separated and immediately stored at −20 °C to minimize potential ex vivo conversion of 3 to 1. All plasma samples were subsequently assayed for 3 and 1 concentration using a validated LCMS method, with a lower limit of quantification of 1 ng/mL for 3 and 0.5 ng/mL for 1. Noncompartmental pharmacokinetic analysis was performed using WinNonLin software (version 4.0, Pharsight Corporation, Mountain View, CA, USA) to estimate the terminal elimination halflife, total plasma clearance, and volume of distribution. For the assessment of 3 and 1 brain uptake, a 200 μL solution of 3 (in the same formulation used for pharmacokinetic studies) was injected as a bolus into the jugular vein (via an indwelling cannula) of rats. At 5, 30, and 240 min postdose, animals were anaesthetized using inhaled isofluorane (3%) and 500 μL of blood was removed by cardiac puncture (n = 3 rats per time point), followed by brain removal. Brain samples were homogenized in 3 parts of water containing stabilization mixture, and both the brain homogenate and plasma supernatant were immediately stored at −20 °C until analysis by LCMS (lower limits of quantitation of 5.0 ng/g brain homogenate for 3 and 0.5 ng/g for 1). All brain homogenate concentrations were corrected for vascular contamination of compound, using a vascular volume of 0.026 mL/g (determined by intravenous administration of the BBB-impenetrable marker 14C-inulin), and brain-to-plasma (B:P) ratios determined at each postdose time point. In Vitro Metabolism in Rat and Human Liver Microsomes. Compound 3 or 1 was incubated at 37 °C with rat or human liver microsomes at an initial concentration of 1 μM (in 0.1 M phosphate buffer, pH 7.4). Microsomal reactions were initiated by the addition of an NADPH-regenerating system (1 mg/mL), and at various time points, the reaction was terminated by the addition of acetonitrile and parent compound concentration determined by LCMS. In order to assess the potential of glucuronidation, additional microsomal samples were incubated with NADPH and UDPGA (1 mg/mL), the cofactor required for conjugative phase II metabolism by UGT enzymes. The concentration of parent compound (3 or 1) versus time was subsequently modeled to an exponential decay function to determine the first order rate constant for parent compound depletion (k), which was ultimately used in the estimation of a hepatic extraction ratio, with the appropriate assumptions.29 LCMS conditions were also optimized to detect the +16 (oxygenated) and +176 (glucuronidated) metabolites of 3 and 1.

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Article

ACKNOWLEDGMENTS This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC), Neurosciences Victoria, and Alzheimer’s Drug Discovery Foundation/Institute for the Study of Aging, USA. Funding was also obtained from the Victorian Government Operational Infrastructure Support Scheme to St. Vincent’s Institute. M.W.P. is an NHMRC Senior Principal Research Fellow, S.Y.C. is an NHMRC Senior Research Fellow, and J.K.H. is a joint Cure Cancer/Leukemia Foundation Post-Doctoral Fellow. We acknowledge the contribution of Dr. Keith Watson in the design some of the analogues of 1.

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ABBREVIATIONS USED APN, aminopeptidase N; Ang IV, angiotensin IV; AVP, arginine8 vasopressin; APCI, atmospheric pressure chemical ionization; BBB, blood−brain barrier; DABCO, 1,4-diazabicyclo-octane; DMSO, dimethyl sulfoxide; ESI, electrospray ion; EtOH, ethanol; EtOAc, ethyl acetate; HRMS, high resolution mass spectrometry; IRAP, insulin-regulated aminopeptidase; Leu-MCA, L-leucine-4-methyl-7-coumarinylamide; Leu-Enk, leu-enkephalin; LTA4H, leukotriene A4 hydrolase; LCMS, liquid chromatography−mass spectrometry; LVV-H7, LVVhemorphin 7; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; NMR, nuclear magnetic resonance; ppm, parts per million; TBAB, tetra-n-butylammonium bromide; TLC, thin layer chromatography; UDPGA, uridine diphosphate glucuronic acid

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*(P.E.T.) Tel: 613-9903-9672. E-mail: philip.thompson@ monash.edu. *(S.Y.C.) Tel: 613-9905-2515. E-mail: [email protected]. Notes

The authors declare no competing financial interest. I



Article

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