Rational tuning of visual cycle modulator pharmacodynamics

Article abstract of DOI:10.1124/jpet.117.240721

Modulators of the visual cycle have been developed for treatment of various retinal disorders. These agents were designed to inhibit retinoid isomerase [retinal pigment epithelium-specific 65 kDa protein (RPE65)], the rate-limiting enzyme of the visual cy

Full text of DOI:10.1124/jpet.117.240721

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TITLE PAGE
Rational tuning of visual cycle modulator pharmacodynamics
Philip D. Kiser, Jianye Zhang, Mohsen Badiee, Junzo Kinoshita, Neal S. Peachey, Gregory P.
Tochtrop and Krzysztof Palczewski
Department of Pharmacology, School of Medicine, Case Western Reserve University,
Cleveland, OH 44106, USA (P.D.K., J.Z., K.P.)
Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH
44106, USA (P.D.K., N.S.P.)
Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA (M.B.,
G.P.T)
Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195, USA (J.K., N.S.P.)
Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western
Reserve University, Cleveland, OH 44195, USA (N.S.P.)
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RUNNING TITLE PAGE
RUNNING TITLE
Rational design of visual cycle modulators
CORRESPONDENCE
Philip Kiser, Dept. of Pharmacology, School of Medicine, Case Western Reserve University,
10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-368-0040; Fax: 216-368-1300; E-mail:
pdk7@case.edu
Krzysztof Palczewski, Dept. of Pharmacology, School of Medicine, Case Western Reserve
University, 10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-368-4631; Fax: 216-368-1300;
E-mail: kxp65@case.edu
MANUSCRIPT STATISTICS
Text Pages: 53
Number of Tables: 1
Figures: 8
References: 66
Abstract Length: 235 words
Introduction Length: 728 words
Discussion Length: 1173 words
ABBREVIATIONS
A2E, N-Retinylidene-N-retinylethanolamine; ABCA4, ATP-binding cassette transporter A4; AMD,
age-related
macular
degeneration;
emixustat
(ACU-4429),
3-amino-1-(3-
(
cyclohexylmethoxy)phenyl)propan-1-ol; CRALBP, cellular retinaldehyde-binding protein; ERG,
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electroretinogram; LRAT, lecithin:retinol acyltransferase; OCT, optical coherence tomography;
ONL, outer nuclear layer; PE, phosphatidylethanolamine; RDH8, retinol dehydrogenase 8; RPE,
retinal pigment epithelium; RPE65, retinal pigment epithelium-specific 65 kDa protein, retinoid
isomerase.
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ABSTRACT
Modulators of the visual cycle have been developed for treatment of various retinal disorders.
These agents were designed to inhibit retinoid isomerase (RPE65), the rate-limiting enzyme of
the visual cycle, based on the idea that attenuation of visual pigment regeneration could reduce
formation of toxic retinal conjugates. Certain of these agents that contain primary amine groups
also can also reversibly form retinaldehyde-Schiff base adducts, which contributes to their retinal
protective activity. Direct inhibition of RPE65 as a therapeutic strategy is complicated by adverse
effects resulting from slowed chromophore regeneration whereas effective retinal sequestration
can require high drug doses with potential off-target effects. We hypothesized that the RPE65-
emixustat crystal structure could help guide the design of retinaldehyde-sequestering agents with
varying degrees of RPE65 inhibitory activity. We found that addition of an isopropyl group to the
central phenyl ring of emixustat and related compounds resulted in agents effectively lacking in
vitro retinoid isomerase inhibitory activity, whereas substitution of the terminal 6-membered ring
with branched moieties capable of stronger RPE65 interaction potentiated inhibition. The
isopropyl derivative series produced discernible visual cycle suppression in vivo, albeit much less
potently than compounds with high affinity for the RPE65 active site. These agents distributed
into the retina and formed Schiff base adducts with retinaldehyde. Except for one compound (MB-
007), these agents conferred protection against retinal phototoxicity suggesting that both direct
RPE65 inhibition and retinal sequestration are mechanisms of potential therapeutic relevance.
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INTRODUCTION
Visual function in vertebrates relies on a vitamin A-derived molecule called retinaldehyde,
which is the chromophore of rod and cone visual pigments (Hubbard and Wald, 1952). The
efficacy of retinaldehyde in triggering light perception depends on it existing in an 11-cis
configuration coupled to rod and cone opsins through a protonated imine (Schiff base) bond.
Absorption of light by 11-cis-retinylidene stimulates its isomerization to an all-trans configuration
with resultant conformational changes in the opsin molecule allowing it to trigger
phototransduction (Palczewski, 2006; Hofmann et al., 2009).
The photoactivated visual pigment eventually releases all-trans-retinaldehyde which
terminates signal transduction and leaves the opsin molecule insensitive to further light
stimulation. Restoration of light sensitivity depends upon the conversion of all-trans-retinaldehyde
back into 11-cis-retinaldehyde (Wald, 1968). The classic version of this process, known as the
visual cycle, is carried out by enzymes and retinoid binding proteins located within and between
the retinal pigment epithelium (RPE) and photoreceptor outer segments (Fig. 1A) (Kiser et al.,
2014b).
Despite its essential function in initiating vision, retinaldehyde can cause retinal toxicity, a
feature implicated in diseases including Stargardt macular dystrophy and age-related macular
degeneration (AMD) (Chen et al., 2012; Maeda et al., 2012b). This toxicity stems in large part
from its aldehyde functionality, which is reactive towards certain cellular nucleophiles (Ben-Shabat
et al., 2002). At concentrations easily achieved in vivo under strong lighting conditions
retinaldehyde induces apoptosis in cultured cell lines (Maeda et al., 2009a; Chen et al., 2012).
Mice deficient in ABCA4 and RDH8, key proteins involved in retinaldehyde clearance, also are
susceptible to light-induced retinal degeneration (Maeda et al., 2008). These mice accumulate
autofluorescent material in their retinas, known as lipofuscin, that is partially composed of
retinaldehyde-PE adducts such as A2E (Sparrow et al., 2003).
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Amongst strategies for preventing retinaldehyde toxicity, inhibition of the visual cycle was
one of the first investigated and has been advanced to clinical testing (Fig. 1A). This approach is
based on the observation that RPE65-null mice fail to develop retinal lipofuscin and A2E (Katz
and Redmond, 2001). RPE65 catalyzes the rate-limiting trans-cis isomerization step of the visual
cycle (Redmond et al., 1998; Redmond et al., 2005). Inhibition of its activity was proposed to slow
visual pigment regeneration with less retinaldehyde being released during light exposure thereby
reducing lipofuscin formation (Maiti et al., 2006). This pharmacological concept was realized with
the development of retinylamine, a transition state analog of the retinoid isomerization reaction
(
Golczak et al., 2005) . A non-retinoid derivative of retinylamine called emixustat (ACU-4429) was
developed later (Bavik et al., 2015) entering clinical trials for the treatment of dry AMD (Kubota et
al., 2012; Kubota et al., 2014). It is currently being evaluated as a treatment for other conditions
including diabetic retinopathy (NCT02753400).
Both retinylamine (Maeda et al., 2012a) and emixustat (Zhang et al., 2015) exert a second
mechanism of protection against retinaldehyde toxicity whereby their primary amine groups
reversibly react with retinaldehyde to form a Schiff base conjugate thus quenching its reactivity
(
Fig. 1A). Manageable amounts of retinaldehyde are subsequently released over time to reenter
the visual cycle. This sequestration concept has been extended to include non-retinoid primary
amine drugs that completely lack RPE65 inhibitory activity (Maeda et al., 2012a).
While compounds exhibiting either of these mechanisms of visual cycle modulation have
potential to ameliorate disease processes, their mechanisms of action make them inherently
susceptible to producing untoward effects. Nyctalopia and dyschromatopsia, two adverse effects
reported in emixustat clinical trials (Kubota et al., 2012; Kubota et al., 2014), are direct extensions
of visual cycle suppression. Long-term suppression of visual cycle activity is also linked to cone
photoreceptor pathology in mice (Rohrer et al., 2005; Maeda et al., 2009b). On the other hand,
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the potentially high doses of primary amine compounds needed for effective retinaldehyde-
sequestration activity have potential to produce off-target effects (Maeda et al., 2012a).
Crystal structures of RPE65 in complex with emixustat and a derivative called MB-001
have revealed molecular interactions governing the binding affinity of these compounds (Kiser et
al., 2015; Zhang et al., 2015). We hypothesized that these structures could inform strategies to
modify the affinity of emixustat derivatives towards the RPE65 active site with maintenance of
retinaldehyde-sequestering activity. To experimentally test this hypothesis, we synthesized
emixustat derivatives with various degrees of predicted affinity for the RPE65 active site and
characterized their effects on the visual cycle and ability to protect against retinal phototoxicity.
METHODS
Analysis of the RPE65 emixustat-binding site
The atomic coordinates for an RPE65-emixustat complex structure were downloaded from the
Protein Data Bank (Accession code 4RSC). The structure was visualized and analysed using
PyMOL (Schrodinger LLC) and COOT (Debreczeni and Emsley, 2012). Cavity volume estimates
were made by manually filling the cavities with water molecules in PyMOL and calculating the
volume of the resulting pseudo-molecule using the Molinspiration web server
(
http://www.molinspiration.com/services/volume.html). The same web service was used to
estimate the volumes of selected moieties within MB compounds.
Synthetic Chemistry
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O
OH
MsCl, Et N
OMs
Cs CO3
3
2
N
HO
DMF
CH Cl
2
2
OH
O
1
O
N H , H O
2
4
2
NH2
N
O
O
EtOH
OH
R
)-MB-004
OH
O
2
(
MsCl : methanesulfonyl chloride
Scheme 1. Synthesis of (R)-MB-004.
2
-Propylpentyl methanesulfonate (1): An oven-dried 100 mL, two-necked, round-bottomed
flask equipped with a magnetic stir bar was sealed under argon with two rubber septa. The flask
was charged with anhydrous CH Cl (32 mL), 2-propylpentan-1-ol (2.5 g, 19.2 mmol, 1.0 eq.) and
2
2
triethylamine (2.9 g, 28.7 mmol, 1.5 eq.). This solution was cooled to 0 °C and then
methanesulfonyl chloride (2.4 g, 21.1 mmol, 1.01 eq.) was added dropwise. The reaction mixture
was warmed to room temperature over a period of 2 h. An aqueous solution of 1N HCl was added
and the layers separated. The organic layer was dried over Na
2
SO and concentrated under
4
reduced pressure. Purification by silica gel column chromatography (EtOAc/Hexanes, 10:90)
1
afforded 3.3 g (83 %) of the desired compound 1 as a pale yellow oil. H NMR (500 MHz, CDCl
3
)
δ 4.12 (d, J = 5.5 Hz, 2H), 3.00 (s, 3H), 1.74 (p, J = 5.5 Hz, 1H), 1.42 – 1.26 (m, 8H), 0.99 – 0.84
13
(
m, 6H); C NMR (125 MHz, CDCl
3
) δ 72.65, 37.45, 37.32, 33.06, 19.87, 14.40.
(
R)-2-(3-Hydroxy-3-(3-((2-propylpentyl)oxy)phenyl)propyl)isoindoline-1,3-dione (2): A dried
round bottom flask equipped with a stir bar was sealed under argon. It was charged with the
succinimide protected phenol, prepared as described (Kiser et al., 2015) (4.3 g, 14.4 mmol, 1.0
eq) in 85 ml anhydrous DMF. Anhydrous Cs
2
CO
3
(6.4 g, 19.7 mmol, 1.4 eq.) was added to a
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stirred solution of phenol. Compound 1 (3.4 g, 16.4 mmol, 1.1 eq.) was added to the phenolate
solution and the reaction mixture was stirred at 60 °C under argon for 24 h. The reaction mixture
was partitioned between EtOAc and 25% NH
extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous
MgSO and concentrated under reduced pressure. Purification by silica gel column
4
Cl (aqueous), and the aqueous layer was further
4
chromatography (EtOAc/Hexanes, 30:70) afforded 2.3 g (40 %) of the desired compound 2 as a
1
colorless oil. H NMR (500 MHz, CDCl
3
) δ 7.86 – 7.82 (m, 2H), 7.74 – 7.70 (m, 2H), 7.18 (t, J =
7.9 Hz, 1H), 6.91 – 6.89 (m, 1H), 6.88 (d, J = 7.8 Hz, 1H), 6.73 (dd, J = 8.2, 2.3 Hz, 1H), 4.66 (dd,
J = 8.1, 5.2 Hz, 1H), 3.91 (t, J = 6.5 Hz, 2H), 3.80 (d, J = 5.6 Hz, 2H), 2.12 – 2.06 (m, 2H), 1.81 –
1
1
3
.74 (m, 1H), 1.45 – 1.30 (m, 8H), 0.94 – 0.88 (m, 6H); 13C NMR (125 MHz, CDCl
) δ 168.94,
3
59.70, 145.29, 134.17, 132.18, 129.55, 123.46, 117.77, 113.76, 111.77, 71.41, 70.91, 37.71,
7.69, 35.05, 33.87, 20.14, and 14.60.
(
R)-3-amino-1-(3-((2-propylpentyl)oxy)phenyl)propan-1-ol ((R)-MB-004): An oven-dried one-
necked, round-bottomed flask equipped with a magnetic stir bar was charged with compound 2
2.3 g, 5.9 mmol, 1.0 eq.), 150 ml EtOH and N .H O (1.6 ml, 50.9 mmol). The reaction mixture
(
H
2 4
2
was stirred at room temperature for 24 h, clarified by passage through filter paper and the resulting
filtrate was concentrated under reduced pressure. Purification by silica gel column
chromatography (NH
4
OH/MeOH/CH
2
Cl
2
4:20:80) afforded 1.18 g (75 %) of the final product as a
1
colorless oil. H NMR (500 MHz, CD
3
OD) δ 7.24 (t, J = 7.9 Hz, 1H), 6.94 (s, 1H), 6.92 (d, J = 7.8
Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 4.76 (t, J = 6.6 Hz, 1H), 3.86 (d, J = 5.4 Hz, 2H), 3.01 – 2.87
(
m, 2H), 1.96 (q, J = 6.9 Hz, 2H), 1.80 (p, J = 5.7 Hz, 1H), 1.49 – 1.34 (m, 8H), 0.96 – 0.90 (m,
1
3
6
3
2
H); C NMR (125 MHz, CD
3
OD) δ 160.99, 147.46, 130.50, 118.86, 114.39, 112.95, 73.26, 71.76,
+
[M+H]+:
9.11, 39.05, 38.85, 34.93, 21.06, 14.78; ESI-HRMS: m/z calculated for C17
H30NO
2
80.22711, found: 280.22708.
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nBuLi
1) iPrMgBr,
tBuOK, CH CN
A
or B
3
O
DMF
RO
Br
5a R= R1
2)
RO
THF
X
Br
X= Br
c X= OH
4
4
6a R= R1
6b R= R2
6c R= R3
5b R= R2
5c R= R3
C or D
THF
NH2
RO
RO
N
OH
OH
7a R= R1
7b R= R2
7c R= R3
MB-005 R= R1
MB-006 R= R2
MB-007 R= R3
R1
R2
R3
A: CuI,
K PO , R OH or R OH.
3
4 1 2
8
3
-Hydroxyquinoline,
B: K CO , compound 3, acetone
2
C: Borane
dimethylsulfide
D: Lithium aluminium
hydride
Scheme 2. Synthesis of MB-005, MB-006, and MB-007.
1. NaBH4
O
Br
2
. PBr3
3
H O
2
2
2B
(HO)
Br
HO
Br
H O/PEG400
2
4c
Scheme 3. Reagents for the synthesis of compound 3 and 4c. Compound 3 was prepared as
described (Crombie et al., 2001).
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3-Bromo-5-isopropylphenol (4c): A 50 mL round bottom flask containing 16.48 mL of
CARBOWAX™ polyethylene glycol 400 was charged with 30% aqueous H (2.52 mL, 12.36
2
O
2
mmol, 1.5 eq.) and the resulting solution was stirred for 5 min at room temperature. Then, (3-
bromo-5-isopropylphenyl) boronic acid (2 g, 8.24 mmol, 1 eq.) was added (note: reaction is highly
exothermic) and stirred at room temperature open to air for a period of 2h. Upon completion of
the reaction, the mixture was extracted by diethyl ether twice. The organic layers were combined,
washed with brine, dried over anhydrous Na
2
SO , and the solvent was removed under reduced
4
pressure (Gohain et al., 2014), and purification by flash chromatography on a silica gel column
1
(
(
EtOAc/Hexanes, 95:5) afforded 1.65 g, (93 %) of the desired phenol 4c as a white solid. H NMR
500 MHz, CDCl
3
) δ 6.95 (s, 1H), 6.83 (s, 1H), 6.63 (s, 1H), 4.79 (s, 1H), 2.82 (sept, J = 7.0 Hz,
1
1
H), 1.21 (d, J = 6.9 Hz, 6H); 13C NMR (125 MHz, CDCl
3
) δ 156.30, 152.72, 122.68, 122.48,
16.25, 112.62, 34.12, 23.83 (Somai Magar and Lee, 2014).
-Bromo-3-(cyclohexylmethoxy)-5-isopropylbenzene (5a): An oven-dried 50 mL, pear-
1
shaped Schlenk flask equipped with a magnetic stir bar was sealed under argon with a rubber
septum (3 cycles of evacuation followed by argon backfilling). The flask was charged with copper
iodide (514.2 mg, 2.7 mmol, 0.15 eq.), 8-hydroxyquinoline (784.2 mg, 5.4 mmol, 0.3 eq.), and
anhydrous potassium phosphate tribasic (7.65 g, 36.0 mmol, 2.0 eq.). The flask was then
evacuated and backfilled with argon (3 cycles). Commercially available 1,3-Dibromo-5-
isopropylbenzene 4 (5 g, 18.0 mmol, 1.0 eq.) and cyclohexylmethanol (R OH: 15 mL) then were
1
added at room temperature via a syringe. The rubber septum stopper was replaced with a screw-
cap and the reaction mixture was placed in a preheated oil bath and stirred at 110 °C. After 24 h,
the reaction mixture was allowed to cool to 40 °C and was then diluted with hexanes (30 mL).
After stir 5 min at room temperature, the slurry was passed through filter paper and the filter cake
was washed with hexanes. The filtrate was concentrated under reduced pressure (Niu et al.,
2009). Purification by flash column chromatography on a silica gel with 100% hexanes afforded
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1
2
6
2
.2 g (40 %) of the desired ether 5a as a colorless oil. H NMR (500 MHz, CDCl
3
) δ 6.94 (s, 1H),
.85 (s, 1H), 6.69 (s, 1H), 3.71 (d, J = 6.2 Hz, 2H), 2.81 (sept, J = 7.1 Hz, 1H), 1.89 – 1.82 (m,
13
H), 1.82 – 1.67 (m, 4H), 1.37 – 1.15 (m, 3H), 1.22 (d, J = 6.8 Hz, 6H), 1.09 – 1.00 (m, 2H); C
) δ 160.22, 152.24, 122.68, 121.97, 114.73, 112.40, 73.79, 37.83, 34.28,
0.02, 26.64, 25.93, 23.90.
-Bromo-3-isopropyl-5-((2-propylpentyl)oxy)benzene (5b): The same procedure was followed
NMR (125 MHz, CDCl
3
3
1
as described for 5a. In brief, the argon-filled flask was charged with copper iodide (514.2 mg), 8-
hydroxyquinoline (784.2 mg), and anhydrous potassium phosphate tribasic (7.65 g). The flask
was then evacuated and backfilled with argon (3 cycles). Commercially available 1,3-Dibromo-5-
isopropylbenzene 4 (5 g) and 2-propylpentan-1-ol (R OH: 15 mL) were added. Purification by
2
flash column chromatography on silica gel with 100% hexanes afforded 1.95 g (33 %) of the
1
desired ether 5b as a colorless oil. H NMR (500 MHz, CDCl
3
) δ 6.94 (s, 1H), 6.86 (s, 1H), 6.69
(
s, 1H), 3.79 (d, J = 5.6 Hz, 2H), 2.83 (sept, J = 7.2 Hz, 1H), 1.79 (quint, J = 5.6 Hz, 1H), 1.47 –
13
1
.31 (m, 8H), 1.23 (d, J = 6.9 Hz, 6H), 0.92 (t, J = 6.9 Hz, 6H); C NMR (125 MHz, CDCl ) δ
3
160.27, 152.21, 122.67, 121.92, 114.80, 112.35, 71.16, 37.67, 34.30, 33.82, 23.91, 20.14, 14.58.
1-Bromo-3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)benzene (5c): An oven-
dried 100 mL, three-necked, round-bottomed flask equipped with a magnetic stir bar and sealed
under argon. The flask was charged with phenol 4c (1.83 g, 8.5 mmol, 1 eq.) and anhydrous
potassium carbonate (4.71 g, 34 mmol, 4 eq.). The flask was evacuated, filled up with argon twice
and then charged with dry acetone (60 mL). Compound 3 (1.85 g, 8.5 mmol, 1.2 eq.) was then
added this mixture. After refluxed under argon atmosphere for 24 h, the reaction mixture was
cooled to room temperature, then filtered and concentrated under reduced pressure. Purification
by flash chromatography on a silica gel column (100% Hexanes) afforded 2.4 g, (80 %) of the
1
desired ether 5c as a colorless oil. H NMR (500 MHz, CDCl
3
) δ 6.96 (s, 1H), 6.93 (t, J = 2.0 Hz,
1H), 6.75 (t, J = 1.9 Hz, 1H), 4.39 (s, 2H), 2.83 (sept, J = 6.9 Hz, 1H), 2.05 (t, J = 6.3 Hz, 2H),
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1
3
1
.70 (s, 3H), 1.68 – 1.62 (m, 2H), 1.53 – 1.48 (m, 2H), 1.23 (d, J = 6.9 Hz, 6H), 1.04 (s, 6H); C
NMR (125 MHz, CDCl ) δ 160.12, 152.08, 136.07, 132.83, 122.50, 121.92, 114.63, 112.61, 64.66,
9.24, 34.15, 34.10, 32.88, 28.43, 23.77, 19.82, 19.27.
-(Cyclohexylmethoxy)-5-isopropylbenzaldehyde (6a): An oven-dried 50 mL, two-necked,
3
3
3
round-bottomed flask was equipped with a magnetic stir bar and sealed under argon. The flask
was charged with the aryl bromide 5a (2.5 g, 8.0 mmol, 1.2 eq.) in anhydrous THF (8 mL). The
reaction mixture was cooled to 0 °C, and a 3 M solution of isopropylmagnesium bromide in 2-Met-
THF (3.3 mmol, 0.5 eq.) was added over 5 min and stirred for an additional 10 min. Then a 2.16
M solution of nBuLi in hexanes (6.7 mmol, 1.0 eq.) was added dropwise over 10 min while
maintaining the temperature below 5 °C. The resulting mixture was stirred at 0 °C for 1 h, cooled
to -10 °C, and dry DMF (670 uL, 8.9 mmol, 1.3 eq.) in dry THF (9 mL) was added dropwise over
10 min. The resulting mixture was warmed to rt over 1 h, then was added to a 0.5 M citric acid
solution (30 mL). After 10 min stirring, the phases were separated and the aqueous phase was
extracted one additional time with toluene. The combined organic phases were concentrated and
water was removed azeotropically with toluene (Gallou et al., 2008). Purification by flash
chromatography on a silica gel column (Et
2
O/Hexanes 7:93) afforded 1.9 g, (90%) of the desired
1
aldehyde 6a as a colorless oil. H NMR (500 MHz, CDCl
3
) δ 9.94 (s, 1H), 7.32 (s, 1H), 7.19 (s,
1H), 7.05 (s, 1H), 3.80 (d, J = 6.3, 2H), 2.95 (sept, J = 7.2, 6.8 Hz, 1H), 1.92 – 1.85 (m, 2H), 1.84
1
3
–
1.67 (m, 4H), 1.36 – 1.17 (m, 3H), 1.28 (d, J = 6.9 Hz, 6H), 1.12 – 1.02 (m, 2H); C NMR (125
) δ 192.72, 160.10, 151.52, 137.92, 121.84, 120.66, 110.20, 73.87, 37.83, 34.12,
0.03, 26.63, 25.93, 23.92.
-Isopropyl-5-((2-propylpentyl)oxy)benzaldehyde (6b): The same procedure was followed as
MHz, CDCl
3
3
3
described for 6a. At 0 °C, a 3 M solution of isopropylmagnesium bromide in 2-Met-THF (2.9 mmol,
.5 eq.) was added to a solution of the aryl bromide 5b (2.27 g, 6.94 mmol, 1.2 eq.) in anhydrous
THF (6.94 mL). Then, a 2.16 M solution of nBuLi in hexanes (5.9 mmol, 1.0 eq.) was added
0
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followed by addition a solution of dry DMF (584 uL, 7.74 mmol, 1.3 eq.) in dry THF (7.74 mL) at -
1
1
1
1
0 °C. Purification by flash chromatography on a silica gel column (Et
2
O/Hexanes 7:93) afforded
1
.7 g, (90%) of the desired aldehyde 6b as a colorless oil. H NMR (500 MHz, CDCl
3
) δ 9.95 (s,
H), 7.32 (s, 1H), 7.21 (s, 1H), 7.05 (s, 1H), 3.88 (d, J = 5.3 Hz, 2H), 2.95 (sept, J = 7.1 Hz, 1H),
13
.82 (p, J = 5.8 Hz, 1H), 1.50 – 1.32 (m, 8H), 1.28 (d, J = 7.0 Hz, 6H), 0.95 – 0.89 (m, 6H); C
) δ 192.64, 160.14, 151.47, 137.92, 121.74, 120.58, 110.27, 71.25, 37.66,
4.13, 33.82, 23.91, 20.13, 14.56.
-Isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)benzaldehyde (6c): The same
NMR (125 MHz, CDCl
3
3
3
procedure was followed as described for 6a. At 0 °C, a 3 M solution of isopropylmagnesium
bromide in 2-Met-THF (1.75 mmol, 0.5 eq.) was added to a solution of the aryl bromide 5c (1.47
g, 4.2 mmol, 1.2 eq.) in anhydrous THF (5 mL). Then, a 2.16 M solution of nBuLi in hexanes (3.5
mmol, 1.0 eq.) was added and followed by addition of a solution of dry DMF (343 uL, 4.54 mmol,
1.3 eq.) in dry THF (4.5 mL) at -10 °C. Purification by flash chromatography on a silica gel column
1
(
Et
2
O/Hexanes 5:95) afforded 1.1 g, (87%) of the desired aldehyde 6c as a colorless oil. H NMR
(
500 MHz, CDCl
3
) δ 9.96 (s, 1H), 7.34 (t, J = 1.5 Hz, 1H), 7.29 (dd, J = 2.6, 1.3 Hz, 1H), 7.11 (t,
J = 2.1 Hz, 1H), 4.48 (s, 2H), 2.96 (sept, J = 6.9 Hz, 1H), 2.06 (t, J = 6.3 Hz, 2H), 1.72 (s, 3H),
.69 – 1.63 (m, 2H), 1.54 – 1.49 (m, 2H), 1.28 (d, J = 6.9 Hz, 6H), 1.06 (s, 6H); 13C NMR (125
1
3
MHz, CDCl ) δ 192.74, 160.13, 151.50, 137.91, 136.29, 132.99, 122.11, 121.06, 110.04, 64.89,
39.39, 34.27, 34.13, 33.03, 28.58, 23.93, 19.98, 19.41.
3-(3-(Cyclohexylmethoxy)-5-isopropylphenyl)-3-hydroxypropanenitrile (7a): An oven-dried
50 mL, two-necked, round-bottomed flask equipped with a magnetic stir bar and sealed under
argon. The flask was charged with anhydrous THF (9 mL) and anhydrous acetonitrile (290 uL,
.2 mmol, 1.01 eq.), cooled to -42 °C, and treated dropwise with a solution of potassium tert-
butoxide (630 mg, 5.7 mmol, 1.12 eq.) in THF (6 mL). After stirring for 45 min, benzaldehyde 6a
1.3 g, 5 mmol, 1.0 eq.) in THF (2.7 mL) also was added dropwise. The reaction was allowed to
5
(
14

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warm to -15°C over a 4 h period and was quenched by the slow addition of 25% NH
1 mL). The layers were partitioned and the aqueous phase was extracted twice with EtOAc. The
combined organic layers were washed twice with H O, once with brine, dried over MgSO , and
4
Cl (aqeuous)
(
2
4
concentrated under reduced pressure. Purification by flash column chromatography on silica gel
1
(
EtOAc/Hexanes 35:65) afforded 1.3 g (86%) of the nitrile 7a as a pale yellow oil. H NMR (500
MHz, CDCl ) δ 6.80 (s, 1H), 6.75 (s, 2H), 4.99 (t, J = 6.3 Hz, 1H), 3.75 (d, J = 6.1 Hz, 2H), 2.87
3
(
(
sept, J = 6.6 Hz, 1H), 2.78 – 2.74 (m, 2H), 1.90 – 1.83 (m, 2H), 1.81 – 1.66 (m, 4H), 1.36 – 1.25
m, 3H), 1.23 (d, J = 6.7 Hz, 6H), 1.11 – 1.01 (m, 2H); 13C NMR (125 MHz, CDCl
3
) δ 160.03,
151.56, 142.52, 117.48, 115.89, 113.49, 108.61, 73.67, 70.59, 37.93, 34.41, 30.08, 28.04, 26.65,
25.95, 24.06, 24.01.
3-Hydroxy-3-(3-isopropyl-5-((2-propylpentyl)oxy)phenyl)propanenitrile (7b): The same
procedure was followed as described for 7a. A solution of potassium tert-butoxide (770 mg, 6.9
mmol, 1.12 eq.) in THF (7.5 mL) was added to a solution of anhydrous acetonitrile (356 uL, 6.4
mmol, 1.04 eq.) in anhydrous THF (9 mL) at -42 °C. Then, benzaldehyde 6b (1.7 g, 6.2 mmol, 1.0
1
eq.) in THF (6 mL) was added dropwise yielding the nitrile 7b (1.7 g, 87%) as a pale yellow oil. H
NMR (500 MHz, CDCl
3
) δ 6.80 (s, 1H), 6.75 (s, 2H), 4.99 (t, J = 6.4 Hz, 1H), 3.82 (d, J = 5.5 Hz,
2
1
1
2
H), 2.88 (sept, J = 7.3 Hz, 1H), 2.79 – 2.75 (m, 2H), 2.30 (bs, 1H), 1.86 – 1.75 (m, 1H), 1.48 –
13
.32 (m, 8H), 1.24 (d, J = 6.7 Hz, 6H), 0.92 (t, J = 6.8 Hz, 6H); C NMR (125 MHz, CDCl ) δ
3
59.95, 151.39, 142.37, 117.35, 115.71, 113.30, 108.55, 70.89, 70.47, 37.60, 34.29, 33.72, 27.90,
3.93, 23.88, 20.03, 14.45.
3-Hydroxy-3-(3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-
yl)methoxy)phenyl)propanenitrile (7c): The same procedure was followed as described for 7a.
A solution of potassium tert-butoxide (485 mg, 4.32 mmol, 1.12 eq.) in THF (10 mL) was added
to a solution of anhydrous acetonitrile (222 uL, 4.2 mmol, 1.1 eq.) in anhydrous THF (8.4 mL) at
-
42 °C. Then, benzaldehyde 6c (1.16 g, 3.9 mmol, 1.0 eq.) in THF (4 mL) was added dropwise.
15

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Purification by flash column chromatography on silica gel (EtOAc/Hexanes 35:65) afforded 0.9 g
1
(
69%) of desired nitrile 7c as a pale yellow oil. H NMR (500 MHz, CDCl
3
) δ 6.84 – 6.82 (m, 2H),
6.82 – 6.80 (m, 1H), 5.01 (t, J = 6.3 Hz, 1H), 4.43 (s, 2H), 2.89 (sept, J = 6.8 Hz, 1H), 2.80 – 2.76
(
m, 2H), 2.29 (s, 1H), 2.05 (d, J = 6.3 Hz, 2H), 1.71 (s, 3H), 1.69 – 1.62 (m, 2H), 1.54 – 1.48 (m,
1
3
2
1
2
H), 1.25 (d, J = 6.9 Hz, 6H), 1.05 (s, 6H); C NMR (125 MHz, CDCl ) δ 160.08, 151.53, 142.47,
3
36.06, 133.13, 117.51, 116.00, 113.81, 108.75, 70.66, 64.64, 39.40, 34.44, 34.26, 33.02, 28.60,
8.04, 24.08, 24.03, 19.99, 19.42.
3-Amino-1-(3-(cyclohexylmethoxy)-5-isopropylphenyl)propan-1-ol (MB-005): An oven-dried
50 ml, two-necked, round-bottomed flask equipped with a magnetic stir bar was sealed under
argon with two rubber septa, one of which contained a needle adapter to an argon inlet. The flask
was charged with anhydrous THF (14 mL) and nitrile 7a (1.3 g, 4.3 mmol, 1.0 eq.). To the stirred
reaction mixture at room temperature, a 5.0 M solution of borane dimethyl sulfide complex in
diethylether (10.8 mmol, 2.5 eq.) was added slowly. Then the reaction mixture was heated under
reflux for 4h. The reaction mixture was cooled to 0 °C and methanol was slowly added to
decompose the unreacted borane complex. The organic solvent was evaporated under reduced
4
pressure. Purification by silica gel column chromatography (NH OH/MeOH/CHCl
3
4:20:80)
1
afforded 0.6 g (46%) of MB-005 as a pale yellow oil. H NMR (500 MHz, CD
3
OD) δ 6.81 (s, 1H),
6
2
1
1
2
3
.74 (s, 1H), 6.67 (s, 1H), 4.74 (t, J = 6.4 Hz, 1H), 3.76 (d, J = 6.1 Hz, 2H), 3.01 – 2.90 (m, 2H),
.85 (sept, J = 7.3 Hz, 1H), 1.95 (q, J = 6.8 Hz, 2H), 1.91 – 1.84 (m, 2H), 1.82 – 1.68 (m, 4H),
13
.39 – 1.25 (m, 3H), 1.23 (d, J = 6.7 Hz, 6H), 1.15 – 1.04 (m, 2H); C NMR (125 MHz, CDCl
3
) δ
59.63, 150.60, 146.42, 116.21, 111.88, 108.74, 75.67, 73.54, 40.65, 39.50, 38.00, 34.42, 30.14,
+
+
6.71, 26.00, 24.17, 24.10 ; ESI-HRMS: m/z calculated for C19
H
32NO [M+H] : 306.24276; found:
2
06.24576.
3-Amino-1-(3-isopropyl-5-((2-propylpentyl)oxy)phenyl)propan-1-ol (MB-006): The same
procedure was followed as described for MB-005. A 2.0 M solution of borane dimethyl sulfide
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complex in diethylether (10.25 mmol, 2.5 eq.) was added to a solution of the nitrile 7b (1.3 g, 4.1
mmol, 1.0 eq.) in anhydrous THF (10 mL). Purification by silica gel column chromatography
1
(
NH
500 MHz, CDCl
.6 Hz, 2H), 3.23 – 3.14 (m, 1H), 3.12 – 3.01 (m, 1H), 2.84 (sept, J = 7.3 Hz, 1H), 1.94 (q, J = 6.0
Hz, 2H), 1.81 – 1.72 (m, 1H), 1.49 – 1.27 (m, 8H), 1.22 (d, J = 6.6 Hz, 6H), 0.90 (t, J = 6.9 Hz,
4
OH/MeOH/CH
2
Cl
2
4:10:90) afforded 0.63 g (47%) of MB-006 as a pale yellow oil. H NMR
(
3
) δ 6.79 (s, 1H), 6.74 (s, 1H), 6.65 (s, 1H), 4.90 (t, J = 6.2 Hz, 1H), 3.81 (d, J =
5
13
6
H); C NMR (125 MHz, CDCl ) δ 159.74, 150.68, 145.86, 116.17, 111.91, 108.93, 74.99, 70.89,
3
40.09, 38.01, 37.80, 34.42, 33.88, 24.16, 24.11, 20.16, 14.61; ESI-HRMS: m/z calculated for
36NO +
[M+H] : 322.27406; found: 322.27402.
+
20
C H
2
3
-Amino-1-(3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)phenyl)propan-1-ol
MB-007): An oven-dried 50 mL, two-necked, round-bottomed flask equipped with a magnetic stir
bar was sealed under argon with two rubber septa, one of which contained a needle adapter to
an argon inlet. The flask was charged with anhydrous THF (25 mL) and a 2.0 M solution of LiAlH
(
4
in THF (3.68 uL, 7.36 mmol, 2.5 eq). At 0 °C, a solution of nitrile 7c (1g, 2.9 mmol, 1eq) in
anhydrous THF (6 mL) was then added dropwise to this reaction mixture. The resulting yellow
mixture was stirred at 0 °C for 1 h. At 0 °C, the reaction was quenched by slow addition of H O
2
(
(
366 uL) (caution: with evolution of hydrogen gas) followed sequentially by 15% w/v aq. NaOH
366 uL) and H O (1.1 mL). Then, the amorphous mixture was stirred for 10 min at room
Cl . The filtrate
and concentrated under reduced pressure. (Caspi et al., 2004) Purification
2
temperature. The resulting mixture was vacuum filtered and washed well with CH
2
2
was dried over MgSO
4
by flash chromatography on a silica gel column (NH
4
OH/MeOH/CH
2
Cl
2
4:10:90 6:20:80)
) δ 6.87 – 6.79 (m,
1
afforded 0.6 g (60%) of MB-007 as a colorless oil. H NMR (500 MHz, CDCl
3
2
–
3
H), 6.73 (t, J = 1.9 Hz, 1H), 4.92 (dd, J = 8.8, 3.2 Hz, 1H), 4.43 (s, 2H), 3.19 – 3.10 (m, 1H), 3.03
2.94 (m, 1H), 2.87 (sept, J = 6.9 Hz, 1H), 2.04 (t, J = 6.3 Hz, 2H), 1.94 – 1.76 (m, 2H), 1.71 (s,
H), 1.68 – 1.61 (m, 2H), 1.53 – 1.47 (m, 2H), 1.24 (d, J = 6.9 Hz, 6H), 1.05 (s, 6H); 13C NMR
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(
3
125 MHz, CDCl ) δ 159.55, 150.35, 146.26, 135.56, 133.31, 116.20, 112.18, 108.71, 76.77,
75.66, 64.34, 40.64, 39.53, 39.30, 34.29, 34.13, 32.88, 28.46, 24.03, 23.96, 19.86, 19.32. ESI-
36NO +
[M+H] : 346.27406, found 346.27399.
+
HRMS: m/z calculated for C22
H
2
ACN, tBuOK
THF
K
CO , 9
2
3
O
HO
O
O
HO
N
Aceton
N
N
OH
O
OH
OH
1
0
8
O
PPh3
EtO
Pd/ H2
O
N
EtAcO
Benzene
EtO
OH
OH
EtO
12
1
1 E/Z
O
O
O
O
Br / Urea
2
NH2
Br
BMS
THF
O
Acetic acid
HO
9
MB-008
BMS: Borane
dimethylsulfide
Scheme 4. Reagents and conditions for the synthesis of MB-008.
3-Hydroxy-3-(3-hydroxyphenyl)propanenitrile (8): An oven-dried 100 mL, two-necked, round-
bottomed flask was sealed under argon. The flask was charged with anhydrous THF (11 mL) and
anhydrous acetonitrile (465 uL, 8.3 mmol), cooled to -42 °C and a solution of potassium tert-
butoxide (2.06 g, 18.4 mmol) in THF (20 mL) was added dropwise. After stirring for 45 min at -42
°
C, 3-hydroxy-benzaldehyde (1g, 8.2 mmol) in THF (4.5 mL) was added dropwise. The reaction
was allowed to warm to -15 °C over a 4 h period, and then was quenched by the slow addition of
5% NH Cl (aq) (14.5 mL). The layers were partitioned, and the aqueous phase was extracted
twice with EtOAc. The combined organic layers were washed twice with H O, once with brine,
dried over MgSO , and evaporated under reduced pressure. Purification by silica gel column
chromatography (EtOAc/Hexanes 50:50) afforded 1.06 g (80%) of compound 8 as a pale yellow
2
4
2
4
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1
oil. H NMR (400 MHz, CDCl
3
) δ 7.26 (t, 1H, J= 8 Hz), 6.95-6.91 (m, 2H), 6.82 (ddd, 1H, J = 1.2,
1
2
.4, 8.0 Hz), 5.01 (t, 1H, J= 6.4 Hz), 2.77 (d, 2H, J= 6 Hz) ppm; H NMR values matched those in
13
the literature (Caspi et al., 2004). C NMR (100 MHz, CDCl ) δ 156.5, 143.0, 130.4, 117.7, 117.4,
3
116.0, 112.5, 68.1, 25.7 ppm.
1-Bromopentan-2-one (9): An oven-dried 100 mL, two-necked, round-bottomed flask was
charged with urea (9.8 g, 163.2 mmol, 1.63 eq.), acetic acid (39 mL), and 2-pentanone (10.65
mL, 100 mmol, 1 eq.). At 0 °C, Br (5.46 ml, 17.05 g, 107.2 mmol, 1.07 eq.) was added to the
mixture and stirred at 18-20 °C until the red color disappeared (~4.5 h). This mixture was then
diluted with water (150 mL) and extracted with CH CI . The extract was washed with saturated
aqueous sodium carbonate, dried over MgSO , and concentrated under reduced pressure
caution: compound is a lachrymator, and needs to be handled carefully). Purification by silica gel
2
2
2
4
(
column chromatography (Et
2
O/Hexanes 3:97) afforded 4.2 g (26 %) of 1-bromopentan-2-one 9
1
as a pale yellow liquid(Zav'yalov et al., 1989). H NMR (500 MHz, CDCl
3
) δ 3.90 (s, 2H), 2.66 (t,
13
J = 7.2 Hz, 2H), 1.67 (h, J = 7.5 Hz, 2H), 0.96 (t, J = 7.2 Hz, 3H); C NMR (125 MHz, CDCl
3
) δ
202.25, 41.85, 34.44, 17.50, 13.72.
3-Hydroxy-3-(3-((2-oxopentyl)oxy)phenyl)propanenitrile (10): An oven-dried 100 mL, two-
necked, round-bottomed flask equipped with stir bar was sealed under argon. The flask was
charged with the substituted phenol 8 (566 mg, 3.5 mmole, 1.1eq.), 1-bromo-2-pentanone 9 (520
mg, 3.2 mmole, 1eq.), anhydrous potassium carbonate (646.8 mg. 4.68 mmol, 1.3 eq.), and dry
acetone (32 mL). The reaction mixture was refluxed for 24 h under argon atmosphere. The
resulting slurry was cooled down, treated with water, and extracted with ether (3x). The combined
ether extracts were washed with 2.5M NaOH aq solution, brine, dried over MgSO
4
and evaporated
under reduced pressure. Purification by silica gel column chromatography (EtOAc/Hexanes
50:50) afforded 570 mg (73 %) of compound 10 as a pale yellow oil (White and Norcross, 1961).
1H NMR (500 MHz, CDCl
3
) δ 7.32 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.97 (s, 1H), 6.85
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(
dd, J = 8.2, 2.6 Hz, 1H), 5.03 (t, J = 6.2 Hz, 1H), 4.57 (s, 2H), 2.76 (d, J = 6.1 Hz, 2H), 2.56 (t, J
7.3 Hz, 2H), 1.67 (h, J = 7.7 Hz, 2H), 0.95 (t, J = 7.7 Hz, 3H); 13C NMR (125 MHz, CDCl
=
3
) δ
2
07.10, 158.28, 142.91, 130.29, 118.70, 117.05, 114.68, 111.94, 72.74, 69.94, 40.96, 27.95,
6.69, 13.73.
1
Ethyl-3-((3-(2-cyano-1-hydroxyethyl)phenoxy)methyl)hex-2-enoate (11): An oven-dried 100
mL, two-necked, round-bottomed flask equipped with a magnetic stir bar was sealed under argon.
The flask was charged with compound 10 (389.0 mg, 1.57 mmol, 1eq.) and anhydrous benzene
(
40 mL), followed by addition of (ethoxycarbonylmethylene)triphenyl phosphorane (75.9 mg,
.218 mmol, 1.09 eq.). This reaction mixture was refluxed under argon for 8 h at 84 °C. Then, the
0
solution was cooled down to room temperature and concentrated under reduced pressure.
Purification by silica gel column chromatography (EtOAc/Hexanes 35:65) afforded 274 mg (55%)
of E/Z mixture of compound 11 as a colorless oil. This was used for the next step without further
characterization.
Ethyl 3-((3-(2-cyano-1-hydroxyethyl)phenoxy)methyl)hexanoate (12): An oven-dried 25 mL,
two-necked, round-bottomed flask equipped with a magnetic stir bar was sealed under argon. The
flask was charged with EtOAc (12 mL) and compound 11 (223 mg, 0.7 mmol, 1eq.). To the stirred
mixture, palladium on carbon (75 mg, 10% wt, 0.07 mmol, 0.1 eq.) was added. The flask was
purged under mild vacuum and backfilled three times with hydrogen. Hydrogenation was carried
out via 2-ply balloon pressure overnight at 25 °C. Then, the reaction mixture was filtered through
a pad of celite and the filtrate was concentrated under reduced pressure. Purification by silica gel
column chromatography (EtOAc/Hexanes 30:70) afforded 200 mg (89 %) of compound 12 as a
1
colorless oil (Kimura et al., 2002). H NMR (500 MHz, CDCl
3
) δ 7.28 (t, J = 7.8 Hz, 1H), 6.94 (s,
2
H), 6.85 (dd, J = 8.3, 2.5 Hz, 1H), 4.99 (t, J = 6.2 Hz, 1H), 4.12 (q, J = 7.2 Hz, 2H), 3.96 – 3.91
m, 1H), 3.90 – 3.84 (m, 1H), 2.75 (d, J = 5.5 Hz, 2H), 2.63 (brs, 1H), 2.52 – 2.44 (m, 1H), 2.41 –
.31 (m, 2H), 1.55-1.45 (m, 1H), 1.41 – 1.34 (m, 3H), 1.24 (t, J = 7.1 Hz, 3H), 0.92 (t, J = 6.8 Hz,
(
2
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3
H); 13C NMR (125 MHz, CDCl
) δ 173.07, 159.44, 142.70, 129.98, 117.70, 117.29, 114.75,
3
111.62, 70.18, 70.06, 60.41, 36.66, 35.07, 33.53, 27.91, 20.01, 14.24, 14.22.
3-((3-(3-Amino-1-hydroxypropyl)phenoxy)methyl)hexan-1-ol (MB-008): An oven-dried 10 ml,
two-necked, round-bottomed flask was sealed under argon. The flask was charged with
anhydrous THF (5 mL) and compound 12 (40 mg, 0.125 mmol, 1 eq.). To the stirred reaction
mixture at room temperature, borane methyl sulfide complex 5M in ether (125 uL, 0.626 mmol, 5
eq.) was slowly added. After complete addition, the reaction mixture was refluxed (2-4 h) and then
cooled to 0 °C. The unreacted borane complex was decomposed via addition of methanol (Brown
et al., 1982). The organic solvent was evaporated under reduced pressure, and purification of the
residue by silica gel column chromatography (NH
4
OH/MeOH/CH
2
Cl
2
10:20:80) afforded 20.5 mg
1
(
90%) of MB-008 (a mixture of four stereoisomers) as a pale yellow oil. H NMR (500 MHz,
CD OD) δ 7.22 (t, J = 7.9 Hz, 1H), 6.97 – 6.88 (m, 2H), 6.80 (dd, J = 8.2, 2.4 Hz, 1H), 4.71 (dd, J
3
=
7.9, 5.3 Hz, 1H), 3.90 (d, J = 5.4 Hz, 2H), 3.66 (t, J = 6.9 Hz, 2H), 2.84 – 2.71 (m, 2H), 1.96 (h,
J = 5.6 Hz, 1H), 1.92 – 1.81 (m, 2H), 1.77 – 1.59 (m, 2H), 1.55 – 1.35 (m, 4H), 0.94 (t, J = 6.9 Hz,
3
H); 13C NMR (125 MHz, CD
OD) δ 160.85, 147.98, 130.36, 119.08, 114.29, 112.98, 73.44,
3
71.68, 61.04, 41.97, 39.50, 36.03, 35.67, 35.07, 21.05, 14.73. ESI-HRMS: m/z calculated for
28NO +
[M+H] : 282.20637, found 282.20633.
+
16
C H
3
Retinoid isomerase activity assays. Bovine RPE microsomes were prepared as previously
described to provide the isomerase for the assay (Stecher et al., 1999). Synthesized primary
amine (1 μl in DMF, with a final concentration ranging between 50 nM and 50 μM) was added to
10 mM Bis-Tris propane/HCl buffer, pH 7.4, containing 150 μg RPE microsomes, 1% BSA, 1 mM
disodium pyrophosphate, and 20 μM cellular retinaldehyde-binding protein (CRALBP). The
resulting mixture was pre-incubated at room temperature for 5 min. Then, all-trans-retinol (1 μl in
DMF, at a final concentration of 20 μM) was added. The resulting mixture was incubated at 37 °C
for 1 h. The reaction was quenched by adding 300 μl methanol, and products were extracted with
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300 μl hexanes. Production of 11-cis-retinol was quantified by normal-phase HPLC with 10% (v/v)
EtOAc in hexanes as the eluent at a flow rate of 1.4 ml/min. Retinoids were detected by monitoring
their absorbance at 325 nm and quantified based on a standard curve representing the
relationship between the amount of synthetic 11-cis-retinol standard and the area under the
corresponding chromatographic peak.
RPE65 purification and crystallization. RPE65 was purified from bovine RPE microsomes
using the detergent C
8
E (Anatrace, Mowmee, OH) as previously described (Kiser et al., 2009).
6
Inhibitors were added to the protein sample at the time of detergent solubilization as well as after
concentrating the purified protein to a final concentration of 1 μM. Inhibitors were delivered to the
samples as 100 mM DMSO stock solutions. Crystallization was accomplished by mixing 1-2 μL
of the concentrated protein sample with 1-2 μL of crystallization solution consisting of 40% (v/v)
PEG 300, 100 mM CHES-NaOH, pH 9.5, and 200 mM NaCl or 30% v/v PEG 400, 100 mM CAPS-
NaOH, pH 10.5, 500 mM (NH
4
)
2
SO and 10% v/v glycerol (Conditions #26 and #27, respectively
4
of the Wizard Cryo screen, Rigaku). These conditions reliably yield untwinned hexagonal RPE65
crystals in which residues constituting the active site cavity were previously shown to adopt active-
like conformations in the presence of emixustat-related molecules (Kiser et al., 2015). Crystals
appeared in one or two days and grew to maximal dimensions of 75 x 75 x 300 µm within one
month. Mature crystals were harvested with Microloops (Mitegen), flash cooled and stored in
liquid nitrogen prior to X-ray exposure.
Collection and processing of X-ray diffraction data and structure refinement. X-ray
diffraction data sets were collected at the NE-CAT ID-E beamline at the Advanced Photon Source.
Diffraction data were indexed, integrated and scaled with XDS (Kabsch, 2010). Data from three
separate crystals of the RPE65/MB-004 complex were merged using XSCALE (Kabsch, 2010) to
boost the signal-to-noise ratio and extend the high resolution limit. Initial models were generated
through rigid body refinement of a previously reported RPE65 crystal structure (PDB accession
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code 4RYY) using the program REFMAC (Murshudov et al., 2011). Ligand and water atoms were
removed from the model prior to conducting the initial refinement. The model was then completed
and optimized by multiple rounds of manual real space model building informed by
MOLPROBITY(Chen et al., 2010) close contact and geometry analyses in COOT (Emsley et al.,
2010) followed by restrained reciprocal space refinement in REFMAC. Atomic coordinates and
geometric restraint files for ligands were generated with the Grade Web Server. Some bond angle
restraint weights were manually adjusted to ensure ideal ligand stereochemistry. Residues 109-
126 and 197-201 in the RPE65/MB-004 complex and 110–126, 197–201 and 267–271 in the
RPE65/MB-008 complex were excluded from the final models owing to their poorly resolved
electron densities. The soundness of the final models was assessed with the MOLPROBITY
(
Chen et al., 2015) and wwPDB (Read et al., 2011) structure validation servers.
−
/−
−/−
Mouse handling and drug administration. Abca4 Rdh8 mice on a C57BL/6J background
were generated as previously described (Maeda et al., 2009a). Male and female WT mice with
−
/−
−/−
C57BL/6J backgrounds were obtained from The Jackson Laboratory. Abca4 Rdh8 mice used
in this study were homozygous for the Leu450 allele of Rpe65 as determined by a genotyping
protocol published previously (Grimm et al., 2004) and free of Crb1/rd8 and rd/rd mutations.
Animals were housed either in the animal facility at the Case Western Reserve University School
of Medicine, where they received a standard chow diet (LabDiet 5053) and were maintained under
the standard 12-h light (~300 lux) and 12-h dark cycle or at the Cole Eye Institute animal facility
(
animals used for ERG experiments) where they received LabDiet 2918 and were maintained
under the standard 14-h light (~100 lux) and 10-h dark cycle. Mice were dark adapted overnight
–/–
–/–
prior to all experiments. A cohort of evenly divided male and female Rdh8 Abca4 or C57BL/6J
mice was used when they reached the age of 1 to 4 months. All tested primary amines were
suspended in 100 μl soybean oil with less than 10% (v/v) DMSO and administered by oral gavage
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with a 22-gauge feeding needle. Experimental manipulations in the dark were done under dim red
light transmitted through a Kodak No. 1 Safelight Filter (transmittance >560 nm).
−
/−
−/−
Induction of acute retinal degeneration in Abca4 Rdh8 mice. Following vehicle- or test
−
/−
−/−
compound-administration by oral gavage, 1-2 month old Abca4 Rdh8 mice were dark adapted
for 24 h. Following induction of mydriasis with 1% tropicamide the mice were exposed to
fluorescent light (10,000 lux; 150 W spiral lamp; Commercial Electric) for 30 min in a white paper
bucket (Papersmith), a treatment previously shown to generate retinal damage in the absence of
protective agents (Zhang et al., 2015). Mice were then kept in the dark for an additional 3 days
before examination of their retinal structure, which was carried out with ultra-high-resolution
spectral domain OCT (SD-OCT; Bioptigen) as previously reported ((Zhang et al., 2015)).
−
/−
−/−
Identification of retinaldehyde conjugates with MB compounds in Abca4 Rdh8 mice.
MB-005 or MB-006 (1 mg/mouse) were administrated by oral gavage to 1-2 month old
−
/−
−/−
Abca4 Rdh8 mice 24 h prior to light exposure. After exposure to fluorescent light for 10 min,
animals were euthanized and their eyes were collected and homogenized in acetonitrile. After
centrifugation, the supernatant was concentrated to 400 µl under vacuum. Extracts (100 µl) were
injected into an LXQ linear ion trap mass spectrometer (Thermo Scientific) coupled to an Agilent
1100 HPLC system (Agilent Technologies). Separation of retinylidene Schiff bases was achieved
on a reverse phase C18 Phenomenex HPLC column (250 × 4.60 mm; 5 μm) by a linear gradient
of acetonitrile in water of 50% to 100% within 15 min at a flow rate of 1.5 ml/min. All solvents
contained 0.1% formic acid (v/v). The HPLC effluent was directed into the mass spectrometer via
an atmospheric pressure chemical ionization (APCI) interface operated in the positive ionization
mode. Identification of a retinylidene Schiff base was achieved by comparing its mass spectrum
with that of an authentic standard.
Quantification of MB-005 in mouse eyes. MB-005 (1 mg/mouse) was administrated by oral
gavage to 1-2 month old C57BL/6J mice. The mice were then placed back in their home cages
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and kept in standard lighting conditions for either 6 or 24 h. Then, mice were euthanized, and their
eyes were homogenized in 1 ml of 10 mM sodium phosphate buffer, pH 7.4, containing 50%
methanol (v/v). The resulting mixture was extracted with 4 ml EtOAc. Extracts were dried in vacuo
and reconstituted in 400 μl ethanol containing 1 nmol emixustat as the internal standard. This
solution (100 μl) was analyzed by liquid chromatography–electrospray ionization–MS/MS (LC-
ESI-MS/MS) (4.6 mm × 150 mm; Agilent ZORBAX Eclipse XDB-C18) with a gradient of
acetonitrile in water (0%–100% over 30 min) as the eluent at a flow rate of 0.5 ml/min. Amounts
of amines were quantified based on a standard curve representing the relationship between the
amount of MB-005 and the ratio of corresponding peak areas of MB-005 to emixustat.
Visual chromophore recovery assay. C57B/6J mice (1-2 months of age) were administered
test compounds or vehicle control by oral gavage and then allowed to dark adapt for 24 h.
Following induction of mydriasis with 1% tropicamide, the mice were exposed to bright light
resulting in photoactivation of > 90% of rhodopsin and then kept in the dark for 6 h to allow visual
pigment regeneration. Afterwards, animals were euthanized and their eyes were collected and
homogenized in 10 mM sodium phosphate buffer, pH 7.4, containing 50% methanol (v/v) and 40
mM hydroxylamine. The resulting mixture was extracted with 4 mL of hexanes. Extracts were
dried in vacuo, reconstituted in 300 μL of hexanes, and 100 μL of the extract was injected into an
HPLC system for analysis with 10% (v/v) EtOAc in hexanes as the eluent. Amounts of 11-cis-
retinal were quantified based on a standard curve representing the relationship between the
amount of 11-cis-retinyl oxime and the area under the corresponding chromatographic peak.
ERG recordings. C57BL/6J mice aged 1.5-4 months were anesthetized with ketamine (80
mg/kg) and xylazine (16 mg/kg). Mydriatic eye drops were used to dilate the pupil (1%
tropicamide; 2.5% phenylephrine). ERGs were obtained with an active electrode that contacted
the corneal surface; needle electrodes placed in the cheek and tail provided reference and ground
2
leads, respectively. Strobe stimuli ranging from -3.6 to 1.2 log cd s/m were presented in an LKC
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(
Gaithersburg, MD) ganzfeld. Responses were amplified, averaged and stored with an LKC UTAS
E-3000 signal averaging system.
Baseline dark-adapted ERGs were recorded from each mouse after overnight dark
adaptation. After a minimum one-day interval, MB compounds or vehicle were administered by
gavage at approximately 9 a.m. The next morning, mydriatic eye drops were used to dilate the
pupils and mice were then exposed to a 10-minute 10,000 lux bleach. Mice were dark-adapted
for 6 hours, after which their ERGs were recorded. The amplitude of the a-wave was measured
from the prestimulus baseline to 8 ms after the stimulus flash. The amplitude of the b-wave was
measured to the b-wave peak either from the a-wave trough or, if no a-wave was present, from
the baseline.
Study approval. All animal procedures and experiments were approved by the Institutional
Animal Care and Use Committees of Case Western Reserve University or the Cleveland Clinic,
and conformed to recommendations of the American Veterinary Medical Association Panel on
Euthanasia and the Association for Research in Vision and Ophthalmology.
RESULTS
Identification of potential sites for emixustat modification based on RPE65 crystal
structures. The emixustat molecule can be divided into three basic regions: a polar
aminopropanol moiety, a lipophilic cyclohexyl methoxy ring and a central phenyl ring to which
both the substituents are attached (Fig. 1A). MB-001 differs from emixustat by the substitution of
a trimethyl hexenyl ring in place of the cyclohexyl moiety. These molecules reside in the region of
the RPE65 active site cavity that is proximal to its membrane embedded entrance (Kiser et al.,
2015). The aminopropanol moiety, located deepest within the binding pocket of the molecule,
forms electrostatic interactions with Thr147 and Glu148 as well as with an iron-bound fatty acid
carboxylate. Such interactions appear to contribute substantially to the binding affinity making
alterations to this part of the molecule that disrupt active site binding affinity easily envisioned, for
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example by removal or alkylation of the hydroxyl moiety. However, because such changes could
influence the reactivity of the amino group towards retinaldehyde, we chose not to attempt
modifications of this region of the molecule. Notably, we showed previously that emixustat forms
a retinaldehyde Schiff base more efficiently than retinylamine (Zhang et al., 2015), possibly due
to the nearby hydroxyl moiety that can intramolecularly interact with the iminium functionality.
The central phenyl ring is involved in π−π interactions with Phe103 as well as van der
Waals contacts with a number of other active site residues. Notably, the active site region just
above the phenyl ring is relatively polar with two well-ordered water molecules engaged in
hydrogen bonding interactions with its constituent residues. These water molecules are located ~
3.3 Å away from C5 of the emixustat molecule (Fig. 1B). We reasoned that introduction of a bulky
lipophilic group to the region of the phenyl ring in close proximity to this polar pocket could
effectively prevent binding of the derivative molecule to the RPE65 active site as a result of steric
and electrostatic repulsive forces (Fig. 1C).
The terminal aliphatic ring moieties of these molecules, located closest to the active site
entrance, are the most mobile regions of the molecule as evidenced by their higher average
atomic displacement parameters (ADPs) and multiple conformations observed in different crystal
forms (Kiser et al., 2015; Zhang et al., 2015). The long range van der Waals interactions formed
between this portion of the molecules and residues forming the active site opening are expected
to minimally contribute to the binding affinity of emixustat and MB-001. This region thus appeared
to be a strong candidate for structural modification to improve active site binding affinity. Two
binding cavities are present on either side of the cyclohexyl moiety, one polar and filled with water
molecules and the other a small hydrophobic cavity formed by residues Ile259, Phe264, Tyr275,
Cys278 and Phe279 that could serve as a site of interaction for suitable derivatives (Fig. 1B). In
particular, we hypothesized that replacement of the terminal 6-membered ring with short linear
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moieties could result in molecules with the ability to engage these unexploited potential binding
cavities (Fig. 1C).
Thus, we developed novel synthetic routes for the generation of two classes of
compounds; one series in which the terminal 6-membered ring of emixustat/MB-001 was
substituted by either heptan-4-yl (MB-004) or 1-hydroxyhexan-3-yl (MB-008) groups and another
in which an isopropyl moiety was added on the meta position of the phenyl ring with the terminal
lipophilic moiety consisting of either cyclohexyl- (MB-005), heptan-4-yl (MB-006) or trimethyl
hexenyl (MB-007) groups (Fig. 2, A and B, left panels). Based on the structural features outlined
above, we hypothesized that the latter group of compounds would exhibit impaired inhibitory
activity towards RPE65 whereas the inhibitory activity of the former group could be enhanced
owing to potentially reinforced binding interactions. Both sets of compounds contained unmodified
primary amine functionalities and thus were anticipated to retain their retinaldehyde sequestration
activity (Fig. 1C).
Changes to the emixustat/MB-001 core structure alter in vitro retinoid isomerase inhibitory
activity. To test these structural predictions, we examined the inhibitory activity of candidate
compounds against RPE65 activity in an in vitro retinoid isomerase assay using bovine RPE
microsomes as the enzyme source. Consistent with our hypothesis, both MB-004 and MB-008
exerted more potent RPE65 inhibitory effects relative to emixustat and MB-001. Compared to
emixustat (Kiser et al., 2015), the two compounds MB-004 and MB-008 were about 2.2 and 1.2
times more potent with IC50 values of 106 ± 16 nM and 189 ± 31 nM, respectively (Fig. 2A).
Because racemic forms of emixustat and MB-008 were used in these experiments, whereas MB-
004 was synthesized as the pure R isomer, stereochemical differences must be accounted for
when comparing the potencies of these compounds. We previously reported that (R)-emixustat
is ~1.6 times more potent than the S-isomer (Zhang et al., 2015). The estimated IC50 of (R)-
emixustat in this assay is therefore around 175 nM, making MB-004 approximately 1.7 times more
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potent in this head to head comparison. The four possible isomeric forms of MB-008 make head
to head potency comparisons to emixustat more difficult, but the racemic mixture is at least as
potent as (R)-emixustat and the possibility remains that one particular isomer could have even
higher potency. Conversely, MB-005, MB-006 and MB-007 all exhibited little or no inhibition
against retinoid isomerization in this assay up to concentrations of 50 μM, consistent with their
inability to access the RPE65 active site and block retinyl ester uptake (Fig. 2B) Thus, these
structure-guided modifications to the emixustat molecule modulated RPE65 inhibition in a manner
consistent with our expectations.
MB-004 AND MB-008 engage binding sites in the RPE65 active site cavity that are not used
by emixustat and MB-001. To test our hypothesis that binding of MB-004 and MB-008 to the
RPE65 active site could be strengthened by interactions with cavities located near the
cyclohexanyl ring of emixustat, we determined crystal structures of bovine RPE65 in complex with
both molecules. The RPE65-MB-004 and RPE65-MB-008 structures, refined against data
extending to 2.2 and 2.1 Å resolution, respectively, were obtained by co-crystallization with the
compounds present in an approximately 5-fold molar excess over the protein under conditions
similar to those used in prior RPE65-emixustat structure determinations (Table 1). In both cases,
the ligands were clearly identified from their initial residual electron density maps (Fig. 3, A and
B). The 3-amino-1-phenylpropan-1-ol moieties, common to MB-004, MB-008 and emixustat, were
found in essentially identical conformations in all structures (Fig. 4). The (R)-isomer of the 3-
amino-1-hydroxypropylphenyl moiety of MB-008 appeared to be the dominant form of the
molecule occupying the active site, in agreement with our previous study on the binding of
emixustat isomers to the RPE65 active site (Zhang et al., 2015).
The dipropanyl tail of MB-004 adopted an extended conformation with one half of the
moiety occupying the abovementioned hydrophobic cavity in van der Waals contact with the side
chains of residues Ile259, Phe264, Tyr275 and Phe279 (Fig. 4B). The other half of this moiety
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chain was also found in an extended conformation near the active site tunnel entrance, but was
less restrained, being held in place only by a few relatively less favorable van der Waals contacts
with the Glu148 backbone carbonyl oxygen, backbone atoms of Gly176 and a nearby water
molecule. This difference in binding interactions was reflected in the ~11% lower average B factor
2
observed for the chain occupying the hydrophobic cavity (44 Å ) compared to the other half of the
2
substituent with sparser interaction partners (49 Å ).
The 7-membered tail of MB-008 adopted a conformation very similar to that of MB-004
with the exception of a slight rotation of the hydroxyethyl moiety that allowed formation of a
hydrogen bond with a water molecule located in the polar cavity mentioned above (Fig. 4C). Trial
refinements with the molecule modeled in the four possible isomers suggested that the R,S form
of the molecule is the dominant form bound in the active site. Despite the apparently more
favorable interaction made by the hydroxyethyl side chain, its B-factors remained elevated
compared to the rest of the molecule, similar to the analogous region of MB-004.
In summary, replacement of the emixustat cyclohexyl tail with a short linear alkane moiety
appeared to strengthen binding affinity owing to the fulfillment of favorable van der Waals and
hydrophobic interactions with a lipophilic pocket located near the active site entrance. The
presence of a second linear moiety with a capability to form hydrogen bonding interactions with
polar residues and solvent molecules located across from the lipophilic pocket appeared to
contribute minimally to the binding affinity of MB-008.
Effects of MB compounds on visual cycle function in vivo. Next, we tested the effects of the
MB compound series on retinoid cycle function in living mice. For these experiments, mice were
given a single dose of the test compound by oral gavage and 24 h later they were subjected to
light exposure that bleached > 90% of rhodopsin. After light treatment, mice were allowed to dark
adapt for 6 h before their ocular retinoid content was determined by HPLC. MB-004, at a dose of
10 μg/mouse dramatically suppressed visual chromophore regeneration to at least the same
30