Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma treatment

Article abstract of DOI:10.1016/j.bmc.2018.11.003

Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based ¹H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6 h. This interconversion process “intrinsic reversibility” was exploited by modifications of the ligand's size (length and bulkiness) to generate analogs with “tunable’ adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by ～4.5 mmHg in a sustained manner for at least 12 h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.

Full text of DOI:10.1016/j.bmc.2018.11.003

Accepted Manuscript
Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma
treatment
Shakiru O. Alapafuja, Michael S. Malamas, Vidya Shukla, Alexander Zvonok,
Sally Miller, Laura Daily, Girija Rajarshi, Christina Yume Miyabe, Honrao
Chandrashekhar, JodiAnne Wood, Sergiy Tyukhtenko, Alex Straiker,
Alexandros Makriyannis
PII:
S0968-0896(18)31589-X
https://doi.org/10.1016/j.bmc.2018.11.003
BMC 14607
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 September 2018
26 October 2018
2 November 2018
Please cite this article as: Alapafuja, S.O., Malamas, M.S., Shukla, V., Zvonok, A., Miller, S., Daily, L., Rajarshi,
G., Miyabe, C.Y., Chandrashekhar, H., Wood, J., Tyukhtenko, S., Straiker, A., Makriyannis, A., Synthesis and
evaluation of potent and selective MGL inhibitors as a glaucoma treatment, Bioorganic & Medicinal Chemistry
(2018), doi: https://doi.org/10.1016/j.bmc.2018.11.003
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Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma treatment
Shakiru O. Alapafuja 2, Michael S. Malamas *1, Vidya Shukla 1, Alexander Zvonok 1, Sally
Miller 3, Laura Daily 3, Girija Rajarshi 1, Christina Yume Miyabe 1, Honrao Chandrashekhar 1,
JodiAnne Wood 1, Sergiy Tyukhtenko 1, Alex Straiker 3 and Alexandros Makriyannis *1.
1Center for Drug Discovery and Departments of Chemistry and Chemical Biology and
Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, USA;
2MAK Scientific LLC, 151 South Bedford Street, Burlington, MA 01803 USA;
3Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47405,
USA.
Abstract
Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma
through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1
receptors. Herein, we report the discovery of new series of carbamates as highly potent and
selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity
against recombinant human and purified rat MGL, were selective (>1000-fold) against serine
hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and
1
CB2. Protein-based H NMR experiments indicated that inhibitor 2 rapidly formed a covalent
adduct with MGL with a residence time of about 6 hours. This interconversion process “intrinsic
reversibility” was exploited by modifications of the ligand’s size (length and bulkiness) to
generate analogs with tunable adduct residence time (). Inhibitor 2 was evaluated in a
normotensive murine model for assessing intraocular pressure (IOP), which could lead to
glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by ~4.5
mmHg in a sustained manner for at least 12 hours after a single ocular application, underscoring
the potential for topically-administered MGL inhibitors as a novel therapeutic target for the
treatment of glaucoma.
Keywords: Glaucoma, monoacylglycerol lipase (MGL), fatty acid amide hydrolase (FAAH),
CB1, CB2 cannabinoid receptors, 9-THC, (−)-Δ9-tetrahydrocannabinol, N-
arachidonoylethanolamide, anandamide (AEA), 2-arachidonoylglycerol (2-AG).

1. Introduction
Glaucoma is characterized as a progressive ocular neuropathy that affects the optic nerve and
represents the leading cause of irreversible blindness. Ocular hypertension is considered the
primary risk factor for developing glaucoma and studies have shown that the risk for glaucoma
increases by 12% with every 1 mmHg increase in intraocular pressure (IOP). Currently, there are
six main classes of topical ocular medications which reduce IOP to treat glaucoma by either
enhancing aqueous humor outflow or suppressing aqueous humor production in the eye. The
commonly used drugs for the reduction of IOP comprise of cholinergic agonists, 2-adrenergic
antagonists, 2-adrenergic agonists, carbonic anhydrase inhibitors, prostaglandins analogs, and
most recently rho kinase inhibitors.1-6 While these drugs lower IOP, not all patients respond to
the treatments. For many patients, these medications in monotherapy do not effectively control
IOP, and approximately half of patients with elevated IOP are treated by co-administration of
two or more glaucoma medications.7 Further, these medications are associated with ocular and
central nervous system (CNS), cardiovascular, gastrointestinal, pulmonary and haematologic
side-effects that can impair the effectiveness of the treatment.6
1.1 The endocannabinoid system as a therapeutic target for glaucoma: There is increasing
evidence that modulation of the endocannabinoid system plays a key role in the eye.8, 9 The
endocannabinoid system consists of cannabinoid receptors (CB1, CB2), the endogenous ligands
N-arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) and their
hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL).
CB1 is widely distributed in several regions of the eye, including ciliary epithelium and
trabecular meshwork,10-12 which are implicated in regulation of IOP. Smoking marijuana
cigarettes or inhaling the principal active ingredient in marijuana, Delta-9-tetrahydrocannabinol
(9-THC; a partial agonist of the CB1 receptor) lowers intraocular pressure (IOP) by about 40%
13, endorsing the role of cannabinoids in glaucoma treatment. Subsequent studies found that the
CB1 agonist WIN55212-2 lowered IOP in human patients who were unresponsive to available
glaucoma treatments14, and that prolonged treatment was both well-tolerated in rabbits and did
not result in desensitization.15 Discovery efforts to advance CB1 agonists as a glaucoma therapy

have not been pursued partly due to early reports that the effect of topical cannabinoid
application is brief and ineffective16, 17 and also to concern over the psychotropic side-effects
associated with CB1 activation.18-20
1.2 MGL inhibitors as potential therapy for glaucoma: As an alternate strategy to activate CB1
receptors, MGL inhibitors were pursued as potential medications to glaucoma by enhancing the
endogenous levels of 2-AG, a substrate of CB1. Recent studies showed that administration of 2-
AG lowered IOP in a mouse normotensive model.9, 21 This effect was found to be absent in
transgenic knockout CB1-/- mice, but present in the CB2-/- mice,9 indicative of the role of the CB1
receptor in IOP. However, this effect was short-lived due to the rapid degradation of 2-AG by its
hydrolytic enzyme MGL. In order to circumvent this deficit, we used MGL inhibitors to block
the degradation of 2-AG in ocular tissues. A recent publication also supports the role of MGL
inhibition in glaucoma treatment.9 MGL is the primary hydrolytic enzyme of 2-AG and accounts
for about 85% of its degradation. MGL is expressed prominently in the non-pigmented ciliary
epithelium of the retina, a key regulatory region of the eye controlling IOP via production of
aqueous humor. Persistent and complete inhibition of MGL for extended periods have been
associated with CB1 receptor desensitization, tolerance and hypomotility. These deleterious
effects are not observed with partial MGL blockage using lower doses of specific inhibitors.22,
23,24
Thus, modulating CB1 through inhibition of the endocannabinoid-degrading enzyme MGL
may be able to increase duration of action in controlling IOP, reduce desensitization, and limit
psychotropic effects associated with direct CB1 activation.
2. Chemistry
2.1 Design of MGL inhibitors: MGL is a membrane bound serine hydrolase enzyme. The crystal
structure of human MGL showed a cap domain covering the structurally conserved active site
containing the catalytic triad made up of residues Ser122, Asp239 and His269. The triad
activates Ser122, which nucleophilically attacks the carbonyl of the endocannabinoid 2-AG. An
oxyanion hole stabilizes the tetrahedral anionic intermediate during hydrolysis, while allowing
the interaction with cellular membranes. The distal region of the substrate binding site contains a
large hydrophobic tunnel that accommodates the acyl-chain of 2-AG. This hydrophobic tunnel’s
regional structural conformation is different compared to that of FAAH and we have explored

this region through
the introduction of
stereochemical
features (relative and
absolute
configuration as well
as
conformational
within
restriction)
individual analogs to
achieve selectivity against FAAH. Many designed MGL inhibitors (Fig. 1) contain a carbamate
pharmacophore, which covalently binds to Ser122 forming a covalent adduct intermediate as an
approach to offer sustained MGL inhibition and prolonged pharmacodynamic (PD) effect. A
typical structural feature of these inhibitors is an amine core linking the carbamate
pharmacophore to the lipophilic tail. Upon binding of the inhibitor to the MGL active site,
Ser122 attacks the carbonyl carbon of the carbamate group, triggering the formation of a
covalent adduct. This results in a stable enzyme-ligand adduct rendering the enzyme incapable of
further hydrolytic effect. However, this newly formed carbamate adduct, in a second phase
hydrolysis process, is converted back to the active enzyme, while in the process a structurally
modified ligand is released as a hydrolytic product. This interconversion process “intrinsic
reversibility” can be exploited by modifications of the ligand’s size (length and bulkiness) and
generate analogs with “tunable” adduct residence time (). This unique reversible mode of MGL
inhibition represents a powerful mechanism for abrogating enzyme activity, and it is considered
the most pharmacologically tractable mechanism of choice for enzyme inhibition. We have used
1
a protein-based real-time H NMR spectroscopy technology to accurately measure the residence
time of structurally diverse MGL inhibitors.
This strategy on targeting “reversible covalent” drugs offers the advantage of “tuning-up” a
desirable biological engagement and effect of a molecular target and mimic the long-lasting
activity of irreversible inhibitors, with dissociation rates in vivo that approach the rate of target
degradation and re-synthesis. Notably, fully irreversible adducts have raised serious potential
safety concerns in drug development because of the relationship between covalent drug binding
and the potential of immunogenic-driven allergic reactions and idiosyncratic drug toxicity.25, 26

Also, this strategy offers an additional advantage in
minimizing potential “drug-drug” interactions, since the drug
could rapidly clear from the circulation (short PK effect),
while still maintains a lasting target engagement effect (long
PD effect).
In an attempt to design potent and selective MGL inhibitors,
we have examined piperazine and piperidine carbamates (Fig. 2) with bulky “benzhydryl-like”
tails occupying the hydrophobic tunnel of the binding pocket of MGL, which accommodates the
acyl chain of 2-AG. The bulky lipophilic-tail of the ligand was essential to achieve high
selectivity of the new MGL inhibitors compared to FAAH and other serine hydrolases. Structure-
activity relationship studies have produced a large number of compounds and in this paper we
outline a selected group in Tables 1 and 2. The new MGL inhibitors were prepared according to
synthetic methods A-C.
Method A: Syntheses of substituted piperazine carbamates
Commercially
available
substituted aldehyde
1
(scheme 1) was treated with
alkyl, cycloalkyl or aryl
Grignard reagents to give
substituted benzyl alcohol
2, which was converted to
the corresponding chloride
3
upon treatment with
thionyl chloride. Chloride 3 was treated with piperazine to produce alkylated piperazine 4.
Commercially available phenol 5 was converted to chloroformate 6 upon treatment with
triphosgene and N, N-diisopropylethylamine. Finally, piperazine carbamate 7 was formed by
reaction of chloroformate 6 with piperazine 4 in the presence of triethylamine.
Method B: Syntheses of substituted piperidine carbamates

Commercially available 1-(tert-
butyl) 4-methyl piperidine-1,4-
dicarboxylate 1 (scheme 2) was
treated with phenyl magnesium
bromide to afford benzhydrol 2.
Intermediate
2
was
simultaneously dehydrated and
deprotected with trifluoroacetic
acid to afford piperidinium salt
3. Compound 3 was further reduced to piperidinium salt 4 using catalytic hydrogenation (H2/Pd-
C). Finally, carbamate 5 (Y = bond) was obtained by treating piperidine 4 with aryl
chloroformate 6 (scheme 1, described in Method A) in the presence of triethylamine.
Benzhydroxy piperidine 7 was prepared from benzhydrol 6 and 4-hydroxyl-piperidine under
acidic conditions (p-toluenesulfonic acid). Carbamate 5 (Y = O) was obtained by reaction of
benzhydroxy piperidine 7 and aryl chloroformate 6 (scheme 1, described in Method A) as above.
Methods C: Synthesis of benzhydrylsulfonyl-piperidine carbamates
tert-Butyl
4-((4-bromophenyl)thio)
piperidine-1-carboxylate 1 (scheme 3)
was oxidized to sulfone 2 with 3-
chloroperbenzoic acid. Deprotection of
the N-BOC group with trifluoroacetic acid
afforded piperidinium salt 3. Intermediate
3 was then treated with aryl chloroformate
6 (scheme 1, described in Method A) to
give sulfonyl piperidine carbamate 4 (Y =
C). 1-(Arylsulfonyl) piperazine carbamate
4 (Y = N) was prepared as following.
Aryl sulfonyl chloride 5 was treated with piperazine to produce 1-(arylsulfonyl) piperazine 6,
which was then converted to carbamate 4 (Y = N) as described above.
3. Results and Discussion

All synthesized compounds were tested for their ability to inhibit recombinant human MGL
(hMGL) and purified rat MGL (rMGL) using the fluorogenic substrate arachidonoyl, 7-hydroxy-
6-methoxy-4-methylcoumarin ester (AHMMCE). We used both species (human and rat) MGL to
assess potential inhibitory differences, which have been previously identified by various
structural chemotypes. Also, all compounds were tested against hydrolytic enzymes recombinant
human fatty acid amide hydrolase (hFAAH) and human alpha/beta-hydrolase domain containing
6 (hABHD6) for selectivity. Furthermore, selected compounds were evaluated for their ability to
bind to CB1 and CB2 receptors using rat brain27 or HEK293 cell membranes expressing mouse
CB2 (mCB2) or human CB2 (hCB2),28-30 respectively, via competition-equilibrium binding
29-31
using [3H]CP-55,940
to minimize their involvement due to cross-reactivity of potential
common pharmacophoric features.
Our design strategy was directed to optimize
structural features based on existing MGL
templates in the literature or generated in our
own lab. Computational methods supported by
available X-ray crystallographic data for MGL,
and molecular modeling studies applied to pre-
evaluate proposed synthetic targets. To that end,
we have established a three-dimensional binding
Figure 3. Schematic docking of compound 2 at
MGL’s active site
pocket (Fig. 3) based on human MGL (PDB 3PE6, 1.35 Å) cocrystal with a reversible MGL
inhibitor.32 This strategy allowed us to construct preconvalent MGL-ligand complexes using
Glide33-35 to determine inhibitor fit into binding site with correct binding geometry. For
molecules that geometrically fit in the MGL binding pocket, covalency was carried out to
calculate lowest free-binding energy using the well-established YASARA program.36 In addition,
in silico chemoinformatics calculations (i.e., MW, LE, LipE, ClogP, and tPSA) guided the choice
and priority for compounds to be synthesized in order to introduce desirable pharmacokinetic
characteristics. Full assessment of the in vitro potency and selectivity as well ADME compound
profiling (i.e., plasma stability, microsomal stability and solubility) was performed for selected
molecules to first achieve compounds with good aqueous solubility for topical eye drops
application, and secondly good oral bioavailability as potential systemic drugs.

A selection of compounds of our structure-activity relationship (SAR) studies are shown in
Tables 1-2. In support to our initial design strategy to introduce a bulky lipophilic-tail and
differentiate the ligand’s binding mode between MGL and FAAH, analog 1 with a benzyl group
attached to the piperidine nucleus was found to have similar potency for both MGL and FAAH,
while the bulkier benzhydryl analog 2 was about 4-fold more potent for MGL, but lacked affinity
for FAAH at concertations >10 uM. Armed with these findings, we have prepared a variety of
closely related “benzhydryl-like” analogs. The dichloro-substituted benzhydryl analog 3 was 3-
fold weaker in hMGL, while modifications of the linker between the benzhydryl group and the
piperidine nucleus produced variable potency results for MGL. For example, the oxygen-linked
analog 4 was 6-fold more potent for hMGL, while the amino-linked 5 and sulfonyl-linked 6
analogs resulted in significant loss of potency. We have also explored piperazine-based analogs
which appeared to follow a similar activity pattern to that of the piperidines. Analog 7 was found
to be the most active hMGL inhibitor of this group, while the more hydrophilic pyridine analog 9
lost about 10-fold in potency. The bulkier 9H-fluorene was similar to benzhydryl 7. The
isopropyl and cycloalkyl analogs 11-14, which represent bulky lipophilic tails appear to be more
potent for FAAH. These findings were rather unexpected and we can postulate that a bi-
aromatic group at the lipophilic tail of the ligand represents a critical structural feature for
achieving selectivity against FAAH. Noteworthy, the phenyl carbamate 15 was practically
inactive for MGL, when compared to the cyano-substituted analog 7, indicative of the essential
presence of the cyano group for MGL activity. This finding encouraged us to further explore the
carbamate pharmacophore region of the ligand. Towards this goal, we have introduced several
modifications at the carbamate phenyl group by incorporating various electron donating and
withdrawing groups (Table 2). Analogs 17-19 with fluorine, methyl and methoxy substituents
were almost equipotent to 16, while the hydroxyl analog 20 was 2-fold better (20 vs 19) for
hMGL. Also, the more hydrophilic pyridyl-analogs 21 and 22 were found to be more potent for
hMGL. We have judged that the increased polar nature of analogs 21-22 was well tolerated in
this region of the binding pocket, where the polar glycerol moiety of 2-AG could bind. All
reported analogs were evaluated against human alpha/beta-hydrolase domain containing 6
(hABHD6) and found to be inactive at concertations >10 uM. Furthermore, all compounds
showed no affinity for the cannabinoid receptors CB1 and CB2 at concertations >1 uM.

Table 1. In vitro MGL inhibitory potency of piperidine/piperazine carbamates
IC50 (nM)a
hMGL rFAAH
Compd
1
R1
X
rMGL
157
41.9
8.9
44.7
C
C
C
36.5
32.5
>1000
2
3
29.6
>1000
C
C
C
N
120
21.7
250
>1000
>1000
ntb
4
5
6
7
1,000
~1,000 ~1,000
108
10.6
>1000

N
98.2
72.8
>1000
8
N
N
360
100
>1000
>1000
9
52.3
18.2
10
N
N
N
780
150
890
224
89
68
63
56
11
12
13
200
N
367
127
965
67
nt
14
15
>1,000
a
IC50 values were measured by MGL and FAAH inhibition assays. Activity
measured after 15 min pre-incubation with compound using the fluorogenic
substrates arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester for MGL
and arachidonoyl 7-amino-4-methylcoumarin amide for FAAH. IC50 values were
calculated by using Prism software (GraphPad); b nt = Not tested
Table 2. In vitro MGL inhibitory potency of piperidine carbamates

IC50 (nM)
hMGL
Compd
16
R2
rMGL
rFAAH
112
25
>1000
105
150
98
34
>1000
>1000
>1000
>1000
17
18
19
20
67
39
22
98.2
20.1
75.8
3.0
8.5
>1000
>1000
21
22
a
IC50 values were measured by MGL and FAAH inhibition
assays. Activity measured after 15 min pre-incubation with
compound using the fluorogenic substrates arachidonoyl-7-
hydroxy-6-methoxy-4-methylcoumarin ester for MGL and
arachidonoyl-7-amino-4-methylcoumarin amide for FAAH. IC50
values were calculated using Prism software (GraphPad).

3.1 Protein-based real-time 1H NMR spectroscopy to determine ligand residence time
MGL inhibitors with prolonged on-target residence time
(t) as potential glaucoma treatment offer the advantage of
superior biochemical efficacy and patient compliance by
requiring lower drug doses and reduced eye applications,
thus minimizing local irritable side-effects. In our
strategy to design MGL inhibitors that form a reversible
covalent bond with the catalytic serine residue of MGL,
we have applied protein-based real-time 1H NMR
38
spectroscopy37,
otpimized for straighforward
determination of t values for different types of covalent
hMGL inhibitors. The ‘residence time by design’ and the
ability to modulate and improve the duration of target
engagement in vivo was based on the initial covalent
interaction between the nucleophilic catalytic serine
Figure 4. Inhibitor 2 covalently
modifies catalytic Ser122 and
induces perturbations on the low-
field region of the 1D 1H NMR
spectrum of hMGL. Initially,
catalytic His269 shifts upfield (arrow
indicator) and as adduct hydrolyzes,
it recovers to the original position.
residue of MGL and the carbamate pharmacophore region
1
of the ligand. Using the protein-based real-time H NMR spectroscopy, we have observed that
biochemical  values of structurally diverse covalent inhibitors can differ from a few hours to
1
several days (collective data will be presented elsewhere). Figure 4 illustrates the real-time H
NMR spectra of the interaction between inhibitor 2 and hMGL. Addition of 2 led to the
formation of covalent adduct, which was stable for about 20 min and then completely hydrolyzed
1
within ~6 hours. The real-time H NMR spectroscopy enabled the unambiguous recognition of
free and bound enzyme, quantification of mixtures and allowed the monitoring of time-
dependent events like slow dissociation of ligands from the binding site. In order to rationally
modulate , structural changes and conformational dynamics as well as electronic and steric
complementarity between ligand and hMGL binding site were evaluated (detailed findings of
1
this H NMR technology will be presented elsewhere). Results from these studies supported our
design strategy for tuning the residence time of MGL inhibitors for a prolonged
pharmacodynamic effect.
3.2 ADME Properties

Preliminary assessment of the ADME properties suggest that compound 2 represented a
promising druggable target. Compound 2 showed good microsomal stability in liver preparations
of various species with t1/2 min 122 (human) 43 (rat) 20 (mouse). Also 2 showed good plasma
stability with t1/2 min >2 h (mouse, rat and human plasma). In a pharmacokinetic study with
CD1-mice (iv 2 mg/kg. po 10 mg/kg) compound 2 showed moderate drug exposure with oral
bioavailability about 15%, and good brain penetrability ~65%. Based on the long residence time
(~ 6 hours) amongst several tested MGL inhibitors and favorable ADME properties, compound 2
was selected for pharmacological assessment in the normotensive IOP model as a potential drug
for glaucoma.
3.3 Pharmacology Data
We have completed a pivotal
study assessing IOP in the eye in
a normotensive murine model
with our selective MGL inhibitor
2 (Fig. 5). This study sought to
investigate the effect of MGL
inhibition,
the
enzyme
responsible for 2-AG hydrolysis
in ocular tissues, and a potential
therapeutic target for glaucoma.
In our experiments, IOP was measured in mice by rebound tonometry using a Tonolab device. In
order to obtain reproducible IOP measurements, the mice were briefly anesthetized with
isoflurane during drug administration and pressure measurements. The drug was administered as
a single topical treatment and IOP was then measured in the eyes of drug-treated mice and
vehicle-treated mice. We have found a single topical treatment at the eye significantly lowered
IOP at 4, 8 and 12 hr post-treatment (Fig. 5; IOP at 4 hrs (mmHg + SEM) in vehicle treated
animals: 20.0 ± 0.5; compound 2 (3 mM) at 4 hrs: 15.4 ± 0.8; vehicle at 8 hrs: 19.2 ± 0.6;
compound 2 at 8 hrs: 15.8 ± 0.8; vehicle at 12 hrs: 22.2 ± 1.1; compound 2 at 12 hrs: 14.9 ± 0.6;
n=8; **, p<0.01, ***, p<0.001 1 way ANOVA with Sidak’s post-hoc test vs. corresponding

control time point) . By 28 hours no drug effect was seen (vehicle IOP at 28 hrs (mmHg): 20.1 ±
1.1; compound 2 (3 mM): 19.9 ± 1.5; sample size and statistics as above). The largest drops in
IOP were observed at 4, 8 and 12 hr time points, with drops of 4.6, 3.9 and 4.7 mmHg relative to
vehicle treated controls. As such these represent substantial, sustained reductions, particularly for
a normotensive model. The effect was absent at 28 hours, but the substantial inhibition at 12
hours suggests that the drug’s effect is likely to continue at least several hours longer. It was
gratifying to observe, as was anticipated from a covalent inhibitor, to have a prolonged
pharmacodynamic response for 12 hours far exceeding the projected pharmacokinetic effect. We
consider that assessment of residence time represents a valuable selection criterion to advance
compounds to in vivo assays, supported by the strong relationship between the prolonged
ligand/protein interactions, the observed increased biochemical efficiency on target engagement
and the sustained pharmacodynamic effect that often outlasts the pharmacokinetic parameters.
4. Conclusions
We have discovered new series of potent and selective carbamate MGL inhibitors. MGL is an
enzyme implicated in 2-AG hydrolysis in ocular tissues, where rise of intraocular pressure could
lead to glaucoma, a major cause of blindness. Thus, inhibition of MGL represents a potential
therapeutic target for glaucoma treatment. The new inhibitors showed potent nanomolar
inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold)
against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid
1
receptors CB1 and CB2. Protein-based H NMR experiments indicated that inhibitor 2 rapidly
formed a covalent adduct with MGL with a residence time of about 6 hours. This interconversion
process “intrinsic reversibility” was exploited by modifications of the ligand’s size (length and
bulkiness) to generate analogs with tunable adduct residence time (). ADME assessment of 2
showed good microsomal and plasma stability and pharmacokinetic properties. Inhibitor 2 was
evaluated in a normotensive murine model for assessing IOP and it was found to decrease ocular
pressure by ~4.5 mmHg in a sustained manner for at least 12 hours after a single ocular
application. This represents a substantial improvement over our previous results using MGL
blocker KML299 and underscores the potential for topically-administered MGL inhibitors as a
novel therapeutic target for the treatment of glaucoma. With such a strong effect in normotensive
model, hypertensive eyes may see an even larger effect and it may be possible to optimize

treatment conditions to make this a once-daily treatment, thereby offering advantages over
glaucoma therapies that require multiple treatments per day.
5. Experimental section
5.1. Chemistry
Proton nuclear magnetic resonance spectra were obtained on a VARIAN 400 spectrometer at 500
MHz. Spectra are given in ppm () and coupling constants, J values, are reported in hertz.
Splitting patterns are designated as follows: s, singlet; brs, broad singlet; d, doublet; t, triplet; q,
quartet; m, multiplet. Tetramethylsilane was used as an internal reference standard. Mass
spectra were obtained on a Waters Micromass ZQ spectrometer. HPLC techniques and high
resolution mass spectrometry were used to determine compound purity. All reagents and solvents
were obtained from commercial suppliers and used without further purification. All non-
aqueous reactions were carried out in oven-dried glassware under an atmosphere of dried argon
or nitrogen. The reactions were monitored by thin layer chromatography (TLC plates F254,
Merck) or LC-MS analysis. All products, unless otherwise noted, were purified by flash
chromatography using a Biotage Isolera purification system with pre-packed silica cartridges.
Purity of all final products was > 96% as determined by LC-MS using the following protocol.
Mobile Phase A = water, B = acetonitrile solvent gradient 95/5 to 5/95 A:B in 11 min; flow rate
1.5 mL/min; Waters XTerra MS C8 column (4.6 × 50 mm) with UV detection at 190-400 nm
wavelength. The following abbreviations are used: CDCl3, deuterated chloroform; EtOAc, ethyl
acetate; CH2Cl2, dichlorometane; MgSO4, magnesium sulfate; THF, tetrahydrofuran; NH4Cl,
ammonium chloride; MeOH, methanol; NaHCO3, sodium bicarbonate; TFA, trifluoroacetic acid;
LC-MS, liquid chromatography with mass spectrometry; MS, mass spectrometry; NMR, nuclear
magnetic resonance spectrometry; TLC, thin layer chromatography.
Method A (scheme 1)
5.1.1. 2-Methyl-1-phenylpropan-1-ol (2, scheme 1, R1 = H, R2 = isopropyl)
Step a). To a solution of commercially available benzaldehyde 1 (1.3 g, 12.3 mmol) in THF (25
o
mL) at -20 C under a nitrogen atmosphere was added isopropyl magnesium bromide (4.1 mL,
3.0 M solution in diethyl ether) over 15 min period with stirring. The reaction mixture was

stirred for 1 h at -20 o C, and upon completion (monitored by TLC) was diluted with saturated
aqueous NH4Cl solution (10 mL). The aqueous layer was extracted with EtOAc and the
combined organic layer was dried over MgSO4 and concentrated under vacuum. The crude
material was purified by column chromatography on silica gel to give alcohol 2 (1.73 g, 94%
yield) as colorless liquid; 1H NMR (400 MHz, CDCl3) δ ppm 7.35-7.25 (m, 5H), 4.12 (d, 1H, J =
6.0 Hz,), 2.05-1.90 (m, 1H), 1.92 (brs, 1H), 1.31 (d, 3H, J = 6.0 Hz), 1.19 (d, 3H, J = 6.0 Hz).
5.1.2. (1-Chloro-2-methylpropyl)benzene (3, scheme 1, R1 = H, R2 = isopropyl)
Step b). To a solution of benzyl alcohol 2 (900 mg, 6.0 mmol) in dichloromethane (50 mL) at
room temperature was added thionyl chloride (1.1 mL, 15 mmol) dropwise over a 10 min period.
The reaction was refluxed for 1 hour and then concentrated under vacuum to give 0.96 g, 95%
yiled of (1-chloro-2-methylpropyl)benzene 3 as a colorless viscous liquid, which was used to the
next step without any further purification; 1H NMR (400 MHz, CDCl3) δ ppm 7.36-7.27 (m, 5H),
4.67 (d, 1H, J = 6.0 Hz), 2.05-1.90 (m, 1H), 1.92 (brs, 1H), 1.31 (d, 3H, J = 6.0 Hz), 1.19 (d, 3H,
J = 6.0 Hz).
5.1.3. 1-(2-methyl-1-phenylpropyl)piperazine (4, scheme 1, R1 = H, R2 = isopropyl)
Step c). To a solution of (1-chloro-2-methylpropyl)benzene 3 (0.84 g, 5.0 mmol) in ethanol (10
mL) was added piperazine (2.15 g, 25.0 mmol) and the mixture was heated in a sealed tube at 80
oC for 2 hours. The reaction mixture was cooled to room temperature and the solvent was
removed under vacuum. The residue was partitioned between dichloromethane and saturated
aqueous sodium bicarbonate solution. The mixture was extracted with dichloromethane three
times and the combined organic extracts were washed with brine and dried over MgSO4. The
solvent was removed under vacuum to give 0.98 g, 90% yield of 1-(2-methyl-1-
1
phenylpropyl)piperazine 4 as pale yellow viscous liquid. H NMR (400 MHz, CDCl3) δ ppm
7.36-7.27 (m, 5H), 4.67 (d, 1H, J = 6.0 Hz), 3.07 (d, 1H, J = 8.0 Hz), 2.50-2.40 (m, 4H), 2.40-
2.30 (m, 4H), 2.05-1.90 (m, 1H), 2.10 (brs, 1H), 1.31 (d, 3H, J = 4.0 Hz), 1.19 (d, 3H, J = 8.0
Hz).
5.1.4. 3-Cyanophenyl chloroformate (6, scheme 1, R3 = meta-CN, X = C)
Step d). To a stirred solution of triphosgene (0.5 g, 1.70 mmol) in dichloromethane (15 mL) at 0
oC under an argon atmosphere was added dropwise a mixture of 3-cyanophenol (0.58g, 4.87
mmol) and N,N-diisopropylethylamine (0.87 mL, 5.0 mmol) in methylene chloride (15 mL). The

reaction mixture was stirred at 0 o C for 1 h and then diluted with ice-cold water and extracted
with methylene chloride (2 x 10 mL). The combined organic extracts were dried over MgSO4.
The solvent was removed under reduced vacuum to give 0.75 g, 84% yiled of 3-cyanophenyl
chloroformate 6 as a pale yellow solid, which was used without any further purification.
5.1.5. 3-Cyanophenyl 4-(2-methyl-1-phenylpropyl)piperazine-1-carboxylate (7, scheme 1, R1 =
H, R2 = isopropyl, R3 = meta-CN, X = N)
Step e). To a stirred solution of 3-cyanophenyl chloroformate 6 (0.55 g, 3.0 mmol) in methylene
chloride (20 mL) at room temperature under an argon atmosphere were added 1-(2-methyl-1-
phenylpropyl)piperazine 4 (0.79 g, 3.6 mmol) and triethylamine (0.61 mL, 4.2 mmol). The
resulting mixture was stirred at room temperature for 2 hours and then the mixture was diluted
with water and extracted with methylene chloride (2 x 10 mL). The combined organic extracts
were dried over MgSO4. The solvent was removed under reduced pressure to give crude
material, which purified by column chromatography on silica gel to give 0.8 g, 72% yiled of 3-
1
cyanophenyl 4-(2-methyl-1-phenylpropyl)piperazine-1-carboxylate 7 as a white solid. H NMR
(400 MHz, CDCl3) δ ppm 7.45 (d, 1H, J = 8.0 Hz), 7.42 (t, 2H, J = 8.0), 7.37 (t, 1H, J = 2.0 Hz),
7.34 (t, 2H, J = 8.0) overlapping with (m, 1H), 7.30-7.27 (m, 1H), 7.13 (d, 2H, J = 8.0 Hz), 3.70-
3.40 (m, 4H), 3.07 (d, 1H, J = 8.0 Hz), 2.50-2.40 (m, 2H), 2.40-2.30 (m, 2H), 2.32-2.20 (m, 1H),
1.02 (d, 3H, J = 4.0), 0.74 (d, 3H, J = 8.0); (ES) m/z [M+1]: 364.19.
The following compounds shown in Table 1 were prepared according to Method A.
5.1.6. 3-Cyanophenyl 4-(benzhydrylamino)piperidine-1-carboxylate (5)
1H NMR (400 MHz, CDCl3) δ ppm 7.49-7.46 (m, 1H), 7.45-7.35 (m, 4H), 7.34-7.28 (m, 6H),
7.27-7.20 (m, 3), 5.05 (s, 1H), 4.15 (t, 2H, J = 12.0 Hz), brs, 2H), 3.3, (t, 1H, J = 12.0 Hz), 2.91
(t, 1H J = 12.0 Hz), 2.70-2.53 (m, 1H), 2.07-2.02 (m, 2H), 1.99 (brs, 1H), 1.5-1.32 (m, 2H); (ES)
m/z [M+1]: 412.57; purity: >96%, retention time: 5.8 min.
5.1.7. 3-Cyanophenyl 4-benzhydrylpiperazine-1-carboxylate (7)
1H NMR (400 MHz, CDCl3) δ ppm 7.52-7.49 (m, 2H), 7.4 (t, 1H, J = 8.0 Hz), 7.39 (td, 1H, J =
8.0 Hz, J = 2.0 Hz), 7.37-7.34 (m, 5H), 7.31 (m, 1H), 3.69 (brs, 2H), 3.58 (s, 2H), 3.55 (brs,

2H), 3.26 (s, 2H), 2.53 (t, 4H, J = 5.0 Hz); (ES) m/z [M+1]: 398.5; purity: >96%, retention time:
5.250 min.
5.1.8. 3-Cyanophenyl 4-(bis(4-fluorophenyl)methyl)piperazine-1-carboxylate (8)
1H NMR (400 MHz, CDCl3) δ ppm 7.48 (d as m, 1H, J = 8.0 Hz), 7.44 (t, 1H, J = 8.0 Hz); 7.42
(d, 1H, J = 2.0 Hz), 7.36 (d as m, 3H, J = 8.5 Hz), 7.35 (d, 2H, J = 8.5 Hz), 6.99 (t, 7.36 4H, J =
8.5 Hz), 3.69 (brs, 2H); 4.29 (s, 1H), 3.66 (brs, 2H), 3.57 (brs, 2H), 2.43 (t, 4H, J = 5.0 Hz); (ES)
m/z [M+1]: 433.16.
5.1.9. 3-Cyanophenyl 4-(phenyl(pyridin-3-yl)methyl)piperidine-1-carboxylate (9)
1H NMR (400 MHz, CDCl3) δ ppm 8.8-8.7 (m, 1H), 8.55-8.45(m, 1H), 8.1-7.75 (m, 1H), 7.52-
7.28 (m 11H) 4.49 (s, 1H), 3.90-3.58 (m, 4H), 2.72-2.65 (m, 4H); (ES) m/z [M+1]: 398.5; purity:
>98%, retention time: 4.8 min.
5.1.10. 3-Cyanophenyl 4-(9H-fluoren-9-yl)piperazine-1-carboxylate (10)
1H NMR (400 MHz, CDCl3) δ ppm 8.58 (d, 1H J = 4.5 Hz); 7.85 (d, 1H, J = 8.0 Hz); 7.79-7.77
(m as d overlapping with triplets, 2H), 7.65 (d, 1H, J = 7.5 Hz, 2H), 7.41 (t, 2H, J = 7.5 Hz,),
7.34 (t, 2H, J = 7.5 Hz,), 5.02 (s, 1H), 3.58-3.53 (m, 2H), 3.46-3.41 (m, 2H), 2.65-2.55 (m, 4H);
(ES) m/z [M+1]: 396.16.
5.1.11. 3-Cyanophenyl 4-(cyclopentyl(phenyl)methyl)piperazine-1-carboxylate (13)
1H NMR (400 MHz, CDCl3) δ ppm 7.44 (d, 1H, J = 8.0 Hz), 7.41 (t, 2H, J = 8.0 Hz), 7.36 (t, 1H,
J = 2.0 Hz), 7.34 (t, 2H, J = 8.0 Hz) overlapping with (m, 1H), 7.29-7.26 (m, 1H), 7.13 (d, 2H, J
= 8.0 Hz), 3.70-3.40 (m, 4H), 3.25 (d, 1H, J = 7.0 Hz), 2.60-2.45 (m, 3H), 2.40-2.30 (m, 2H),
1.95-1.85 (m, 1H), 1.52-1.48 (m, 6H); (ES) m/z [M+1]: 398.5.
Method B (scheme 2)
5.1.12. tert-Butyl 4-(hydroxydiphenylmethyl)piperidine-1-carboxylate (2, scheme 2)
Step a). To a cold 0 oC solution of 1-(tert-butyl) 4-methyl piperidine-1,4-dicarboxylate 1 (4.86 g,
20 mmol) in THF (100 mL) was added phenyl magnesium bromide (1M solution in THF, 80
mL) over 15 min period. The mixture was stirred at room temperature for 1 hour and then was

quenched with saturated aqueous NH4Cl solution (20 mL). The aqueous layer was extracted with
EtOAc and the combined organic layer was washed with brine, and dried over MgSO4. The
crude material was purified by column chromatography on silica gel to give tert-butyl 4-
(hydroxydiphenylmethyl)piperidine-1-carboxylate 5.6 g, 76% yield as white solid. 1H NMR (400
MHz, CDCl3) δ ppm 7.47(d, 4H, J = 8.0 Hz), 7.31 (t, 4H, J = 8.0 Hz), 7.20 (t, 2H, J = 8.0 Hz),
4.25-4.05 (m, 2H), 2.71 (t, 1H, J = 12.0 Hz), 2.55 (dt, 1H, J = 12.0 Hz, J = 4.0 Hz), 2.12 (s, 1H),
1.55 (d, 2H, J = 12.0 Hz), 1.35 (dq 2H, J = 12.0 Hz, J = 4.0 Hz).
5.1.13. 4-(Diphenylmethylene)piperidinium trifluoro acetate (3, scheme 2)
Step b). A mixture of tert-butyl 4-(hydroxydiphenylmethyl)piperidine-1-carboxylate (5.5g, 15.0
mmol) and TFA (5.8 mL, 75 mmol) in CH2Cl2 (100 mL) was stirred overnight at room
temperature and the mixture was concentrated. The residue was triturated with 10% EtOAc /
hexane and the precipitated salt was filtered and dried to give 5.4 g, 97% yiled of 4-
(diphenylmethylene)piperidinium trifluoro acetate as a white solid. 1H NMR (400 MHz, CDCl3)
δ ppm 8.61 (brs, 1H), 8.34 (brs, 1H), 7.40-7.10 (m, 10H), 3.40-3.35 (m, 4H), Hz, J = 4.0 Hz),
2.55-2.45 (m, 4H)
5.1.14. 4-(Diphenylmethyl)piperidinium trifluoro acetate (4, scheme 2)
Step c). A solution of 4-(diphenylmethylene)piperidinium trifluoroacetate 3 (5.30 g, 14.52
mmol) in methanol was flushed with argon and then 10% palladium on carbon (0.8 g, 15%
wt/wt) was added followed by treatment with hydrogen at atmospheric pressure overnight. The
mixture was filtered over a celite pad and the celite was further washed with MeOH. The
combined organics were concentrated under vacuum to afford 4.98 g, 93% yield of 4-
(diphenylmethyl)piperidinium trifluoro acetate as a white solid. It was used to the next step
1
without further purification. H NMR (400 MHz, CDCl3) δ ppm 8.61 (brs, 1H), 8.34 (brs, 1H),
7.37-7.32 (m, 4H), 7.30-7.25 9m, 4H), 7.17-7.10 (m, 2H), 3.59 (d, 1H, J = 8.0 Hz), 3.25-3.18
(m, 2H), 2.85-2.80 (m, 2H), 2.60-2.48 (m, 1H), 1.54 (d, 2H, J = 12.0 Hz), 1.23 (q, 2H, J = 12.0
Hz)
5.1.15. 3-Cyanophenyl 4-benzhydrylpiperidine-1-carboxylate (2, Table 1)
Step d). To a stirred solution of 3-cyanophenyl chloroformate (Method A, step (d), 0.64 g, 3.5
mmol,) in methylene chloride (50 mL) at room temperature under an argon atmosphere were

added 4-(diphenylmethyl)piperidinium trifluoro acetate (1.53 g, 4.2 mmol) and triethylamine
(0.5 mL, 3.60 mmol). The mixture was stirred at room temperature for 2 hours and then diluted
with water and extracted with methylene chloride (2 x 20 mL). The combined organic layer was
dried with MgSO4. The solvent was removed under reduced pressure to give a crude product,
which was purified by column chromatography on silica gel to afford 0.9 g, 64% yield of 3-
1
cyanophenyl 4-benzhydrylpiperidine-1-carboxylate as white solid. H NMR (400 MHz, CDCl3)
δ ppm 7.47-7.41 (m, 3H), 7.36 (dt, 1H, J = 8.0 Hz, J = 2.0 Hz), 7.32- 7.25 (m, 9H), 7.22-7.15
(m, 2H), 4.20 (d, 2H, J = 13.0 Hz), 3.54(d, 1H J = 6.8 Hz), 2.96 (dt, 2H, J = 13.0 Hz, J = 2.0
Hz), 2.42-2.30 (m, 1H), 1.67 (d, 2H, J = 13.0 Hz), 1.20 (dq, 2H, J = 13.0 Hz, J = 2.0 Hz); (ES)
m/z [M+1]: 397.5; purity: >98%, retention time: 5.30 min.
The following compounds shown in Table 1 were prepared according to Method B.
5.1.16. 3-Cyanophenyl 4-(bis(4-chlorophenyl)methyl)piperidine-1-carboxylate (3)
1H NMR (400 MHz, CDCl3) δ ppm 7.5-7.4(m, 4H), 7.38-7.25(m, 8H), 7.22-7.15 (m, 2H), 4.21
(t, 2H, J = 12.0 Hz), 3.53 (d, 1H, J = 12.0 Hz), 2.97 (t, 1H, J = 12.0 Hz), 2.87 (t, 1H, J = 12.0
Hz), 2.40-2.35 (m, 1H), 1.67 (d, 2H, J = 12.0), 1.25-1.17 (m, 2H); (ES) m/z [M+1]: 466.4; purity:
>96%, retention time: 5.3 min.
5.1.17. 4-Cyanopyridin-2-yl 4-benzhydrylpiperidine-1-carboxylate (1)
1H NMR (400 MHz, CDCl3) δ ppm 8.52 (d, 1H, J = 8.0), 7.38 (d, 2H J = 4), .032-7.27 (m, 8H),
7.22-7.15 (m, 2H), , 1H), 4.22 (t, 2H, J = 12.0 Hz) 3.53 (d, 1H J = 8.0), 3.0, (t, 1H, J = 12.0 Hz),
2.98 (t, 1H J = 12.0 Hz), 2.39-2.36 (m, 1H), 1.75-1.6 (m, 2H), 1.33-1.2 (m, 2H); (ES) m/z [M+1]:
398.57; purity: >96%, retention time: 5.1 min.
5.1.18. 3-Cyanophenyl 4-(benzhydryloxy)piperidine-1-carboxylate (4)
1H NMR (400 MHz, CDCl3) δ ppm 7.49-7.46 (m, 1H), 7.45-7.41 (m, 2H), 7.39-7.33 (m, 8H),
7.32-7.27 (m, 3H), 5.60 (s, 1H), 3.95-3.75 (m, 2H), 3.74-3.68 (m, 1H), 3.55-3.40 (m, 2H), 1.93-
1.87 (m, 2H), 1.84-1.74 (m, 2H); (ES) m/z [M+1]: 413.5; purity: >96%, retention time: 5.9 min.
5.1.19. 5-Cyano-2-fluorophenyl 4-benzhydrylpiperidine-1-carboxylate (16)

1H NMR (400 MHz, CDCl3) δ ppm 7.53 (dd, 1H, J = 7.0 Hz, J = 2.0 Hz), 7.49 (ddd, 1H, J = 7.0
Hz, J = 2.0, J = 2.0), 7.35-7.25 (m, 8H), 7.25- 7.22 (m, 1H), 7.22-7.15 (m, 2H), 4.22 (d, 1H, J =
13.0 Hz), 4.17 (d, 1H, J = 13.0 Hz), 3.54 (d, 1H, J = 7.0 Hz), 3.0 (t, 1H, J = 13.0 Hz), 2.86 (t,
1H, J = 13.0 Hz), 2.40-2.30 (m, 1H), 1.67 (d, 2H, J = 13.0), 1.25 (dq, 2H, J = 13.0 Hz, J = 2.0
Hz); [M+1]: 415.7; purity: >98%, retention time: 5.96 min.
5.1.20. 3-Cyano-5-hydroxyphenyl 4-benzhydrylpiperidine-1-carboxylate (20)
1H NMR (400 MHz, CDCl3) δ ppm 7.35-7.27 (m, 8H), 7.22-7.15 (m, 2H), , 6.89 (s, 1H), 6.89 (s,
1H), 6.79 (s, 1H), 4.19 (d, 2H J = 13.0), 3.56 (d, 1H J = 7.0), 3.05-2.75 (m, 2H), 2.37-2.31 (m,
2H), 1.67 (d, 2H, J = 13.0), 1.30 (dq, 2H, J = 13.0, J = 2.0)
5.1.21. 6-Cyanopyridin-2-yl 4-benzhydrylpiperidine-1-carboxylate (21)
1H NMR (400 MHz, CDCl3) δ ppm 7.9 (t, 1H, J = 8.0), 7.57 (d, 1H J = 4), 7.36 (d, 1H J = 8.0
Hz), 732-7.27 (m, 8H), 7.22-7.15 (m, 2H), 4.22 (t, 2H, J = 12.0 Hz), 3.53 (d, 1H J = 12.0 Hz),
3.0, (t, 1H, J = 12.0 Hz), 2.98 (t, 1H J = 12.0 Hz), 2.39-2.36 (m, 1H), 1.75-1.6 (m, 2H), 1.33-1.2
(m, 2H); (ES) m/z [M+1]: 398.5; purity: >96%, retention time: 5.1 min.
Method C (scheme 3)
5.1.22. tert-Butyl 4-((4-bromophenyl)sulfonyl)piperidine-1-carboxylate (2, scheme 3)
Step a). To a solution of tert-butyl 4-((4-bromophenyl)thio)piperidine-1-carboxylate 1 (2.1 g, 5.6
mmol) in dichloromethane (50 mL) at 0 °C was added of 3-chloroperbenzoic acid (2.1g, 12.4
mmol) and the solution was stirred at room temperature overnight. The solution was then diluted
with water and washed with 10 percent aqueous sodium sulfite solution and saturated aqueous
NaHCO3 solution. The organic layer was dried over magnesium sulfate and the solvent was
removed under reduced pressure to afford 2.1 g, 89% yield of tert-butyl 4-((4-
bromophenyl)sulfonyl)piperidine-1-carboxylate as a white solid. 1H NMR (400 MHz, CDCl3) δ
ppm 7.71 (s, 4H), 4.35-4.25 (m, 2H). 3.10-2.90 (m, 1H), 2.70-2.55 (m, 2H), 1.96 (d, 2H, J = 8.0
Hz), 1.58 (d, 2H, J = 12.0 Hz). 1.43 (s, 9H); (ES) m/z [M+1]: 449.31
5.1.23. 4-((4-Bromophenyl)sulfonyl)piperidinium trifluoroacetate (3, scheme 3)

Step b) A solution of tert-butyl 4-((4-bromophenyl)sulfonyl)piperidine-1-carboxylate (2.0 g, 4.96
mmol) and TFA (1.9 mL, 24.8 mmol) in CH2Cl2 (20 mL) at room temperature was stirred
overnight and then concentrated under vacuum. The residue was triturated with 10% EtOAc /
hexane and filtered and dried to give 1.9 g, 92% yield of 4-((4-bromophenyl)sulfonyl)
1
piperidinium trifluoro acetate as a white solid. H NMR (400 MHz, CDCl3) δ ppm 8.60 (brs,
1H), 8.33 (brs, 1H), 7.82 (d, 2H, J = 8 Hz), 7.78 (d, 2H, J = 8 Hz), 3.46 (d, 2H, J = 8.0 Hz), 2.97
(t, 1H, J = 12.0 Hz), 2.16 9(d, 2H, J = 12.0 Hz), 1.90 (d, 2H, J = 8.0 Hz).
5.1.23. 3-Cyanophenyl 4-(4-bromophenylsulfonyl)piperidine-1-carboxylate (6, Table 1)
To a stirred solution of the of 3-cyanophenyl chloroformate (Method A, step d, 0.10 g, 0.55
mmol)
in CH2Cl2 at room temperature under argon atmosphere were added 4-((4-
bromophenyl)sulfonyl)piperidinium trifluoroacetate (0.27 g, 0.66 mmol) and triethylamine (0.18
g, 1.32 mmol). The resulting mixture was stirred at room temperature for 2 hours, diluted with
water and extracted with CH2Cl2. The combined organic layer was dried over MgSO4, and the
solvent removed under reduced pressure to give a crude material which was purified by column
chromatography on silica gel to give 3-cyanophenyl 4-(4-bromophenylsulfonyl)piperidine-1-
carboxylate 0.19 g, 76% yields as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.72 (s, 4H),
7.55-7.45 (m, 2H), 7.43-7.40 (m, 1H), 7.35 (d, 1H, J = 8.0 Hz), 4.5-4.30 (m, 4H), 3.20-3.12 (m,
1H), 3.10-3.0 (m, 1H), 2.92-2.80 (m, 2H), 2.20-2.05 (m, 2H), 1.90-1.70 (m, 2H).
6. Pharmacology
6.1. Intraocular Pressure (IOP)
6.1.1. Animals: Experiments were conducted at the Indiana University campus. All mice used
for IOP experiments were handled according to the guidelines of the Indiana University animal
care committee. Mice (age 3-8 months) were kept on a 12 h (06:00-18:00) light dark cycle, and
fed ad libitum. Male C57BL/6J (C57) mice were obtained from Charles River Laboratories
International Inc. (Wilmington, MA).
6.1.2. Intraocular pressure measurements: IOP was measured in mice by rebound tonometry,
using a Tonolab device (Icare Finland Oy, Helsinki, Finland). This instrument uses a light

plastic-tipped probe to briefly make contact with the cornea; after the probe encounters the eye
the instrument measures the speed at which the probe rebounds in order to calculate IOP. To
obtain reproducible IOP measurements, mice were anesthetized with isoflurane (3% induction).
The anesthetized mouse was then placed on a platform in a prone position, where anesthesia was
maintained with 2% isoflurane. Baseline IOP measurements are taken in both eyes.
A
‘measurement’ consisted of the average value of six readings. One eye was then treated with
drug (dissolved in Tocrisolve, a soya-based solvent, 5 μL final volume applied topically) while
the other eye was treated with vehicle. The animal was then allowed to recover. After an hour
the animal was again anesthetized as above. IOP was then measured in the drug-treated and
vehicle-treated contralateral eye.
7. Biochemical Pharmacology
7.1. MGL and FAAH Inhibitor Fluorescent Assay: Human-recombinant FAAH (hFAAH) and
MGL (hMGL) were expressed in E. coli and purified as described.37 A high-throughput
fluorometric screening assay for FAAH inhibition using a fluorescent substrate, arachidonoyl 7-
amino-4-methylcoumarin amide (AAMCA), was performed as reported.38 MGL assays use the
fluorescent
substrate
arachidonoyl,
7-hydroxy-6-methoxy-4-methylcoumarin
ester
(AHMMCE).37 IC50 values were calculated using Prism software (GraphPad).
7.2. rCB1, hCB2, and mCB2 Binding Assay: Selected compounds were tested for their ability
to bind to CB1 and CB2 receptors using rat brain27 or HEK293 cell membranes expressing
mouse CB2 (mCB2) or human CB2 (hCB2),28-30 respectively, as described via competition-
equilibrium binding using [3H]CP-55,940. 29-31, 39
7.3. Stability in plasma and buffer: Compound solutions (200 M) were made in mouse, rat or
human plasma, buffer containing 0.1% BSA or artificial gastric juice. After quenching with
acetonitrile, the samples were analyzed by HPLC to predict in vivo plasma half-lives.40, 41
7.4. Stability towards rat liver microsomal preparations: Compounds were pre-incubated
with rat liver microsomal protein (Celsis) at 37C before the reaction is initiated with NADPH or

buffer (control).42, 43 Following protein precipitation, the samples will be analyzed using a LC-
MS/MS in SRM mode.
7.5. Pharmacokinetic study: CD-1 mice treated iv 2 mg/kg and po 10 mg/kg followed by blood
collection in multiple time points, sacrificed by decapitation for tissue dissection and analysis
following published procedures.44-46
8. Computational Studies
8.1. Methods. The computational studies were conducted in Schrodinger Suite (Version 2017-2).
The prepared ligands were docked into the AM4301 defined binding pocket using Glide module.
The docking process was conducted with XP (extra precision) mode in OPLS3 force field47.
MM-GBSA rescoring was conducted using Prime module with the implicit membrane and
relaxation of residues within 5 Å from the ligand.
8.1. Computational (In silico) Analysis of Physicochemical Properties of Ligands: In silico
ligand screening with commercial computational software (Schrodinger Suite 2017-2) used to
assess early ADME/pharmacokinetic properties of the proposed compounds in areas as Lipinski
rules, ligand efficiency, topological polar surface area (tPSA), cytochrome P450 stability profile,
pKa, aqueous solubility, and lipophilicity (ClogP).
Funding: This work was supported by grants from the National Institute of Health (R01
DA03801 (AM), EY024625 (AS).
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Figure 1. Representative carbamate MGL inhibitors
Figure 2. MGL carbamates
Figure 3. Schematic docking of compound 2 at MGL’s active site
Figure 4. Compound 2 covalently modified catalytic Ser122 and induced perturbations on the
low-field region of the 1D 1H NMR spectrum of hMGL. Upon carbamylation catalytic His269
shifted upfield (arrow indicator) and as adduct hydrolyzed it fully recovered to the original
position.
Figure 5. MGL inhibitor 2 lowers ocular pressure for at least 12 hours with a single topical
treatment in a normotensive murine model. In normotensive mice MGL inhibitor 2 applied
topically at 3 mM substantially lowered pressure at 4, 8 and 12 hours (n=8) relative to vehicle-
treated mice. Effect dissipated after 28 hours. **, p<0.01, ***, p<0.001 1 way ANOVA with
Sidak’s posthoc vs. corresponding time point.

Scheme 1. Reagents: (a) R2 = alkyl, cycloalkyl, or aryl-MgBr, THF; (b) SOCl2, CH2Cl2; (c)
piperazine, EtOH; (d) (COCl3)2CO, DIEA, CH2Cl2; (e) piperazine 4, Et3N, CH2Cl2.
Scheme 2. Reagents: (a) PhMgBr, THF; (b) TFA, CH2Cl2; (c) H2, Pd/C, MeOH; (d) 6 (scheme
1), Et3N, CH2Cl2; (e) p-TSA, 4-hydroxypiperidine, PhCH3
Scheme 3. Reagent: (a) m-CPBA, CH2Cl2; (b) TFA, CH2Cl2; (c) 6 (scheme 1), Et3N; (d)
piperazine, CH2Cl2.

Graphical Abstract
Docking of carbamate inhibitors at MGL’s active site