Rational Alteration of Pharmacokinetics of Chiral Fluorinated and Deuterated Derivatives of Emixustat for Retinal Therapy

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Rational Alteration of Pharmacokinetics of Chiral Fluorinated and

Deuterated Derivatives of Emixustat for Retinal Therapy

Eliav Blum,^∇Jianye Zhang,^∇Jordan Zaluski, David E. Einstein, Edward E. Korshin, Adam Kubas,

Arie Gruzman,* Gregory P. Tochtrop,* Philip D. Kiser,* and Krzysztof Palczewski *

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ABSTRACT: Recycling of all-trans-retinal to 11-cis-retinal through the visual cycle is a

fundamental metabolic pathway in the eye. A potent retinoid isomerase (RPE65) inhibitor,

(R)-emixustat, has been developed and tested in several clinical trials; however, it has not

received regulatory approval for use in any speciﬁc retinopathy. Rapid clearance of this drug

presents challenges to maintaining concentrations in eyes within a therapeutic window. To

address this pharmacokinetic inadequacy, we rationally designed and synthesized a series of

emixustat derivatives with strategically placed ﬂuorine and deuterium atoms to slow down

the key metabolic transformations known for emixustat. Crystal structures and quantum

chemical analysis of RPE65 in complex with the most potent emixustat derivatives revealed the structural and electronic bases for

how ﬂuoro substituents can be favorably accommodated within the active site pocket of RPE65. We found a close (∼3.0 Å) F−π

interaction that is predicted to contribute ∼2.4 kcal/mol to the overall binding energy.

Various therapeutic strategies have been developed to

combat the aberrant metabolism of retinoids including all-

trans-retinal. One of these involves the inhibition of retinoid

isomerase (RPE65), a key enzyme of the visual cycle (Figure

1A), to slow down the generation of all-trans-retinal without

signiﬁcantly impairing vision. (R)-Emixustat, derived from

retinylamine (Figure 1B),⁵is currently undergoing clinical

development as a potent RPE65 inhibitor.⁶⁻¹⁴However, the

complete suppression of RPE65 activity has undesirable

blinding consequences, thus only a narrow concentration

range of the drug is tolerable. Eﬀective use of emixustat is

further complicated because it is rapidly metabolized,¹⁵⁻¹⁸

limiting the duration of its pharmacological eﬀects. A second

strategy is to lower the toxic eﬀects of unbound all-trans-retinal

and temporarily sequester it by condensation with a primary

amine.^19,20Because most RPE65 inhibitors contain an amino

group, they can play a dual role of slowing down the

metabolism of all-trans-retinal as well as sequestering it.¹⁹

Here, we describe the synthesis of several derivatives of

emixustat, involving the strategic incorporation of deuterium

or ﬂuorine to investigate three areas of the emixustat structure

that could modulate its potency and metabolism (Figure 1C).

Regioselective incorporation of ﬂuorine is known to have a

broad range of potential impacts on the properties of small

INTRODUCTION

■

Retinal photoreceptor cells can respond to light throughout life

because they continuously regenerate a light-sensitive

chromophore and photoreceptor structures. Defects in various

proteins involved in these processes cause photoreceptor

degeneration.¹Light detection is mediated by a group of G

protein-coupled receptor proteins called opsins located in rod

and cone photoreceptor cells of the retina. The light-absorbing

chromophore of most vertebrate opsins is 11-cis-retinal.

Absorption of a photon by an opsin pigment causes

isomerization of the chromophore to all-trans-retinal. Regen-

eration of the visual chromophore following light exposure is

dependent upon an enzymatic pathway referred to as the visual

cycle (Figure 1A).²To understand why human vision declines

with age, considerable research has been focused on the retina,

especially the layer of rod and cone photoreceptor cells that

convert light into electrical signals. However, age-related

decreases in retinal photoreceptor cell function cannot be

explained alone by rod/cone cell loss, abnormal retinal

plasticity, or any acute signs of retinal disease. Rather, there

are pathological events that take place over time, including the

aberrant metabolism of all-trans-retinal that can interfere with

normal photoreceptor function (Figure 1A). All-trans-retinal,

when released from rhodopsin, primarily re-enters the visual

cycle. However, in some individuals, it can also persist as an

unbound potentially toxic aldehyde or react with other

molecules to create toxic compounds such as N-retinylidene-

N-retinylethanolamine (A2E) and retinal dimers.³These

events are thought to contribute to the etiology of blinding

diseases such as age-related macular degeneration (AMD) and

Stargardt disease.⁴

Received: February 13, 2021

Published: June 3, 2021

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Figure 1. Pharmacodynamics and pharmacokinetics of visual cycle modulators. (A) Visual cycle modulators such as emixustat inhibit RPE65, thus

blocking the key trans/cis isomerization step of the visual cycle and reducing the formation of toxic retinaldehyde condensation products. (B) Sites

of emixustat modiﬁcation in vivo and strategies to modify its metabolism. (C) Three strategies for modifying the emixustat metabolism investigated

in this work.

molecules. These include pK_amodulation; alteration of target

selectivity through conformational variations or changes in

speciﬁc hydrophobic interactions; and alteration of tissue-

speciﬁc penetration (e.g., CNS), through modiﬁcation of

lipophilicity.²¹These eﬀects of ﬂuorination are in addition to

the well-established strategy of replacing metabolically labile

hydrogens with C−F bonds.²²Regioselective incorporation of

deuterium was utilized to investigate the role of amine

oxidation in the rapid metabolic elimination of emixustat.

We hypothesized that we could attenuate this metabolic

process via engineering a localized primary isotope eﬀect.²³

Collectively, we envisioned a collection of rationally designed

compounds that could improve the potency, absorption,

selectivity, and metabolism of drug candidates to mitigate

the toxicity of all-trans-retinal in age-related blindness.

conventional alkylation of the phenolic hydroxyls with the

corresponding alkyl bromides or mesylates.

Synthesis of non-racemic (2-propylpentyl)oxyﬂuorophenyl

series of γ-aminoalcohols 23−30 by means of capitalization of

the chiral removable appendage for the resolution of the

diastereomeric intermediates is shown in Scheme 1. Indeed,

recently developed for synthesis of enantiopure γ-oxo-α-amino

acids^24,25and β-aminoketones, three-component Mannich

reaction with chiral benzylamines²⁶being applied to

ﬂuorinated in the benzene ring alkoxyacetophenones 1−4,

(S)-(−)-α-methylbenzylamine and formaldehyde, precursors

aﬀorded a suitable access to the corresponding β-aminoketones

5−8. The HCl-catalyzed Mannich reactions of ﬂuorinated

acetophenones 1−4 with (S)-(−)-α-methylbenzylamine lead-

ing to the chiral β-aminoketones 5−8, were initially performed

using paraformaldehyde and microwave (MW) irradiation

(method 1).²⁷While this method provided a reasonable yield

of β-aminoketones 5 and 7 (47% in both cases) from ketones 1

and 3, respectively, the reactions of acetophenones 2 and 4

with the ortho-positioned ﬂuoro- and alkoxy-substituents

under the MW conditions repeatedly resulted in insuﬃcient

production of aminoketones 6 and 8 (<20%). To our delight,

the more traditional method of Mannich condensation

(heating of 2 and 4 with 1,3,5-trioxane in a sealed pressure

tube), method 2,²⁸was found to be more eﬃcient for the

preparation of chiral β-aminoketones 6 and 8 (34 and 78%,

respectively).

RESULTS

■

Synthesis. Here, we report three families of molecules

where we employed a common strategy, constructing a variety

of alkoxy substituents of a central aryl core initially, and then

adding a γ-hydroxyalkylamine moiety, using both established

and novel methods. We considered two general approaches

that are strategically diﬀerentiated by the directionality of

molecule assembling with respect to the aryl core (Schemes 1

and 2). Additionally, a possibility of the construction of the

chiral γ-hydroxyalkylamine framework with the assistance of

removable chiral auxiliary (Scheme 1) as well as an option for

intentional α-deuteration toward the amino group (Scheme 2)

were considered. In the very beginning of both general

synthetic routes, the alkyl substituents were installed onto

either a properly ﬂuorinated hydroxyacetophenone (Scheme

1) or a hydroxybenzaldehyde backbone (Scheme 2) through

The chiral β-aminoketones 5−8, prepared through the

Mannich reaction, were reduced by sodium borohydride in

MeOH to give the corresponding N-protected γ-aminoalcohols

9−12 as a mixture of diastereomers in each case. All of the

mixtures of diastereomeric γ-aminoalcohols 9−12 could be

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a

Scheme 1. Synthesis and Asymmetric Resolution of (2-Propylpentyl)oxyﬂuorophenyl Analogues of Emixustat 23−30

a

Reagents and conditions: (a) 2-Propylpentyl mesylate, K₂CO₃, and DMF, 85 °C, 2−4 h. (b) Paraform, (S)-(−)-methylbenzylamine, conc. HCl

(cat.), and 1,4-dioxane, MW, 100−130 °C, 5 min and (c) 1,3,5-trioxane, (S)-(−)-methylbenzylamine, conc. HCl (cat.), and 1,4-dioxane, sealed

tube, 110 °C, overnight. (d) NaBH₄and MeOH, r.t., 1 h. (e,i) CDI and THF, reﬂux, overnight; (ii) separation of diastereomers by FC on SiO₂. (f)

KOH and EtOH, reﬂux, overnight. (g) Separation of diastereomeric mixtures 10−12 by chiral HPLC. (h) Ammonium formate, Pd/C (cat.), and

MeOH, reﬂux, 30 min.

Scheme 2. Synthesis of Racemic Fluorinated Emixustat Analogues 49−59 via the Addition of Acetonitrile to Functionalized

a

Benzaldehydes

a

Reagents and conditions: (a) R³-Br or R³-OMs, K₂CO₃, and DMF, 80−90 °C, overnight. (b) MeCN, t-BuOK, and THF, −50 °C, or LDA and

THF, −78 °C; 1.5−3.0 h. (c) LAH and THF, 0 °C, 0.5−1.0 h. (d) LAD and THF, 0 °C, 1 h.

separated to single diastereomers 15−22 using preparative

chiral high-performance liquid chromatography (HPLC) as

detailed in the Supporting Information. The ﬁnal N-

deprotection of individual diastereomers 15−22 through Pd/

C-catalyzed transfer hydrogenation with ammonium formate as

a hydrogen source aﬀorded the target γ-hydroxyalkylamines

23−30 as single enantiomers, which were studied biologically

without further characterization of absolute stereochemistry.

For a more detailed biological evaluation, the most active chiral

3-alkyloxy-4-ﬂuoro derivative 24 as well as its enantiomer 23

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Figure 2. (A) Structures of visual cycle modulators used for pharmacokinetic studies. (B) Evaluation of the inhibitory eﬀects of selected visual cycle

modulators on 11-cis-retinol production by bovine RPE microsomes. Data points are shown as mean s.d.; n = 3. (C,D) Progress of visual cycle

modulator elimination in the mouse serum (C) and eyes (D) after a single dose of intraperitoneal injection. Data points are shown as mean s.d.;

n = 4−5. (E) Primary amine levels in mouse eyes on day 7 after the treatments.

Table 1. Mannich-Derived γ-Hydroxyamines

compound

*

R

IC₅₀(nM)

50

295 132

132 19

274 47

compound

*

R

IC₅₀(nM)

24

25

28

30

R

4-F

5-F

6-F

2-F

9

23

26

27

29

S

4-F

5-F

6-F

2-F

177 11

184 24

157 24

287 28

R/S

were synthesized by a more practical method, avoiding

laborious and time-consuming preparative HPLC separation.

Thus, a mixture of diastereomeric N-benzylated γ-amino-

alcohols 9 was treated with 1,1′-carbonyldiimidazole (CDI)²⁹

giving almost quantitatively a mixture of chiral diastereomeric

cyclic carbamates, which was readily separated to single

components 13 (52%) and 14 (39%) by routine ﬂash

chromatography (FC) on silica gel. The cyclic carbamates 13

and 14 were separately hydrolyzed with KOH,³⁰thus resolving

acyclic N-benzylated γ-aminoalcohols 15 (93%) and 16 (71%).

The γ-aminoalcohols 15 and 16 were N-deprotected as above

(ammonium formate in MeOH, Pd/C-catalyst) giving the

corresponding enantiomeric γ-aminoalcohols 23 (91%) and 24

(93%). A comparison of the duplicated speciﬁc rotation values

[α]²_D⁵−21.32° (c = 0.0003, CH₂Cl₂) in 23 and +21.56° (c =

0.0002, CH₂Cl₂) in 24 with the reported data [α]²_D^6.7+ 19.66

(c = 0.01125, EtOH) for (R)-emixustat³¹tentatively favor the

assignment of the absolute R-conﬁguration for 24 and the

opposite S-conﬁguration for 23. However, for a better

understanding of the SAR additional structural study of the

chiral γ-aminoalcohols 23−30 would be desirable.

The synthesis of the racemic ﬂuorinated emixustat analogues

49−59 was performed through an alternative three-step

synthetic route (Scheme 2) similar to the method elaborated

for the parent emixustat.^5,19,31−33After the alkylation of

ﬂuorinated hydroxybenzaldehydes to give the alkoxy deriva-

tives 31−39, treatment of the latter with deprotonated

acetonitrile led to a three-atom elongation with the formation

of the corresponding β-hydroxy-β-arylpropionitriles 40−48

(38−84%). The nitrile groups in these advanced intermediates

40−48 were either reduced with LAH to give racemic γ-

aminoalcohols 49−57 or reduced and α-bis-deuterated with

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Table 2. α-Cyano Alcohol-Derived γ-Hydroxyamines

lithium aluminum deuteride (LAD) to give the bis-deuterio

analogues 58 and 59 in variable yields. Although β-

hydroxypropionitriles 40−48 might be potentially converted

to an enatioenriched or enantiopure state using an additional

oxidation/asymmetric reduction sequence as described for

emixustat,^31,34in this study the ﬂuorinated emixustat analogues

49−59 were preliminary evaluated in the racemic form, as

mixtures of their (S)- and (R)-enantiomers.

Oxidative deamination and hydroxylation on the cyclohexyl

ring are two pathways for emixustat elimination in vivo;¹⁷the

former being more dominant in the vasculature as compared to

the liver.¹⁷By analogy to the studies of oxidative deamination

of other compounds,³⁵deuteration at the position α to the

amino group of emixustat would be predicted to slow down its

metabolic elimination due to a primary kinetic isotope eﬀect.

We explored this possibility by comparing the rate of oxidative

deamination of emixustat and its α-deuterated derivative of

emixustat (compound 58) in an HPLC-based activity assay

using mouse aorta homogenates as the source of the vascular

adhesion protein-1 (VAP-1) enzyme (Figure 3A). We

monitored the formation of the dehydrated aldehyde product

of VAP-1 catalysis (ACU-5201) by reverse-phase HPLC

(Figure 3B) whose identity and absolute amount were

determined by comparison to an authentic synthetic standard

(Figure 3B,C). As compared to emixustat, the formation of

ACU-5201 from compound 58 was reduced by approximately

66% at both time points examined (Figure 3D). These data are

consistent with the idea that proton abstraction from amine α-

carbon is a rate-limiting step in the catalytic mechanism of

emixustat deamination by VAP-1.

Inhibitory Properties and Pharmacokinetics of the

Tested Compounds. A total of 19 novel emixustat

derivatives were synthesized, and their inhibitory eﬀects on

RPE65 in vitro were characterized by the decrease of 11-cis-

retinol production by bovine RPE microsomes (Figure 2A,B,

Table 1, Supporting Information Table S1).⁵All of the

synthesized primary amines showed an intense inhibitory

activity toward RPE65 at submicromolar concentrations.

Compared to emixustat and MB-001, a single ﬂuorination at

C4 of the phenyl ring signiﬁcantly increases the inhibitory

potency. Pronounced enhancement is seen with compound 24

(IC₅₀= 50 9 nM), the most potent RPE65 inhibitor that we

have identiﬁed to date, with an inhibitory potency 3 times

greater than that of the racemic emixustat (IC₅₀= 172 29

nM) and twice that of (R)-MB-004 (IC₅₀= 106 16 nM),³³

which is the parent molecule of compound 24 lacking the 4-

ﬂuorine substitution. The gem-diﬂuorination of the cyclohexyl

ring in 57 also enhanced the inhibition of 11-cis-retinol

production. The inhibition curve of 57 was found to exhibit a

double sigmoidal feature indicative of two distinct modes of

inhibitor binding to RPE65 (Table 2).

To investigate the impact of ﬂuorination and deuteration on

the elimination of emixustat in mouse eyes, a single dose of

380 nmol of emixustat, 24, 49, 57, 58, or 59 was individually

administered to wild-type mice by intraperitoneal injection.

The concentrations of the administered compounds found in

mouse eyes were quantiﬁed by a mass spectrometry (MS)

method (Figure 2A,D,E).¹⁹Most compounds were monitored

for the loss of water (−18 Da) in the liquid chromatography

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time after administration. Interestingly, we observed that

compounds 24 and 49, which displayed the highest initial

plasma concentrations, exhibited the poorest distribution

within the ocular tissue. These data point to a possible ocular

uptake mechanism for emixustat that is disrupted by 4-

ﬂuorination. Scrutiny of the mass spectra for 24 and 59

standards revealed that their peak intensities in methanol were

much weaker than (about 5−10%) the peak intensities of

emixustat and 57 standards in methanol at the same

concentration. In the tandem mass spectra, 24 and 49 were

also resistant to losing the methylene imine fragment. The

diﬃculty of ionization and fragmentation of 24 and 49 in MS

implies that 4-ﬂuorination on the phenyl ring might intensify

the intramolecular N−H−O hydrogen bonding interaction and

reduce the molecular ﬂexibility to hamper drug delivery to the

eye. In our previous studies, most drug candidates against light-

induced retinal degeneration decayed to negligible levels just

24 h after a single-dose intraperitoneal injection.³⁶By contrast,

emixustat and most of its ﬂuorinated derivatives only

moderately declined to around 50 pmol/eye during the same

period, approximating 10% of the rhodopsin content. On day

7, the level of emixustat further dropped to about 4 pmol/eye,

which is too low for all-trans-retinal sequestration but still

suﬃcient to inhibit RPE65 activity and visual chromophore

recovery.¹⁹

The level of deuterated emixustat, 58, in the eye was

approximately twofold higher than that of emixustat. In light of

the plasma level data presented above, we cannot attribute this

elevation to high circulating concentrations favoring distribu-

tion into the ocular tissue. Instead, it is possible that

deuteration could positively impact ocular drug retention or

aﬀect metabolism within the eye by as-yet undeﬁned processes.

On the other hand, compound 57, in which hydroxylation was

partially blocked due to the gem-diﬂuoro substitution on the C-

5″ position, was found at a concentration of 21 5.7 pmol in

mouse eyes even after 7 days of a single intraperitoneal

injection (Figure 2D,E). This amount is 5 times higher than

that for emixustat and 10 times higher than that of compounds

24 and 49; and it is an eﬀective amount for the sequestration

of excessive all-trans-retinal.³⁶These results suggest that

compound 57 is a promising next generation visual cycle

modulator against retinal degeneration due to its higher

inhibition potency, more speciﬁc delivery, and slower decay in

the eye.

Crystal Structures of RPE65 in Complex with

Fluorinated Visual Cycle Modulators. To gain an under-

standing of the factors underlying the enhanced potency of

compounds 24, 49, and 57 compared to their parent

molecules, we determined the crystal structures of RPE65 in

complex with each of these compounds. The crystals were

isomorphous to prior RPE65 structures in space group P6₅and

diﬀracted X-rays to resolutions of 1.95, 2.15, and 1.90 Å,

respectively (Supporting Information Table S2). The struc-

tures were reﬁned to overall R_freevalues of 21.1, 21.7, and

20.1% with excellent geometrical and clash score statistics

(Supporting Information Table S2). A 2.1 Å resolution

structure of apo-RPE65 was also determined for comparison

(Supporting Information Table S2). Unbiased residual maps

obtained after the ﬁrst rounds of reﬁnement in the absence of

modeled ligands revealed a clear |F_o|−|F_c| density for the

bound visual cycle modulators in the proximal active site

region, including well-deﬁned features corresponding to the

ﬂuoro substituents (Figure 4). Residual electron density was

Figure 3. Impact of emixustat deuteration on VAP-1 metabolic

susceptibility. (A) Scheme showing the oxidation of emixustat or 58

(²H-emixustat) by VAP-1 present in the aorta homogenates used for

the assay. (B) HPLC chromatographs showing the formation of ACU-

5201 (right red arrow) over time, after incubation of emixustat or 58

(left red arrow) with mouse aorta homogenates containing VAP-1.

The HPLC traces are split at 10 min with two diﬀerent scales used to

aid in visualization. (C) Identities of the product peaks veriﬁed by

comparison of their retention times and UV/vis absorbance spectra to

that of the authentic ACU-5201 standard. (D) Quantiﬁcation of

product formation showing that the deuteration at the 3-position

reduced product formation nearly 3-fold at both 1 and 2 h time

points. Mean values SDs and individual data points are shown in

the graph.

(LC)−MS/MS−electrospray ionization (ESI) fragmentation

spectra. An exception was 58, in which the loss of water plus

methylene imine (−47 Da) was monitored due to a high

background eﬀect of the fragmentation at −18 Da.

We ﬁrst measured the total plasma concentrations of these

compounds at 3 h, 1 d, or 7 d after compound administration

(Figure 2C). Interestingly, we observed that compounds 24

and 49, which both contain 4-ﬂuoro substituents, exhibited the

highest initial plasma levels suggesting that this modiﬁcation

can deactivate elimination pathway(s) of relevance in mice.

2

Unexpectedly, we observed that H₂emixustat had an initial

serum concentration comparable to that of emixustat, which

suggests that VAP-1 may not be a major pathway of emixustat

clearance in the mouse. 1 d after injection, all compounds

beside 24 were below the limits of detection. The longer

plasma half-life of compound 24 is likely due to its more

ﬂexible alkyl substituents, which may be less susceptible to

oxidation.

Next, we measured the distribution and retention of these

compounds into the mouse eye. Emixustat was eﬃciently

distributed to the ocular tissue reaching 88 43 pmol/eye at 3

h after i.p. drug administration, nearly equal to 15% of the

rhodopsin content in mouse eyes. 5″-gem-Diﬂuorination of

emixustat cyclohexyl (i.e., compound 57) further enhanced

ocular distribution. Compound 57 reached a concentration of

176 93 pmol/eye, almost twice that of emixustat at the same

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Figure 4. Crystal structures of bovine RPE65 in complex with (A) emixustat (PDB accession code 4RSC),³²(B) MB-004 (PDB accession code

5UL5³³), (C) C-4-ﬂuoro-emixustat (compound 49), (D) C-4-ﬂuoro-MB-004 (compound 24), (E) C-4-diﬂuoro-emixustat (compound 57), and

(F) detergent hexaoxyethylene monooctyl ethyl (C₈E₆). The green mesh represents unbiased sigma-A weighted |F_o|−|F_c| electron density

contoured at 3 rmsd calculated prior to modeling the ligand.

also present in the distal cavity corresponding to a palmitate

molecule forming a coordinate bond with the iron prosthetic

group via its carboxylate moiety, consistent with prior

ﬁndings.^32,33Electron density maps following the inclusion of

the inhibitor ligand in the model showed a well-deﬁned density

for the aryl γ-hydroxypropylamine moiety in all cases. The β,β-

dipropylethoxy group of 24 was similarly well deﬁned, whereas

the densities for the cyclohexylmethoxy groups of 49 and 57

were comparatively much weaker (Figure 4). The relative

quality of the electron density support for these diﬀerent

moieties is consistent with prior studies on emixustat and MB-

004.^32,33The crystal structure of apo-RPE65 featured an active

site |F_o|−|F_c| density consistent with hexaethylene glycol

monooctyl ether (C₈E₆).

site provides evidence that such inhibition can be partially

attributed to direct active site binding.³⁷Despite the high

concentration of the detergent in the crystallization mother

liquor (>16 mM), compounds 24, 49, and 57 were able to

outcompete the detergent for binding to the RPE65 active site,

consistent with their strong active site-binding aﬃnities.

The binding modes for compounds 24, 49, and 57 largely

overlap with those of MB-004 and emixustat with a few notable

exceptions (Figure 4). The binding mode of the aryl γ-

hydroxypropylamine moiety of compound 57 was similar to

that of emixustat (Figure 4A,E), whereas its terminal ring was

rotated by ∼90° as evidenced by clear electron density for its

gem-diﬂuoro moiety. This change in the cyclohexyl ring

position was likely driven by ﬂuorine-associated steric eﬀects.

Notably, a similar ring positioning was observed previously for

emixustat in a P6₅22 RPE65 crystal form (PDB accession code

4RYX). In this position, the gem-diﬂuoro moiety engages in

Linear detergents are known to inhibit the activity of

RPE65^37,38and related carotenoid oxygenase enzymes.^39,40

The structure of RPE65 solved with C₈E₆bound to its active

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Figure 5. Theoretical analyses of the interactions of compound 49 (A) and compound 24 (B) with Tyr²⁷⁵, using a model dimer system.

only a single van der Waals interaction with the Asn¹⁹⁴side

chain oxygen atom. Although the bi-phasic IC₅₀curve for 57

suggested two modes of RPE65 binding (Supporting

Information Table S1), we could not discern such behavior

from the structural data, although it is notable that RPE65 is

more poorly ordered near the active site opening in the

structure of the RPE65 compound 57 complex as compared to

the emixustat-bound structure. It is thus possible that multiple

conformations, one of which has a higher aﬃnity for 57, could

be responsible for the bi-phasic inhibition results. In the case of

49, the C1″-O-C3-C2 dihedral angle is rotated by ∼49°

compared to the corresponding angle in emixustat, which is

nearly planar. The analogous dihedral angle in 24 similarly

deviates from the planarity seen in MB-004 by ∼31°. These

diﬀerences can be attributed to the presence of the nearby 4-

ﬂuoro group giving rise to two eﬀects. First, the presence of the

ﬂuorine causes a ∼0.4 Å downward shift in the binding

position of the aryl ring, which may necessitate a

corresponding rotation in the C3″-O bond to avoid steric

clashes with the oxy-linked alkane moieties. Second, the

rotation could also be driven by electrostatic eﬀects between

the electron-dense ﬂuoro moiety and the lone-pair electrons on

the O atom of the aryl ether. In addition, the structure of the

enzyme compound 24 complex revealed a conformational

diﬀerence in one of the propyl groups of 24 as compared to the

complex with MB-004.

The enzyme environment around the 4-ﬂuoro substituent of

the bound modulator is likely an important factor that could

help explain the greater potency of the 4-ﬂuoro compounds 24

and 49 compared to their parent molecules. The dominant

interaction occurs with Tyr²⁷⁵where the ﬂuoro group makes a

close (∼3−3.1 Å) en face contact with the aromatic ring.

Compared to structures with the parent molecules bound, the

Tyr²⁷⁵side chain is rotated by ∼8° around the Cβ-Cγ bond,

likely to alleviate steric clashes with the ﬂuoro moiety or to

facilitate the en face interaction. Redmond’s lab has

mutagenized position Tyr275 previously.⁴¹Only the aromatic

residue Phe was partly active (44%), whereas other

substitutions were inactive.

Quantum Chemical Analysis of the Aromatic−Fluoro

Interaction Observed in Crystals. To further elucidate the

energetics of the aromatic−ﬂuoro interaction observed in the

crystal structures, we employed quantum chemical calculations.

One would expect the ∼3 Å interaction to be repulsive in the

ﬁrst approximation, as a highly electronegative F atom is

brought in the close vicinity of an electron-rich π cloud.

However, analogous stabilizing Cl−π dispersion-driven inter-

actions are known in protein chemistry.⁴²Their strength is on

the order of 2 kcal/mol and are thought to be dispersion-

driven forces. In contrast, F−π contacts have not been

investigated thoroughly. Experiments based on synthetic

models provide estimates of the stabilizing interaction of

roughly 1.6 kcal/mol.⁴³In recent work, Li and co-workers⁴⁴

found that the stability of the F−π interaction increases with

positive charge accumulation in the π-system. Thus, the

interaction seems to be mostly electrostatically driven.

Using dimer model systems obtained from the crystal

structures (Figure 5), we estimated the strength of the

interaction (ΔE_int) between Tyr²⁷⁵and each of the two

ﬂuorinated compounds 49 and 24 to be −2.34 and −2.69

kcal/mol, respectively, using the state-of-the-art DLPNO-

CCSD(T) method at the basis set limit.^45,46The interaction

energy was broken down into various contributions using the

local energy decomposition (LED) scheme.⁴⁷According to

Table 3, the overall electrostatic interaction has a repulsive

characteristic [ΔE_HF‑CCSD(electro) > 0]. Here, key stabilizing

factors within this contribution are the electrostatic attraction

between the “prepared” wavefunctions of the fragments

ΔE_HF(elstat) and charge-transfer contributions ΔE_CCSD(CT).

Both are signiﬁcantly more negative for compound 24. Within

the latter, 24 → Tyr double excitations have a signiﬁcant

contribution (−3.70 kcal/mol). The overall repulsive electro-

static eﬀects are compensated by the dispersion interactions

ΔE_CCSD(disp) of −4.28 and −8.23 kcal/mol for compounds 49

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The orbital-interaction energies should be attributed mainly to

electrostatic stabilization of the electron density in the dimers

and less to dispersion. The latter is added a posteriori in our

calculations, and it does not inﬂuence the density distribution

directlythe total interaction energy (including dispersion

correction) is −2.3 kcal/mol for 49 and −3.4 kcal/mol for 24.

In this context, up to three signiﬁcant NOCV complementary

pairs yield deformation densities, as shown in Figure 5 ( 0.005

a.u. isosurface), that account for up to 60% of the orbital

interaction energies in both cases. In the case of 24, the key

ingredients are the interactions between the n-propyl chain and

the Tyr²⁷⁵phenyl ring (Δρ₁^orb, Δρ₂^orb). The inspection of Δρ^o₃^rb

shows that the region between the F atom and phenyl ring

gains some electron density. At the same time, electron density

reorganization takes place at both fragments within σ(C−F)/

σ*(C−F)/n(F) and π(phenyl)/π*(phenyl) orbitals of 24 and

Tyr²⁷⁵, respectively. The electron density reorganization for 49

compared to 24 is less pronounced and is reﬂected in the

overall diminished interaction energy.

Table 3. LED Analysis of the DLPNO-CCSD(T) Total

Interaction Energy (ΔE_int) in the Crystal Structure-Derived

a

Dimers of Compounds 49 and 24 with Tyr²⁷⁵

ΔE [kcal/mol]

49

24

ΔE_HF-CCSD(electro)

including

1.87

5.80

ΔE_HF(el-prep)

ΔE_CCSD(el-prep)

ΔE_HF(elstat)

8.46

1.51

27.11

5.33

−4.77

−1.58

−0.17

−4.28

−0.47

0.54

−15.21

−5.71

−3.70

−2.02

−8.23

−1.10

1.01

ΔE_HF(exch)

ΔE_CCSD(CT EmixF → Tyr)

ΔE_CCSD(CT Tyr → EmixF)

ΔE_CCSD(disp)

ΔE(T)

ΔE(CBS)

ΔE_int

−2.34

−2.69

a

All values are in kcal/mol.

and 24, respectively. The magnitude of ΔE_HF(elstat) and

ΔE_CCSD(disp) is similar; thus, one can say that both

electrostatic and van der Waals forces are driving the examined

interactions.

DISCUSSION AND CONCLUSIONS

■

Emixustat, a ﬁrst-in-class visual cycle modulation drug

candidate has displayed promising in vitro and in vivo

properties for the treatment of a variety of retinal diseases.

However, emixustat suﬀers from sub-optimal eﬃcacy, problem-

atic side eﬀects, and rapid metabolism, which cloud its clinical

future.¹⁷

To further investigate the nature of the interaction between

Tyr²⁷⁵and compounds 49 and 24 in 3D space, we performed a

noncovalent interaction (NCI) analysis (Figure 5).⁴⁸Brieﬂy,

the NCI plot shows low-density and low-gradient regions that

are associated with NCIs, colored according to one of the

components of the density Laplacian (λ₂): strong attractive

interactions appear at λ₂< 0 (e.g., H-bonds; blue in Figure 5),

while steric repulsion is associated with positive values of λ₂

(red in Figure 5). Dispersion forces appear with small negative

values around λ₂≈ 0 (green in Figure 5). The examined

systems’ NCI plots revealed a signiﬁcant dispersion interaction

region in the middle between the F atom of both 24 and 49

and the π-plane of Tyr²⁷⁵(Figure 5: isovalue of s = 0.6 a.u.,

colored according to −0.035 < sign(λ₂)ρ < 0.02). Another way

to look at intermolecular interactions is to study natural

orbitals for chemical valence (NOCV)⁴⁹within the density

functional theory (here, we used the ωB97X-D3BJ func-

tional).^50,51Brieﬂy, we start by considering isolated fragments

1 and 2 at the geometry of a dimer. The two are characterized

with the electron densities ρ₁and ρ₂, respectively. A simple

union of these densities yields pro-molecular density ρ^pro= ρ₁

+ ρ₂along with an associated pro-molecular wave function

This work investigates whether ﬂuorination and/or deutera-

tion of emixustat can eliminate the pharmacokinetic short-

comings of this clinical candidate. We developed novel

synthetic approaches to produce speciﬁc chiral products as

emixustat’s inhibitory activity toward RPE65 was previously

shown to depend on the C1 stereochemistry.¹⁹We present an

advanced three-component Mannich reaction utilizing (S)-

(−)-α-methylbenzylamine to produce the desired stereo-

isomer. In this reaction scheme, the resultant from the

subsequent reduction step, γ-hydroxypropylaminobenzyl prod-

uct(s), consists of a mixture of diastereomers that can be

carbamylated and separated using conventional ﬂash column

chromatography on silica gel as opposed to chiral chromatog-

raphy required for the separation of the parent enantiomers.

Facile hydrolysis of the carbamate succeeded by debenzylation

regenerates the key γ-hydroxypropylamine framework with the

deﬁned stereochemistry. This method provides clear advan-

tages in terms of cost and scalability over chiral separation

methods and perhaps can extend to other similar compounds.

We investigated the impact of these emixustat and

emixustat-derived compounds 57, 58, and 59 on RPE65

activity in vitro and in live mice. Our ﬁrst observation was that

RPE65 inhibition does not correlate with accumulation in the

eye. 57, 58, and 59 accumulate more in the eye than emixustat,

as shown in Figure 2, possibly due to an increase in metabolic

stability or selective uptake or retention by an as-yet undeﬁned

ocular component. The half-lives of the gem-diﬂuoro

molecules, 57 and 59, are higher than that of 58, which is at

least in part due to the greater metabolic stability from the

abrogation of known oxidation pathways of the cyclohexyl

moiety, and it is possible that other factors are at play. Another

possible explanation for this prolonged half-life eﬀect is that

the gem-diﬂuoro molecules could be transported to or retained

within ocular tissues (e.g., by promoting melanin binding)

more eﬃciently than their non-ﬂuorinated counterparts.

Practically, 57 or gem-diﬂuoro-emixustat has better pharmaco-

Ψ

^pro. Self-consistent optimization of the latter provides Ψ^opt

with the optimal density ρ^opt. We then deﬁne deformation

density Δρ as the diﬀerence between pro-molecular density

and self-consistently converged density Δρ = ρ^pro−ρ^opt

Eigenorbitals of the corresponding deformation density

operators are called NOCVs, and typical bonding and

antibonding pairs are described with complementary NOCVs

.

(φ ).

n

With each such pair, we associate the orbital deformation

orb

density Δρ_n

Δρ^orb= −v(φ )²+ v(φ)²

n

−n

n

where v is the corresponding NOCV eigenvalue. By using the

extended transition state theory (ETS),⁵²one assigns a

particular energy portion to such orbital interaction. By

summing up all NOCV interaction energies, one obtains the

so-called orbital-interaction energy. In the case of our dimers,

this yields −0.7 and −2.4 kcal/mol for 49 and 24, respectively.

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puriﬁcation. Analytical thin-layer chromatography (TLC) was

performed on 0.25 mm glass-backed EMD Millipore 60 F254 plates.

Visualization of the developed chromatogram was accomplished with

UV light (254 nm) and stained with either ethanolic phosphomo-

lybdic acid, ceric ammonium molybdate, or permanganate (KMnO₄).

LC was performed using a forced air-ﬂow (FC) on silica gel (Merck,

230−400 mesh), using eluting solvents (reported as V/V ratio

kinetic properties than emixustat in terms of elimination half-

life and consequent swings in eye levels, solving some of the

problems associated with the use of emixustat in humans.

VAP-1 is a lesser known phase-1 metabolic pathway⁵³

important for the primary amine oxidation of clinically used

drugs including primaquine⁵⁴and tresperimus⁵⁵in addition to

emixustat.¹⁵Our results here suggest that the deuteration of

primary amines susceptible to VAP-1 oxidation could be a

generally eﬀective approach to prolonging their in vivo activity.

The impact of alpha deuterium substitution of amines on VAP-

1 metabolic susceptibility was previously studied in vitro using

benzylamine and various phenylethylamines as test sub-

strates.⁵⁶A variety of para-substituted phenylethylamines,

whose amine protons are in an environment similar to those

of emixustat, all displayed kinetic isotope eﬀects on k_catof ∼5−

8, which is within the theoretically expected primary kinetic

isotope eﬀect (i.e., KIE = 3−7),⁵⁷⁻⁶⁰for a reaction with a rate-

limiting proton abstraction step. Our data showing impaired

oxidation of deuterated emixustat as compared to emixustat are

consistent with hydrogen abstraction being at least partially

rate-limiting in the mechanism of emixustat oxidation by VAP-

1.

The structure−activity relationships presented here are

largely consistent with previous observations including higher

inhibitory activity for the R-versus S-isomer of the γ-

aminoalcohol (Table S1, compare 24 to 23),¹⁹as well as for

compounds with a β,β-dipropylethoxy substituent as opposed

to a cyclohexyl or β-ionone moiety.³³Our structural biology

results support our previous ﬁndings that the β,β-dipropyle-

thoxy group^19,33can engage binding pockets within the RPE65

active site cavity that are not accessible to a cyclohexyl group,

which likely improves the binding aﬃnity of these compounds.

We expand the known SAR of RPE65 inhibitors to include the

eﬀects of ﬂuorine substitution throughout the base structures

of emixustat, MB-001 and MB-004. We found that ﬂuorine

substitution on the aryl moiety could have either a positive or

negative impact on inhibitory activity. A notable favorable

impact was the 4-ﬂuoro derivative of MB-004 (compound 24)

and 4-ﬂuoro-emixustat (49), which exhibited an IC₅₀value (50

nM) approximately 2-fold lower than MB-004 and emixustat,

respectively. Structurally, the enhanced aﬃnity appears to

result from an energetically favorable interaction between the

ﬂuoro substituent and an active site tyrosyl side chain (Tyr²⁷⁵).

According to our calculations, the key stabilization is provided

by van der Waals⁴²contributions as opposed to electrostatic

interactions consistent with the electron-rich nature of the

phenolic side chain. The introduction of a gem-diﬂuoro group

at the 5″ position of the cyclohexyl ring of emixustat

(compound 57) also strengthened the inhibitory activity,

although the structural basis for this eﬀect was less clear given

that the gem-diﬂuoro group interacts minimally with the

RPE65 active site pocket. The gem-diﬂuoro substitution is both

pharmacokinetically and pharmacodynamically favorable and

should be considered in the future development of other

emixustat derivatives to improve in vivo activity.

61

1

mixture). The H, ¹³C /DEPT, ¹⁹F NMR, and 2D-nuclear magnetic

resonance (NMR) spectra were recorded at 25 °C on Bruker

AVANCE NMR spectrometers operating at 300, 400, 500, 600, and

700 MHz for the ¹H channel and were in accordance with the

assigned structures. ¹⁹F NMR spectra were recorded without

decoupling from protons. Chemical shifts were reported in δ units,

part per million, with reference to the residual solvent peak CDCl₃(δ

1

7.26), CD₃OD (δ 3.31), and C₆D₆(δ 7.16) for H and CDCl₃(δ

77.36), CD₃OD (δ 49.00), and C₆D₆(δ 128.06) for ¹³C spectra or

TMS (δ 0.00). NMR data are presented in the following order:

chemical shift, peak multiplicity (b = broad, s = singlet, d = doublet, t

= triplet, q = quartet, m = multiplet, dd = doublet of doublet, ddd =

doublet of doublet of doublet, ddt = doublet of doublet of triplet, dq

= doublet of quartet, dm = doublet of multiplet, and br = broad),

coupling constant (in Hz). The speciﬁc light rotations were measured

with a JASCO digital polarimeter (Model P-1010, λ = 589 nm, 0.05°

accuracy) using a cylindrical quartz cell (5 mL, l = 5 cm) at 25 °C.

Mass spectra were recorded in positive ionization mode on an Agilent

6545 QTOF mass spectrometer (Agilent Technologies, USA),

equipped with ESI and atmospheric pressure chemical ionization

interfaces coupled to an Agilent 1260 ultrahigh-pressure LC

(UHPLC) (Agilent Technologies, Santa Clara, CA, USA). The

Agilent 1260 series system consists of a G4204A quaternary pump, a

G4226A ALS auto-sampler, and a G1316C column compartment.

UHPLC was carried out on a ZORBAX RRHD Eclipse Plus C18, 95

Å, 2.1 × 50 mm, 1.8 μm column (Agilent Technologies, USA)

column with H₂O (0.1% formic acid)−acetonitrile gradient elution

from 5 to 95% acetonitrile in the course of 10 min at a ﬂow rate of 0.5

mL/min. Preparative and analytical HPLC were performed on a

HPLC system (Young Lin Instruments, Anyang, Korea) with a LUNA

C18(2) (10 μm, 250 mm × 21.2 mm) column and a chiral column

LUX Amylose-1 (5 μm, 250 mm × 21.2 mm), for preparative

puriﬁcation, and (5 μm, 250 × 4.6 mm), for analysis, all from

Phenomenex, Inc. (Torrance, CA). Acetonitrile and double-distilled

water in diﬀerent ratios were used as an eluent. The ﬁnal biologically

tested compounds displayed ≥95% purity (conﬁrmed using analytical

HPLC). Compound 57 was obtained at 90% purity. Synthesis of the

ﬁnal emixustat analogues is detailed herein, while the synthesis of all

the precursors is described in the Supporting Information.

(S)-3-Amino-1-(4-ﬂuoro-3-((2-propylpentyl)oxy)phenyl)propan-

1-ol (23). General Procedure. The N-protected aminoalcohol 15

(280 mg, 0.70 mmol) was suspended with 10% Pd/C (100 mg) in dry

MeOH (4 mL) under a nitrogen atmosphere. After 30 min, solid

ammonium formate (1.29 mmol) was added. The resulting mixture

was reﬂuxed (TLC monitoring), cooled to 0 °C, ﬁltered through a

celite pad, and washed with chloroform. The ﬁltrate was concentrated

under reduced pressure to aﬀord the corresponding title γ-

aminoalcohol 23 (188 mg, 91%) as colorless oil. [α]D25 =

−21.32° (c = 0.0003, CH₂Cl₂). ¹H NMR (300 MHz, CDCl₃): δ

7.23−6.85 (m, 2H), 6.80 (ddd, J = 2.0, 4.0, 8.0 Hz, 1H), 4.82 (dd, J =

3.0, 8.5 Hz, 1H), 3.89 (d, J = 5.5 Hz, 2H), 3.64 (bs, 2H), 3.37−2.96

(m, 1H), 2.96−2.59 (m, 1H), 2.07−1.57 (m, 3H), 1.56−1.19 (m,

8H), 0.91 (t, J = 7.0 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃): δ 151.7

(d, J = 244.5 Hz), 147.2 (d, J = 10.5 Hz), 141.4 (d, J = 3.5 Hz), 117.5

(d, J = 7.0 Hz), 115.5 (d, J = 18.5 Hz), 112.2, 74.3, 72.3, 40.1, 39.8,

37.6, 33.6, 19.9, 14.4. ¹⁹F NMR (376 MHz, CDCl₃): δ −137.5 (m).

HRMS (ESI): calcd for C₁₇H₂₈FNO₂[M + H]⁺, 298.2176; found,

298.2172.

EXPERIMENTAL SECTION

■

Chemistry. All reactions were performed in oven-dried glassware

under a dry argon or nitrogen atmosphere. Synthesis under MW

irradiation was performed with a Biotage, Initiator+ Robot Eight

microwave system (Charlotte, NC). Lithium aluminum deuteride [98

atom% ²H(D)] was purchased from Millipore Sigma Isotopes

(Miamisburg, OH). All other reagents were used as supplied without

(R)-3-Amino-1-(4-ﬂuoro-3-((2-propylpentyl)oxy)phenyl)propan-

1-ol (24). According to the general procedure, N-benzylated

compound 16 (458 mg, 1.14 mmol) was deprotected to give the

title γ-aminoalcohol 24 (314.2 mg, 93%) as colorless oil. [α]D25 =

+21.56° (c = 0.0002, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃): δ 7.06−

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6.94 (m, 2H), 6.90−6.70 (m, 1H), 4.82 (dd, J = 3.0, 8.5 Hz, 1H), 3.89

(d, J = 5.5 Hz, 2H), 3.5 (bs, 2H), 3.20−3.02 (m, 1H), 3.00−2.90 (m,

1H), 1.89−1.80 (m, 2H), 1.78−1.68 (m, 1H), 1.49−1.20 (m, 8H),

0.91 (t, J = 7.0 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 151.8 (d, J

= 244.5 Hz), 147.3 (d, J = 10.5 Hz), 141.2 (d, J = 3.5 Hz), 117.6 (d, J

= 7.0 Hz), 115.6 (d, J = 18.5 Hz), 112.3, 74.7, 72.4, 40.2, 39.4, 37.6,

33.6, 19.9, 14.4. ¹⁹F NMR (376 MHz, CDCl₃): δ −137.5 (m). HRMS

(ESI): calcd for C₁₇H₂₈FNO₂[M + H]⁺, 298.2176; found, 298.2173.

(R/S)-3-Amino-1-(3-ﬂuoro-5-((2-propylpentyl)oxy)phenyl)-

propan-1-ol (25). According to the general procedure, N-benzylated

compound 17 (236 mg, 0.59 mmol) was deprotected giving the chiral

6H). ¹³C NMR (176 MHz, C₆D₆): δ 150.0 (d, J = 244.5 Hz), 147.6

(d, J = 11.0 Hz), 134.6 (d, J = 10.5 Hz), 123.9 (d, J = 4.0 Hz), 119.3

(d, J = 3.5 Hz), 113.2, 72.2, 69.8 (d, J = 2.5 Hz), 40.8, 38.5, 38.0, 34.0,

20.3, 14.6. ¹⁹F NMR (376 MHz, C₆D₆): δ −142.05 (t, J = 7.0 Hz).

HRMS (ESI): calcd for C₁₇H₂₈FNO₂[M + H]⁺, 298.2176; found,

298.2187.

(R/S)-3-Amino-1-(2-ﬂuoro-3-((2-propylpentyl)oxy)phenyl)-

propan-1-ol (30). Following the general procedure, N-benzylated

compound 21 (35 mg, 0.09 mmol) was deprotected giving the chiral

1

γ-aminoalcohol 30 (13.6 mg, 52%) as colorless oil. H NMR (400

MHz, C₆D₆): δ 7.60 (t, J = 1.0, 8.0 Hz, 1H), 7.01 (t, J = 8.0 Hz, 1H),

6.69 (t, J = 8.0 Hz, 1H), 5.44 (dd, J = 2.0, 8.5 Hz, 1H), 3.68 (d, J =

5.5 Hz, 2H), 2.40−2.38 (m, 1H), 2.34−2.27 (m, 1H), 1.82−1.69 (m,

2H), 1.54−1.48 (m, 1H) 1.46−1.21 (m, 8H), 0.88 (t, J = 7.0 Hz,

6H). ¹³C NMR (100 MHz, C₆D₆): δ 150.12 (d, J = 244.5 Hz), 147.6

(d, J = 11.0 Hz), 134.6 (d, J = 10.5 Hz), 123.9 (d, J = 4.0 Hz), 119.3

(d, J = 3.5 Hz), 113.2, 72.2, 69.8 (d, J = 2.5 Hz), 40.8, 38.6, 38.0, 34.0,

20.3, 14.6. ¹⁹F (376 MHz, C₆D₆): δ −142.11 (t, J = 7.0 Hz). HRMS

(ESI): calcd for C₁₇H₂₈FNO₂[M + H]⁺, 298.2176; found, 298.2178.

3-Amino-1-(3-(cyclohexylmethoxy)-4-ﬂuorophenyl)propan-1-ol

(49). General Procedure. To a solution of compound 40 (8 g, 28.8

mmol, 1 equiv) in dry tetrahydrofuran (THF) (55 mL) was added

lithium aluminum hydride (2.46 g, 64.9 mmol, 2.25 equiv) in portions

under nitrogen at 0 °C, and then the reaction mixture was stirred at 0

°C for another 0.5 h. The reaction was quenched with adding water

(2.46 mL), sodium hydroxide solution (10%, 2.46 mL), and water

(7.38 mL) sequentially. The resulting mixture was ﬁltered and

concentrated under vacuum. The residue was puriﬁed by silica gel

chromatography with dichloromethane/methanol (10/1) as the

eluent to give 49 (1.5 g, 18%) as a yellow gum. ¹H NMR (600

MHz, CDCl₃): δ 7.08−6.88 (m, 2H), 6.85−6.72 (m, 1H), 4.82 (d, J =

6.9 Hz, 1H), 4.50 (bs, 3H), 3.77 (d, J = 6.4 Hz, 2H), 3.18−3.00 (m,

1H), 3.00−2.83 (m, 1H), 1.94−1.82 (m, 3H), 1.82−1.76 (m, 2H),

1.76−1.70 (m, 2H), 1.70−1.65 (m, 1H), 1.33−1.22 (m, 2H), 1.22−

1.13 (m, 1H), 1.07−0.94 (ddd, J = 2.4, 12.2, 24.1 Hz, 2H). ¹³C NMR

(150 MHz, CDCl₃): δ 151.8 (d, J = 244.8 Hz), 147.3 (d, J = 10.6 Hz),

141.1 (d, J = 3.1 Hz), 117.6 (d, J = 6.8 Hz), 115.7 (d, J = 18.4 Hz),

112.3 (d, J = 1.0 Hz), 74.9, 74.0, 39.6, 38.6, 37.8, 29.9, 26.5, 25.8. ¹⁹F

(565 MHz, CDCl₃): δ −136.8 to −136.95 (m). HRMS (ESI): calcd

for C₁₆H₂₄FNO₂[M + H]⁺, 282.1869; found, 282.1862.

1

γ-aminoalcohol 25 (170 mg, 97%) as colorless oil. H NMR (600

MHz, C₆D₆): δ 7.10−7.02 (m, 1H), 6.99−6.85 (m, 1H), 6.68−6.54

(m, 1H), 5.14−4.61 (m, 1H), 3.56 (d, J = 5.5 Hz, 2H), 2.50−2.35 (m,

1H), 2.34−2.14 (m, 1H), 1.70−1.62 (m, 1H), 1.49−1.19 (m, 10H),

0.87 (t, J = 7.0 Hz, 6H). ¹³C NMR (150 MHz, C₆D₆): δ 164.3 (d, J =

244.0 Hz), 161.2 (d, J = 11.0 Hz), 150.0 (d, J = 8.5 Hz), 108.3 (d, J =

2.5 Hz), 105.0 (d, J = 22.0 Hz), 100.3 (d, J = 25.0 Hz), 75.1, 70.9,

40.7, 39.7, 37.8, 34.0, 20.3, 14.6. ¹⁹F NMR (376 MHz, C₆D₆): δ

−112.89 (t, J = 10.0 Hz). HRMS (ESI): calcd for C₁₇H₂₈FNO₂[M +

H]⁺, 298.2176; found, 298.2179.

(R/S)-3-Amino-1-(3-ﬂuoro-5-((2-propylpentyl)oxy)phenyl)-

propan-1-ol (26). Deprotection of N-benzylated compound 18 (46

mg, 0.11 mmol) according to the general procedure aﬀorded the

1

chiral γ-aminoalcohol 26 (14.2 mg, 42%) as colorless oil. H NMR

(400 MHz, CDCl₃+ CD₃OD): δ 6.72−6.67 (m, 1H), 6.67−6.59 (m,

1H), 6.54−6.43 (m, 1H), 4.76 (dd, J = 4.0, 8.0 Hz, 1H), 3.80 (d, J =

5.5 Hz, 2H) 3.77 (bs, 2H), 3.07−2.70 (m, 2H), 1.86−1.73 (m, 3H),

1.50−1.24 (m, 8H), 0.91 (t, J = 7.0 Hz, 6H). ¹³C NMR (100 MHz,

CDCl₃+ CD₃OD): δ 162.7 (d, J = 244.0 Hz), 160.7 (d, J = 11.0 Hz),

148.25 (d, J = 8.5 Hz), 107.7 (d, J = 2.5 Hz), 104.5 (d, J = 22.0 Hz),

100.6 (d, J = 25.0 Hz), 73.1, 71.3, 40.0, 39.2, 37.5, 33.7, 20.0, 14.4. ¹⁹F

(376 MHz, CDCl₃+ CD₃OD): δ −112.74 (t, J = 10.0 Hz). HRMS

(ESI): calcd for C₁₇H₂₈FNO₂[M + H]⁺, 298.2176; found, 298.2186.

(R/S)-3-Amino-1-(2-ﬂuoro-5-((2-propylpentyl)oxy)phenyl)-

propan-1-ol (27). According to the general procedure, N-benzylated

compound 20 (30 mg, 0.07 mmol) was deprotected giving the chiral

1

γ-aminoalcohol 27 (13.4 mg, 61%) as colorless oil. H NMR (400

MHz, C₆D₆): δ 7.69 (ddd, J = 1.0, 3.0, 6.0 Hz, 1H), 6.83 (t, J = 9.5

Hz, 1H), 6.70−6.58 (m, 1H), 5.42 (dd, J = 1.0, 8.5 Hz, 1H), 3.70 (d, J

= 5.5 Hz, 2H), 2.51−2.40 (m, 1H), 2.37−2.29 (m, 1H), 1.84−1.76

(m, 1H), 1.75−1.65 (m, 1H), 1.54−1.45 (m, 1H), 1.44−1.21 (m,

8H), 0.87 (t, J = 7.0 Hz, 6H). ¹³C NMR (100 MHz, C₆D₆): δ 156.4,

154.2 (d, J = 236.0 Hz), 134.3 (d, J = 14.5 Hz), 115.6 (d, J = 23.5

Hz), 114.0 (d, J = 8.0 Hz), 113.4 (d, J = 4.5 Hz), 71.3, 70.1, 40.0,

38.6, 38.0, 34.1, 20.3, 14.6. ¹⁹F NMR (376 MHz, C₆D₆): δ −131.25

(m). HRMS (ESI): calcd for C₁₇H₂₈FNO₂[M + H]⁺, 298.2176;

found, 298.2178.

3-Amino-1-(3-(cyclohexylmethoxy)-5-ﬂuorophenyl)propan-1-ol

(50). The general procedure was followed with nitrile 41 (5 g, 35.6

1

mmol, 1.1 eq) yielding a yellow gum (1.60 g, 51%). H NMR (600

MHz, CDCl₃): δ 6.73−6.68 (m, 1H), 6.65 (d, J = 9.3 Hz, 1H), 6.46

(dt, J = 2.2, 10.7 Hz, 1H), 4.87 (dd, J = 2.6, 8.4 Hz, 1H), 3.71 (d, J =

6.4 Hz, 2H), 3.29 (bs, 3H), 3.10−3.03 (m, 1H), 3.00−2.89 (m, 1H),

1.88−1.80 (m, 3H), 1.79−1.73 (m, 3H), 1.73−1.64 (m, 2H), 1.32−

1.24 (m, 2H), 1.23−1.15 (m, 1H), 1.03 (ddd, J = 3.2, 12.3, 24.3 Hz,

2H). ¹³C NMR (150 MHz, CDCl₃): δ 163.6 (d, J = 244.3 Hz), 160.6

(d, J = 11.2 Hz), 148.47 (d, J = 8.6 Hz), 117.6 (d, J = 1.9 Hz), 104.6

(d, J = 22.2 Hz), 100.6 (d, J = 25.0 Hz), 74.8, 73.8, 40.3, 39.2, 37.7,

29.9, 26.6, 25.8. ¹⁹F (565 MHz, CDCl₃): δ −112.20 (t, J = 10.1 Hz).

HRMS (ESI): calcd for C₁₆H₂₄FNO₂[M + H]⁺, 282.1869; found,

282.1874.

(R/S)-3-Amino-1-(2-ﬂuoro-5-((2-propylpentyl)oxy)phenyl)-

propan-1-ol (28). Following the general procedure, N-benzylated

compound 19 (33 mg, 0.08 mmol) was deprotected giving the chiral

1

γ-aminoalcohol 28 (15.2 mg, 61%) as colorless oil. H NMR (700

MHz, C₆D₆): δ 7.71 (dd, J = 3.0, 6.0 Hz, 1H), 6.83 (t, J = 9.5 Hz,

1H), 6.71−6.56 (m, 1H), 5.43 (dd, J = 1.0, 8.5 Hz, 1H), 3.70 (d, J =

5.5 Hz, 2H), 2.52−2.37 (m, 1H), 2.37−2.29 (m, 1H), 1.85−1.75 (m,

1H), 1.75−1.63 (m, 1H), 1.51−1.43 (m, 1H), 1.43−1.20 (m, 8H),

0.86 (t, J = 7.0 Hz, 6H). ¹³C NMR (176 MHz, C₆D₆): δ 156.3, 154.2

(d, J = 236.0 Hz), 134.3 (d, J = 14.5 Hz), 115.6 (d, J = 23.5 Hz),

114.0 (d, J = 8.0 Hz), 113.3 (d, J = 4.5 Hz), 71.3, 70.2, 41.0, 38.56,

38.01, 34.08, 20.35, 14.66. ¹⁹F NMR (376 MHz, C₆D₆): δ −131.25

(m). HRMS (ESI): calcd for C₁₇H₂₈FNO₂[M + H]⁺, 298.2176;

found, 298.2177.

3-Amino-1-(5-(cyclohexylmethoxy)-2-ﬂuorophenyl)propan-1-ol

(51). The general procedure was followed with compound 42 (3.5 g,

1

12.6 mmol, 1 equiv) giving a yellow gum (800 mg, 22%) H NMR

(600 MHz, CDCl₃): δ 7.11 (dd, J = 3.1, 6.0 Hz, 1H), 6.87 (dd, J =

9.0, 9.7 Hz, 1H), 6.69 (dt, J = 3.6, 9.0 Hz, 1H), 5.20 (dd, J = 3.0, 8.4

Hz, 1H), 3.75−3.69 (m, 2H), 3.47−2.98 (m, 4H), 2.98−2.94 (m,

1H), 1.94−1.88 (m, 1H), 1.87−1.82 (m, 2H), 1.80−1.72 (m, 4H),

1.70−1.66 (m, 1H), 1.32−1.24 (m, 2H), 1.23−1.15 (m, 1H), 1.03

(ddd, J = 3.3, 12.3, 24.2 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃): δ

155.7 (d, J = 1.7 Hz), 153.6 (d, J = 237.4 Hz), 132.8 (d, J = 14.9 Hz),

115.5 (d, J = 23.5 Hz), 113.9 (d, J = 8.0 Hz), 112.7 (d, J = 4.5 Hz),

74.2, 69.5 (d, J = 1.6 Hz), 40.5, 38.1, 37.9, 30.0, 30.0, 26.6, 25.9. ¹⁹F

(565 MHz, CDCl₃): δ −130.78 to −130.88 (m). HRMS (ESI): calcd

for C₁₆H₂₄FNO₂[M + H]⁺, 282.1869; found, 282.1864.

(R/S)-3-Amino-1-(2-ﬂuoro-3-((2-propylpentyl)oxy)phenyl)-

propan-1-ol (29). According to the general procedure, N-benzylated

compound 22 (36 mg, 0.09 mmol) was deprotected to give the chiral

1

γ-aminoalcohol 29 (14.1 mg, 52%) as colorless oil. H NMR (700

MHz, C₆D₆): δ 7.60 (t, J = 8.0 Hz, 1H), 7.07 (t, J = 8.0 Hz, 1H), 6.68

(dd, J = 1.0, 8.0 Hz, 1H), 5.44 (dd, J = 2.0, 8.5 Hz, 1H), 3.68 (dd, J =

2.0, 5.5 Hz, 2H), 2.45−2.39 (m, 1H), 2.33−2.29 (m, 1H), 1.80−1.70

(m, 2H), 1.55−1.47 (m, 1H) 1.46−1.23 (m, 8H), 0.88 (t, J = 7.0 Hz,

3-Amino-1-(3-(cyclohexylmethoxy)-2-ﬂuorophenyl)propan-1-ol

(52). The general procedure was followed using nitrile 43 (3 g, 10.8

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mmol, 1 equiv) resulting in the isolation of a yellow gum (2.1 g, 66%).

(m). HRMS (ESI): calcd for C₁₉H₂₈FNO₂[M + H]⁺, 322.2182;

found, 322.2173.

¹H NMR (600 MHz, CDCl₃): δ 7.1 (t, J = 6.7 Hz, 1H), 7 (t, J = 8.0

3-Amino-1-(3-((4,4-diﬂuorocyclohexyl)methoxy)phenyl)propan-

1-ol (57). Using compound 48 (3.0 g, 10.1 mmol, 1 equiv) and

following the general procedure gave amine 57 (580 mg, 17% yield)

as a yellow gum. ¹H NMR (600 MHz, CDCl₃): δ 7.19 (t, J = 7.9 Hz,

1H), 6.92−6.89 (m, 1H), 6.89−6.86 (m, 1H), 6.74 (dd, J = 2.0, 8.2

Hz, 1H), 5.80 (bs, 4H), 4.84 (dd, J = 2.6, 9.0 Hz, 1H), 3.76 (d, J = 6.3

Hz, 2H), 3.12−3.04 (m, 1H), 3.01−2.94 (m, 1H), 2.15−2.07 (m,

2H), 1.97−1.86 (m, 4H), 1.84−1.81 (m, 1H), 1.81−1.64 (m, 2H),

1.43−1.35 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 159.2, 146.3,

129.5, 123.6 (t, J = 240.0 Hz), 118.0, 113.2, 111.8, 73.2, 71.8, 38.8,

37.4, 36.0, 33.2 (dd, J = 23.2, 24.7 Hz), 25.9, 25.8. ¹⁹F (565 MHz,

CDCl₃): δ −91.39 (d, J = 235.8 Hz), −101.99 (d, J = 235.8 Hz).

HRMS (ESI): calcd for C₁₆H₂₃F₂NO₂[M + H]⁺, 300.1775; found,

300.1761.

Hz, 1H), 6.82 (t, J = 8.0 Hz, 1H), 5.25 (d, J = 8.3 Hz, 1H), 4.25 (bs,

3H), 3.77 (d, J = 6.3 Hz, 2H), 3.15−3.06 (m, 1H), 3.06−2.96 (m,

1H), 2.00−1.95 (m, 1H), 1.89−1.84 (m, 3H), 1.83−1.79 (m, 1H),

1.76−1.72 (m, 2H), 1.7−1.66 (m, 1H), 1.32−1.24 (m, 2H), 1.22−

1.14 (m, 1H), 1.07−1.00 (ddd, J = 3.3, 12.4, 24.2 Hz, 2H). ¹³C NMR

(150 MHz, CDCl₃): δ 149.4 (d, J = 244.7 Hz), 147.1 (d, J = 10.7 Hz),

132.6 (d, J = 10.7 Hz), 123.8 (d, J = 4.3 Hz), 118.4 (d, J = 3.2 Hz),

113.4, 74.9, 68.7, 39.8, 37.7, 37.1, 29.1, 26.6, 25.8. ¹⁹F (565 MHz,

CDCl₃): δ −141.76 to −141.84 (m). HRMS (ESI): calcd for

C₁₆H₂₄FNO₂[M + H]⁺, 282.1869; found, 282.1869.

3-Amino-1-(4-ﬂuoro-3-((2,6,6-trimethylcyclohex-1-en-1-yl)-

methoxy)phenyl)propan-1-ol (53). Steps were carried out according

to the general procedure using compound 44 (6 g, 18.9 mmol, 1

1

equiv) to give title compound 53 (1.40 g, 22%) as a yellow gum. H

3-Amino-1-(3-(cyclohexylmethoxy)phenyl)propan-3,3-d₂-1-ol

(58). 3-(3-(Cyclohexylmethoxy) phenyl)-3-hydroxypropanenitrile¹⁹

(200 mg, 0.77 mmol) was reduced with LAD (100 mg, 2.38

mmol). Following the common workup procedure, the crude product

was puriﬁed by FC (silica, 70:30:0−90:10:4−70:30:4 DCM/MeOH/

NH₄OH) giving 58 as yellow oil (69 mg, 34%). ¹H NMR (500 MHz,

CDCl₃): δ 7.23 (t, J = 7.9 Hz, 1H), 6.96 (s, 1H), 6.91 (d, J = 7.6 Hz,

1H), 6.77 (dd, J = 8.4, 2.6 Hz, 1H), 4.94 (dd, J = 8.6, 3.2 Hz, 1H),

3.76 (d, J = 6.4 Hz, 2H), 2.45 (s, 3H), 1.91−1.82 (m, 3H), 1.79−1.65

(m, 5H), 1.35−1.14 (m, 3H), 1.05 (qd, J = 12.2, 3.4 Hz, 2H). ¹³C

NMR (126 MHz, CDCl₃): δ 159.6, 146.9, 129.3, 117.8, 113.2, 111.8,

75.6, 73.5, 39.5, 37.9, 30.1, 26.6, 25.9. HRMS (ESI): m/z calcd for

C₁₆H₂₄D₂NO₂[M + H]⁺266.2084; found, 266.2083.

NMR (600 MHz, CDCl₃): δ 7.12 (dd, J = 1.54, 8.0 Hz, 1H), 6.99

(dd, J = 8.3, 11.0 Hz, 1H), 6.87−6.81 (m, 1H), 4.90 (dd, J = 2.2, 8.8

Hz, 1H), 4.50 (s, 2H), 3.62 (bs, 3H), 3.15−3.06 (m, 1H), 3.01−2.91

(m, 1H), 2.03 (t, J = 6.2 Hz, 2H), 1.89−1.82 (m, 1H), 1.78−1.74 (m,

1H), 1.72 (s, 3H), 1.66−1.61 (m, 2H), 1.53−1.45 (m, 2H), 1.05 (s,

6H). ¹³C NMR (150 MHz, CDCl₃): δ 152.1 (d, J = 244.6 Hz), 147.3

(d, J = 11.1 Hz), 141.4 (d, J = 3.3 Hz), 136.2, 132.9, 118.07 (d, J = 6.7

Hz), 115.7 (d, J = 18.6 Hz), 113.2 (d, J = 1.9 Hz), 74.9, 66.4, 40.4,

39.4, 39.3, 34.1, 33.0, 28.5, 19.9, 19.3. ¹⁹F (565 MHz, CDCl₃): δ

−135.72 to −135.82 (m). HRMS (ESI): calcd for C₁₉H₂₈FNO₂[M +

H]⁺, 322.2182; found, 322.2176.

3-Amino-1-(3-ﬂuoro-5-((2,6,6-trimethylcyclohex-1-en-1-yl)-

methoxy)phenyl)propan-1-ol (54). Utilizing the general procedure

with compound 45 (6 g, 18.9 mmol, 1 equiv) led to the successful

isolation of a yellow gum (1.50 g, 18%). ¹H NMR (600 MHz,

CDCl₃): δ 6.82−6.72 (m, 1H), 6.70−6.62 (m, 1H), 6.57−6.50 (m,

1H), 4.88 (dd, J = 2.5, 8.4 Hz, 1H), 4.59 (bs, 3H), 4.39 (s, 2H),

3.18−3.06 (m, 1H), 3.06−2.84 (m, 1H), 2.07−2.00 (m, 2H), 1.97−

1.86 (m, 1H), 1.83−1.75 (m, 1H), 1.67 (s, 3H), 1.66−1.56 (m, 2H),

1.53−1.42 (m, 2H), 1.02 (s, 6H). ¹³C NMR (150 MHz, CDCl₃): δ

163.6 (d, J = 242.9 Hz), 160.7 (d, J = 10.6 Hz), 148.0 (d, J = 8.4 Hz),

136.1, 132.8, 107.8 (d, J = 1.4 Hz), 104.7 (d, J = 22.7 Hz), 100.9 (d, J

= 24.8 Hz), 74.1, 64.9, 39.6, 39.3, 38.2, 34.1, 32.9, 28.5, 19.9, 19.3. ¹⁹F

(565 MHz, CDCl₃): δ −111.89 (t, J = 9.9 Hz). HRMS (ESI): calcd

for C₁₉H₂₈FNO₂[M + H]⁺, 322.2182; found, 322.2173.

3-Amino-1-(3-((4,4-diﬂuorocyclohexyl)methoxy)phenyl)propan-

3,3-d₂-1-ol (59). The general procedure for the reduction of nitrile

was followed using nitrile 48 (200 mg, 0.77 mmol) and LAD (100

mg, 2.38 mmol). Following puriﬁcation by FC (silica, 90:10:0−

70:30:0−90:10:4−70:30:4 DCM/MeOH/NH₄OH) gave 59 as a

colorless syrup (140 mg, 45%). ¹H NMR (500 MHz, CDCl₃): δ 7.23

(d, J = 7.8 Hz, 1H), 6.97 (s, 1H), 6.93 (d, J = 7.6 Hz, 1H), 6.77 (dd, J

= 8.1, 2.5 Hz, 1H), 4.95 (dd, J = 8.7, 3.0 Hz, 1H), 3.83 (dd, J = 6.5,

2.1 Hz, 2H), 2.18−2.10 (m, 3H), 2.00−1.93 (m, 4H), 1.92−1.84 (m,

4H), 1.81−1.66 (m, 5H), 1.47−1.38 (m, 2H). ¹³C NMR (126 MHz,

CDCl₃): δ 159.2, 147.1, 129.4, 118.2, 113.2, 111.7, 75.6, 71.8 (d, J =

2.8 Hz), 39.5, 36.1, 33.2 (dd, J = 25.5, 22.8 Hz), 25.9 (d, J = 9.7 Hz).

¹⁹F NMR (471 MHz, CDCl₃): δ −91.40 (d, J = 236.4 Hz), −102.00

3-Amino-1-(2-ﬂuoro-5-((2,6,6-trimethylcyclohex-1-en-1-yl)-

methoxy)phenyl)propan-1-ol (55). The general procedure was

carried out with 46 (3.0 g, 9.45 mmol, 1 equiv) giving amine 55

(d, J = 236.2 Hz). HRMS (ESI): m/z calcd for C₁₆H₂₁D₂F₂NO₂

[M]⁺, 301.1820; found, 301.1822.

1

RPE65 Crystallization and Structure Determination. Crystals

of RPE65 in the complex with 4-ﬂuoro-emixustat (49), C-2′-ﬂuoro-

MB-004 (24), or C-4 gem-diﬂuoro-emixustat (57) were obtained

using previously described procedures.^32,33Brieﬂy, isolated bovine

RPE membranes were incubated with 1 mM of each compound

[delivered in dimethylformamide (DMF)] for 15 min prior to

solubilization with 24 mM hexaethylene glycol monooctyl ether

(C₈E₆). After anion-exchange chromatography, puriﬁed RPE65 was

concentrated to 10−15 mg/mL and the test compounds were again to

a concentration of 1 mM prior to crystallization. An RPE65 sample

was also prepared in the absence of added inhibitors. Crystals were

grown by the hanging-drop vapor-diﬀusion method by mixing 2 μL of

a 10 mg/mL RPE65 sample with 2 μL of one of the following

crystallization solutions: 100 mM 2-(cyclohexylamino)ethanesulfonic

acid−NaOH, pH 9.5, containing 40% (v/v) polyethylene glycol 300

and 200 mM NaCl, which was used for the samples containing 49 or

no added inhibitor, or 100 mM Tris−HCl, pH 8.5, containing 30%

(v/v) polyethylene glycol 200 and 200 mM ammonium phosphate

dibasic, which was used for the samples containing 24 or 57. In both

cases, the drops were incubated in a well solution consisting of 100

mM 2-(cyclohexylamino)ethanesulfonic acid−NaOH, pH 9.5, con-

taining 40% (v/v) polyethylene glycol 300 and 200 mM NaCl at 8 °C.

Crystals of approximately 100 × 100 × 300 μm in size were obtained

after 1−2 weeks of incubation. Mature crystals were harvested directly

into liquid nitrogen for X-ray data collection.

(1.1 g, 34%) as a yellow gum. H NMR (600 MHz, CDCl₃): δ 7.18

(dd, J = 3.0, 5.8 Hz, 1H), 6.88 (t, J = 9.4 Hz, 1H), 6.79−6.75 (m,

1H), 5.22 (dd, J = 2.0, 8.3 Hz, 1H), 4.42 (d, J = 9.7 Hz, 1H), 4.38 (d,

J = 9.7 Hz, 1H), 4.14 (bs, 3H), 3.16−3.06 (m, 1H), 3.06−2.95 (m,

1H), 2.03 (t, J = 5.9 Hz, 2H), 1.99−1.93 (m, 1H), 1.82−1.76 (m,

1H), 1.69 (s, 3H), 1.66−1.61 (m, 2H), 1.53−1.45 (m, 2H), 1.03 (s,

6H). ¹³C NMR (150 MHz, CDCl₃): δ 155.8 (d, J = 1.4 Hz), 153.7 (d,

J = 237.4 Hz), 135.7, 133.2, 132.6 (d, J = 14.8 Hz), 115.5 (d, J = 23.7

Hz), 114.4 (d, J = 7.9 Hz), 112.8 (d, J = 4.4 Hz), 69.1, 65.2, 40.1,

39.3, 37.4, 34.2, 32.9, 28.5, 28.5, 19.9, 19.4. ¹⁹F (565 MHz, CDCl₃): δ

−130.41 to −130.48 (m). HRMS (ESI): calcd for C₁₉H₂₈FNO₂[M +

H]⁺, 322.2182; found, 322.2179.

3-Amino-1-(2-ﬂuoro-3-((2,6,6-trimethylcyclohex-1-en-1-yl)-

methoxy)phenyl)propan-1-ol (56). The actions of the general

procedure were carried out with nitrile 47 (6.5 g, 20.5 mmol, 1

1

equiv) to give a brown gum (1.06 g, 16%). H NMR (600 MHz,

CDCl₃): δ 7.14 (t, J = 6.8 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H), 6.94 (t, J

= 8.0 Hz, 1H), 5.24 (d, J = 7.2 Hz, 1H), 4.48 (d, J = 9.8 Hz, 1H), 4.45

(d, J = 9.8 Hz, 1H), 4.24 (bs, 3H), 3.12−3.03 (m, 1H), 3.03−2.92

(m, 1H), 2.03 (t, J = 6.0 Hz, 2H), 1.99−1.92 (m, 1H), 1.86−1.78 (m,

1H), 1.72 (s, 3H), 1.66−1.60 (m, 2H), 1.52−1.46 (m, 2H), 1.05 (s,

6H). ¹³C NMR (150 MHz, CDCl₃): δ 149.8 (d, J = 244.3 Hz), 147.1

(d, J = 11.2 Hz), 136.1, 133.0, 132.9 (d, J = 11.1 Hz), 123.8 (d, J = 4.2

Hz), 118.9 (d, J = 3.2 Hz), 114.5, 68.7, 66.5, 39.9, 39.3, 37.4, 34.1,

33.0, 28.5, 19.9, 19.3. ¹⁹F (565 MHz, CDCl₃): δ −140.35 to −140.46

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X-ray diﬀraction data were collected at the SSRL 12−2, the APS

NE-CAT 24-ID-E, or the NSLS-II FMX beamlines. Data were

processed using XDS,⁶²and the initial model was obtained by direct

reﬁnement using published RPE65 coordinates in which ligands had

been removed³²(PDB accession codes: 4RSE and 4RSC). The

structures were reﬁned by alternating reciprocal space reﬁnement in

REFMAC⁶³and manual building and adjustments in Coot.⁶⁴Ligand

coordinates and geometry dictionary ﬁles were generated using the

Grade server (http://grade.globalphasing.org/cgi-bin/grade/server.

cgi). The models were validated using MolProbity⁶⁵and the

wwPDB validation server.⁶⁶

RPE65 Retinoid Isomerase Activity Assay. Primary amine

listed in Tables 1 and 2 and Supporting Information Table S1 in DMF

(1 μL) was added to a suspension containing 300 μg of RPE

microsomal proteins, 1% bovine serum albumin, 2 mM disodium

pyrophosphate, and 25 μM human apo-cellular retinaldehyde-binding

protein (CRALBP) in 10 mM BTP buﬀer (200 μL) to a ﬁnal

concentration from 0 to 2 μM. After incubation at room temperature

for 5 min, the resulting mixture was mixed with half microliter of all-

trans-retinol (5 mM) in DMF and then incubated at 37 °C for 1 h.

The reaction was quenched by adding 400 μL of methanol (Fisher

Chemical, Fair Lawn, NJ), and the products were extracted with 400

μL of hexanes. Production of 11-cis-retinol was quantiﬁed by normal-

phase HPLC using a Zorbax Rx-SIL column (5 μm, 4.6 × 250 mm,

Agilent, Santa Clara, CA) with 10% (v/v) ethyl acetate in hexanes as

the eluent at a ﬂow rate of 1.4 mL·min⁻¹. Retinoids were detected by

monitoring their absorbance at 325 nm and quantiﬁed based on a

standard curve representing the relationship between the amount of

11-cis-retinol and the area under the corresponding chromatographic

peak.

Quantiﬁcation of Representative Primary Amine Com-

pound Levels in the Serum and Eyes of Mice after

Treatments. 8-week-old BL/6J mice were treated with a 380 nmol

visual cycle modulator 24, 49, 57, 58, 59, or emixustat in dimethyl

sulfoxide (50 μL) by intraperitoneal injection and sacriﬁced at 3 h, 1

day, or 7 days later. Blood and eyeball samples were collected

immediately. After clotting at room temperature for 30 min, the blood

samples were centrifuged for 10 min at 17,000g in a temperature-

controlled benchtop centrifuge (Eppendorf AG). Each serum sample

(100 μL) was carefully removed to avoid disturbing loose clots,

precipitated with 400 μL of pre-cooled methanol, and centrifuged at

17,000g for 15 min at 4 °C. The supernatant was carefully transferred

to a SpinX centrifuge tube ﬁlter with a 0.45 μm cellulose acetate

membrane (Costar, Salt Lake City, UT) and centrifuged at 7000g for

2 min. The ﬁltered samples were dried under vacuum, reconstituted in

100 μL of 50% methanol/water, and centrifuged at 17,000g for 15

min at 4 °C. The resulting supernatants were ready for LC/MS

analyses. The two eyeballs from each mouse were homogenized in

acetonitrile (2 × 800 μL). The resulting mixture was centrifuged at

17,000g for 15 min at 4 °C. The supernatant was dried under vacuum,

reconstituted in 100 μL of 50% methanol/water, and centrifuged at

17,000g for 15 min at 4 °C. Twenty microliters of the supernatant

extracted from the serum or eye samples was injected into an Ultimate

3000 HPLC system coupled with a LXQ mass spectrometer

(ThermoFisher Scientiﬁc, Waltham, MA) with an ESI unit. The

separation was performed on a Proshell EC-18 column (2.7 μm, 3.0 ×

150 mm, Agilent, Santa Clara, CA) using a mobile phase consisting of

0.1% aqueous formic acid (A) and acetonitrile (B) at a ﬂow rate of

600 μL·min⁻¹, and the mobile-phase gradients and time course were

as follows: 0−2 min, 95% A/5% B; 2−10 min, 95−15% A/5−85% B.

The signals were detected in the selected reaction monitoring mode

under conditions described in Supporting Information Table S3 and

quantiﬁed based on the standard curves representing the relationship

between the amounts of primary amine standards and the areas under

the corresponding chromatographic peaks.

saline. Aorta samples were used immediately or stored at −80 °C until

needed. Two aortas were minced using a stainless-steel single-edge

blade and homogenized in a KONTES Potter-Elvehjem tissue

grinder/homogenizer glass pestle in 1 mL of 10 mM HEPES-

NaOH, pH 7.6. The homogenate was collected into a 1.5 mL

Eppendorf tube. Five μl of a 20 mM ethanolic stock solution of

emixustat or d₂-emixustat (58) was added to the aorta homogenate to

give a ﬁnal substrate concentration of 100 μM. The sample was mixed

and then incubated at 28 °C with 300 rpm shaking in an Eppendorf

Thermomixer. 200 μL samples were taken at 0, 1, and 2 h after the

initiation of the reaction. At each time point, the reactions were

immediately quenched with 100 μL of 100% MeOH, vortexed for 3 s,

and stored at −20 °C. After samples from all time points were

collected and frozen, the samples were thawed and centrifuged at

15,000 rpm for 10 min; 250 μL of each supernatant was collected,

placed into a borosilicate tube, and dried in a Speedvac

rotoevaporator. Each dried sample was redissolved in 300 μL of a

1:1 MeOH/H₂O solution, centrifuged to remove particulates, and

then transferred to an HPLC vial. 50 μL of the sample was used for

analysis on an Agilent 1260 Inﬁnity series HPLC equipped with a

Proshell EC-18 column and a diode array detector. The sample was

separated using a mobile phase consisting of 0.1% (v/v) formic acid in

H₂O and acetonitrile at the following ratios and time intervals: 95:5

for 2 min, a gradient from 95:5 to 15:85 over 8 min, a gradient from

15:85 to 2:98 over 0.5 min, continued 2:98 for 4 min, and then a

gradient from 2:98 to 95:5 over 0.5 min. The reaction substrate and

product were assessed by monitoring absorbance at 275 nm.

Emixustat and d₂-emixustat were eluted at ∼8.5 min, while the

assay product (ACU-5201) was eluted at ∼13.25 min. A dilution

series of known concentrations of authentic ACU-5201 in 1:1

MeOH/H₂O was run to generate a standard curve and facilitate the

conversion of product AUCs to absolute mass.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00279.

■

sı

SMILES data and RPE65 EC₅₀values for compounds

(CSV)

Synthesis and characterization of compounds, copies of

NMR spectra, purity HPLC traces for the ﬁnal

synthesized compounds and intermediates, inhibition

of RPE65-mediated isomerization reaction, detection of

primary amines by mass spectroscopy, details of

quantum chemical calculations, and X-ray data collec-

tion, processing, and reﬁnement (PDF)

Accession Codes

The X-ray crystallography data sets reported in this article have

been deposited in the Protein Data Bank [ID codes: 7K88

(C8E6 complex), 7K89 (compound 49 complex), 7K8G

(compound 24 complex), and 7L0E (compound 57

complex)].

AUTHOR INFORMATION

Corresponding Authors

■

Arie Gruzman − Department of Chemistry, Faculty of Exact

Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel;

orcid.org/0000-0002-8006-4201; Email: Aric-

Lev.Gruzman@biu.ac.il

Gregory P. Tochtrop − Department of Chemistry, Case

Western Reserve University, Cleveland, Ohio 44106, United

States; Email: gpt6@case.edu

Philip D. Kiser − Department of Ophthalmology, Gavin

Herbert Eye Institute and Department of Physiology and

Biophysics, University of California, Irvine, California 92697,

VAP-1 Oxidation Assay. Mouse aorta homogenates were used as

the source of VAP-1 for this study. Aortas were removed from mice

(4−6 weeks old) that had been euthanized by CO₂asphyxiation

followed by cervical dislocation. The aorta was dissected and the

blood was removed by rinsing the tissue with phosphate-buﬀered

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United States; Research Service, VA Long Beach Healthcare

System, Long Beach, California 90822, United States;

orcid.org/0000-0003-1184-9539; Email: pkiser@uci.edu

Krzysztof Palczewski − Department of Ophthalmology, Gavin

Herbert Eye Institute, Department of Physiology and

Biophysics, and Department of Chemistry, University of

California, Irvine, California 92697, United States;

orcid.org/0000-0002-0788-545X; Phone: (949)824-

6527; Email: kpalczew@uci.edu

ether; CRALBP, cellular retinaldehyde-binding protein;

DLPNO-CCSD(T), domain-based local pair natural orbital

coupled-cluster with single, double, and perturbative triple

excitation; ETS, transition-state theory; KIE, kinetic isotope

eﬀect; LED, local energy decomposition; NCI, noncovalent

interaction; NOCV, natural orbitals for chemical valence;

RPE65, retinoid isomerase; VAP-1, vascular adhesion protein-

1.

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Eliav Blum − Department of Chemistry, Faculty of Exact

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Jianye Zhang − Department of Ophthalmology, Gavin Herbert

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Jordan Zaluski − Department of Chemistry, Case Western

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David E. Einstein − Department of Physiology and Biophysics,

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Adam Kubas − Institute of Physical Chemistry, Polish

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Author Contributions

^∇E.B. and J.Z. contributed equally to the conduct of the study

and development of the manuscript.

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The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This study was supported by a Bar-Ilan University new faculty

grant (to A.G.). K.P. is the Irving H. Leopold Chair of

Ophthalmology at the Gavin Herbert Eye Institute, Depart-

ment of Ophthalmology, University of California, Irvine. This

research was supported in part by grants to K.P. from the

National Institutes of Health (NIH) (EY009339, EY027283,

and EY030873); to P.D.K. from the U.S. Department of

Veterans Aﬀairs (I01BX004939); and to G.P.T. from the

National Science Foundation (NSF-CHE Award no. 1904530)

and the Department of Defense (DOD-CDMRP award no.

W81XWH-16-1-0699). The authors also acknowledge support

from an RPB unrestricted grant to the Department of

Ophthalmology, University of California, Irvine. The authors

also thank the Interdisciplinary Center for Mathematical and

Computational Modeling in Warsaw, Poland, under grant

GB79-5, for granting access to high-performance computing

resources.

ABBREVIATIONS

■

A2E, N-retinylidene-N-retinylethanolamine; AMD, age-related

macular degeneration; C₈E₆, hexaoxyethylene monooctyl

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J. Med. Chem. 2021, 64, 8287−8302

Article Doi

Rational Alteration of Pharmacokinetics of Chiral Fluorinated and Deuterated Derivatives of Emixustat for Retinal Therapy

DOI: 10.1021/acs.jmedchem.1c00279

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Article abstract of DOI:10.1021/acs.jmedchem.1c00279

Full text of DOI:10.1021/acs.jmedchem.1c00279

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