An effective prodrug strategy to selectively enhance ocular exposure of a cannabinoid receptor (CB1/2) agonist

Article abstract of DOI:10.1021/jm4004939

Glaucoma is a leading cause of vision loss and blindness, with increased intraocular pressure (IOP) a prominent risk factor. IOP can be efficaciously reduced by administration of topical agents. However, the repertoire of approved IOP-lowering drug classes is limited, and effective new alternatives are needed. Agonism of the cannabinoid receptors CB_1/2 significantly reduces IOP clinically and experimentally. However, development of CB _1/2 agonists has been complicated by the need to avoid cardiovascular and psychotropic side effects. 1 is a potent CB_1/2 agonist that is highly excluded from the brain. In a phase I study, compound 1 eyedrops were well tolerated and generated an IOP-lowering trend but were limited in dose and exposure due to poor solubility and ocular absorption. Here we present an innovative strategy to rapidly identify compound 1 prodrugs that are efficiently metabolized to the parent compound for improved solubility and ocular permeability while maintaining low systemic exposures.

Full text of DOI:10.1021/jm4004939

Article
pubs.acs.org/jmc
An Effective Prodrug Strategy to Selectively Enhance Ocular
Exposure of a Cannabinoid Receptor (CB1/2) Agonist
Nello Mainolfi,*,† James Powers,† Jakal Amin,‡ Debby Long,§ Wendy Lee,‡ Margaret E. McLaughlin,§
Bruce Jaffee,§ Christopher Brain,† Jason Elliott,† and Jeremy M. Sivak*,§,∥
†Novartis Institutes for Biomedical Research, Global Discovery Chemistry, 100 Technology Square, Cambridge, Massachusetts 02139,
United States
‡Novartis Institutes for Biomedical Research, Metabolism and Pharmacokinetics, 250 Massachusetts Avenue, Cambridge,
Massachusetts 02139, United States
§Novartis Institutes for Biomedical Research, Ophthalmology, 500 Technology Square, Cambridge, Massachusetts 02139, United
States
S
* Supporting Information
ABSTRACT: Glaucoma is a leading cause of vision loss and blindness, with increased intraocular pressure (IOP) a prominent
risk factor. IOP can be efficaciously reduced by administration of topical agents. However, the repertoire of approved IOP-
lowering drug classes is limited, and effective new alternatives are needed. Agonism of the cannabinoid receptors CB1/2
significantly reduces IOP clinically and experimentally. However, development of CB1/2 agonists has been complicated by the
need to avoid cardiovascular and psychotropic side effects. 1 is a potent CB1/2 agonist that is highly excluded from the brain. In a
phase I study, compound 1 eyedrops were well tolerated and generated an IOP-lowering trend but were limited in dose and
exposure due to poor solubility and ocular absorption. Here we present an innovative strategy to rapidly identify compound 1
prodrugs that are efficiently metabolized to the parent compound for improved solubility and ocular permeability while
maintaining low systemic exposures.
and new and effective alternatives are badly needed to postpone
having to resort to surgical options.
INTRODUCTION
■
Glaucoma is a leading cause of vision loss and blindness,
afflicting more than 60 million people worldwide and rapidly
increasing in prevalence.1,2 The disease is an irreversible
neurodegenerative process, characterized by thinning of the
retinal nerve fiber layer, optic nerve head cupping, and death of
retinal ganglion cells, leading to progressive visual field defects.
A complex combination of risk factors is associated with
glaucoma development and progression, but the most
prominent is increased intraocular pressure (IOP). Primary
open angle glaucoma (POAG), the most common form, is
often asymptomatic during its early stages. While the cause of
increased IOP in POAG remains unclear, substantial and
sustained IOP reduction has been demonstrated to significantly
slow disease progression.3,4 Unfortunately, the repertoire of
approved IOP-lowering drugs remains limited to a few classes,
The G protein-coupled cannabinoid receptors, CB1 and CB2,
are the primary targets of endogenous endocanabinoids, and Δ-
9 tetrahydrocannabinol (THC), one of the several active
ingredients of marijuana (Cannabis sativa). CB1 receptors are
strongly expressed in brain and appear to play an important
role mediating the psychotropic effects of THC. CB2 receptors
are found in a variety of immune cells, including microglia, and
agonists of both receptors are under development for a variety
of indications, including neurodegenerative disease, pain,
nausea, and osteoporosis.5,6 Interest in use of CB1/2 agonists
for treatment of glaucoma stems from studies in the 1970s and
1980s, reporting that smoking marijuana significantly lowers
Received: April 5, 2013
© XXXX American Chemical Society
A
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IOP.7−9 Subsequently, the synthetic CB1/2 agonist, WIN
55,212-2, was shown to significantly reduce IOP in
normotensive rabbits, ocular hypertensive rats and cynomolgus
monkeys, and clinically after a single topical dose.10−13
Additional studies have demonstrated local expression of both
cannabinoid receptors in the eye9,14 and increased aqueous
outflow after exposure to the CB2 agonist JWH015.15 Although
these data are promising, an important concern for the clinical
potential of this class of drugs has been cardiovascular and
psychotropic side effects mediated through systemic and brain
cannabinoid receptor activation. For example, WIN 55,212-2 is
highly CNS penetrant, with a brain to plasma ratio of 1.3−1.9
compared to 1.0 for THC.16 Additionally, the duration of
action for marijuana is only 3−4 h, making distribution and
tissue residency important considerations for any new
therapeutic in this class.
pharmaceutical, and/or pharmacokinetic properties of bio-
logically active compounds delivered systemically21 and, more
recently, also administered topically to the eye.21,22 Ester
prodrugs have been successfully developed in the context of
ocular drug delivery, enabled by the presence of endogenous
esterase activity.21−23 However, use of elaborate permeability
models, Ussing chambers, and large animal studies can make
screening of potential prodrug modifications a difficult and
labor-intensive prospect. We present here an innovative
strategy to rapidly screen potential compound 1 prodrugs
that are efficiently metabolized to the parent compound for
improved solubility and permeability in the eye, while
maintaining low systemic exposures.
CHEMISTRY
■
Compound 1’s pending primary alcohol allowed for a
straightforward synthesis of a diverse set of ester prodrugs.
The main objective of the prodrug design campaign was to
improve parent drug’s solubility (by adding polar groups),
permeability (by adding lipophilic groups, given the lip-
ophilicity of the corneal epithelium), and/or take advantage
of active transport systems to improve ocular bioavailabil-
ity.24,25 In the case of carboxylic esters, the reaction involved
simple EDC mediated coupling. Some esters bearing Boc-
protected amines were subsequently deprotected using TFA
(Scheme 1). The chemistry was performed in a high-
throughput lab, and the final set of compounds is shown in
Table 1.
1 is a potent CB1/2 agonist quinazolinone derivative, with
17−19
IC50s of 94 nM vs CB1 and 3.5 nM vs CB2.
(Figure 1).
In the case of phosphonate ester prodrug 2, the chemistry
was performed with a two-step sequence that involved a
condensation of the acid dibenzyl diisopropylphosphoramidite
1, followed by phosphorus oxidation and a simple hydro-
genolysis of the benzyl protecting groups (Scheme 2).
Figure 1. The preferentially peripherally distributed CB1/2 agonist
from Novartis: 1.
This polar compound (PSA = 166, cLogP = 2.29) is highly
excluded from the brain, with a brain to plasma Cmax ratio of
only 0.02.17 A phase I clinical study was completed in patients
with ocular hypertension, dosing a 0.02% solution of
compound 1 as either a single drop, or a series of four drops
every hour (clinicaltrials.gov I.D. NCT00503360). The drug
was well tolerated, and both treatment groups reduced IOP by
2.3 and 2.4 mmHg, respectively, although these trends did not
reach statistical significance (p = 0.1 and 0.09) (http://www.
novctrd.com, and ref 20). While encouraging, these results
might be dramatically improved if ocular absorption and
residence time were substantially increased.
RESULTS
■
A Rapid Prodrug Screening Strategy to Improve
Anterior Segment Drug Delivery. We designed a stream-
lined strategy to test the series of putative prodrugs synthesized
as chemical modifications of 1. Our simplified scheme is
presented in Figure 2.
As mentioned earlier, the selection of 45 esters for the
formation of corresponding prodrugs of 1 were guided by the
goal of improving aqueous solubility (ability to formulate the
compound at higher concentration than in the original clinical
formulation of 0.02%) (i.e., basic amine containing esters such
as: 5, 6, 7, 8, and 12, etc.), improve intrinsic permeability (i.e.,
lipophilic esters such as: 3, 4, 23, 25, and 28), and explore the
Prodrugs are chemically modified derivatives of pharmaco-
logically active compounds that undergo in vivo trans-
formations to free the active parent. Their development is a
well-established strategy to improve physicochemical, bio-
a
Scheme 1. General Scheme for the Synthesis of Carboxylic Ester Prodrugs of 1
a
Reagents and conditions: (a) EDC (1.35 equiv), DMAP (0.1 equiv), ester (1.5 equiv), DCM (0.1 M), rt, overnight; some ester prodrugs contained
boc-protected amines, which were subsequently deprotected using TFA (10 equiv) in DCM (0.1 M) at rt.
B
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Table 1. Potential Prodrugs of 1 Synthesized Using Conditions in Scheme 1
a
Scheme 2. Synthesis of Compound 1 Phosphonate Ester Prodrug 2
a
Reagents and conditions: (a) 1a (5 equiv), tetrazole (5 equiv), m-CPBA (5 equiv), DCM (0.1 M), rt, overnight; (b) H2, Pd/black (cat.), NaHCO3
(4 equiv), t-BuOH/H2O, rt, overnight.
possibility to identify and exploit active-transport mechanism
(amino acid and/or peptide containing esters such as: 42, 43,
and 44). For prodrugs, resistance to chemical hydrolysis is
necessary for formulation, but rapid enzymatic hydrolysis to
C
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systemic circulation will be rapidly removed.23 Therefore, a
plasma stability assay and a chemical stability assay were
performed on each compound in order to prioritize those that
were chemically stable with improved solubility and which were
rapidly converted to 1 (Table 2).
Of all the potential prodrugs analyzed, only 15 out of 45 were
stable for up to 24 h. Of those, only 11 (2, 9, 12, 16, 24, 32, 36,
40, 44, 45, and 46) showed improved solubility over the one of
compound 1 and a plasma stability profile that predicted rapid
enzymatic cleavage of the ester prodrug to ensure efficacy in
ocular tissues and safety in plasma. Given the similarity between
prodrugs 45 and 46, only one of them (46) was selected for
further studies in vivo. Prodrug 2 showed a suboptimal plasma
stability profile (long half-life), but was included in the subset
of compounds for in vivo PK testing, because it represented the
only example of a different class of potential prodrugs.
Ocular Dosing of 0.02% Compound 1 Solution Is
Poorly Absorbed into the Anterior Chamber and
Rapidly Cleared. To establish a baseline model for
comparison with putative prodrugs, a solution of 0.02% of 1
was administered topically as a single dose to each eye of
pigmented Brown Norway rats. The formulation used for this
initial study was the same as that used for the clinical trial. Dose
volume was determined based on scaling from an estimated 50
μL drop in humans to 4 μL for the rat eye based on average
surface area. Three animals were dosed for each time point,
collected at 0 (untreated control), 0.083, 0.25, 0.5, 1.0, 3.0, 6.0,
and 24 h (0, 5, 15, 30, 60, 180, 360, and 1440 min). At each
time point, cornea, iris and ciliary body, aqueous humor, and
plasma were analyzed for concentrations of 1. Additionally, the
PBS rinse used to briefly wash the eyes after enucleation was
Figure 2. Screening strategy to identify prodrugs of 1 with improved
ocular exposure.
parent compound is desirable, as it suggests enzymatic cleavage
will occur in the eye and ensure that any prodrug which reaches
a
Table 2. Plasma Stability, Chemical Stability (in Vehicle), Solubility Screening, and cLogP
a
Selected compounds in bold.
D
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analyzed in order to control for potential contamination of
tissue dissections from eyedrops. Compound 1 was detected
quickly in all tissues, with a Tmax of 5 min, and an average Cmax
of 82 nM in aqueous humor, 50 nM in cornea, 18 nM in iris
and ciliary body, and 9 nM in plasma. However, drug
concentrations also rapidly decreased to below the lowest
level of quantitation (LLOQ) of 1 nM, generating correspond-
ing low tissue exposures (Figure 3, Table 3).
and ciliary body were collected from each animal, with tissue
from both eyes pooled for analyses. One advantage of this
strategy is that only concentrations of 1 were evaluated,
indicating successful prodrug conversion and greatly simplifying
the analytical effort required. Results from these experiments
are presented in summary form in Table 4, showing AUC’s of 1
in iris and ciliary body and plasma over the critical first three
hours.
Table 4. Summary of Compound 1 Prodrug Abbreviated
a
Ocular PK Studies
compd
AUCICB‑3h (nMh)
AUCplasma‑3h (nMh)
1
BQL
17.0
14
2
10.0
3.9
9
17.4
12
16
24
32
36
40
44
46
BQL
36.7
BQL
11.8
BQL
BQL
BQL
3.6
321.0
BQL
213.0
BQL
40.0
20.0
BQL
Figure 3. Compound 1 is quickly cleared from anterior segment ocular
tissues. A 0.02% clinical formulation of compound 1 was administered
as a single eyedrop to Brown Norway rats in a composite PK study (n
= 3 animals/time point, bars represent SE). Cornea, aqueous humor,
iris/ciliary body, and plasma were collected for analyses over a 24 h
time-course. The PBS rinse following enucleation was also analyzed to
control for potential tissue contamination from the eyedrop. Drug
concentrations were below the lowest level of detection (1 nM) in all
tissues after 3 h, with the iris/ciliary body showing the most rapid
decline. Plasma and PBS rinse remained substantially below ocular
tissue concentrations throughout.
104.0
a
AUC, area under the curve; ICB, iris and ciliary body; BQL, below
quantifiable levels.
All the compounds showed detectable levels of 1 in the eye,
demonstrating that they were functioning as prodrugs. By
comparison, 1 dosed at the same 0.1% concentration resulted in
low or undetectable exposures in the eye. Of the prodrugs, two
compounds in particular, 24 and 36, showed particular promise.
At 3 h, administration of each prodrug resulted in dramatically
improved concentrations of 1 in iris/ciliary body and aqueous
humor compared to 1 itself but maintained low or undetectable
concentrations in plasma (Table 4, Figure 4).
Table 3. PK Parameters of 0.02% Compound 1 Eyedrops
aqueous
parameter
humor
cornea
43
iris
plasma
CONCLUSIONS
■
*AUC(0−last)
[nm·h]
50
not calculated not calculated
A recent position statement from the American Glaucoma
Society on marijuana use and the treatment of glaucoma
highlighted the potential clinical benefits and challenges
associated with targeting the cannabinoid system.26 Our goals
for this project were to increase local exposure of the CB1/2
agonist, 1, by improving solubility and permeability of the
dosed prodrug formulation, without increasing plasma
concentrations of the prodrug or parent compound. Through
a streamlined series of high-throughput and simple chemistry,
in vitro stability and solubility assays, and abbreviated in vivo
PK studies, we have identified two compounds, 24 and 36, with
properties amenable to further study. Each of these compounds
functions as a prodrug, with improved solubility and ocular
permeability, producing dramatically increased exposure of 1 in
the iris and ciliary body and rapid hydrolization in plasma.
The design and development of prodrugs has been
previously used an effective strategy for improving ocular
pharmacokinetics, primarily capitalizing on corneal expression
of esterases,27 to catalyze metabolic conversion to active drug.23
Prodrugs have therefore been designed for a variety of
approved and experimental topical ophthalmic drugs, including:
the β-adrenergic antagonist timolol,28 pilocarpine,29,30 etha-
crynic acid,31 antivirals gancyclovir and acyclovir,32,33 and
prostaglandin receptor agonists.34 However, the process that
has been traditionally used for screening and assessment of
Cmax [nM]
Tmax [h]
82
50
18
9
0.083
0.083 0.083
0.083
No compound was detected beyond 3 h, and concentrations
in the iris and ciliary body, representing target anterior chamber
tissues, were undetectable after 1 h. Plasma and PBS rinse
concentrations were substantially lower than ocular tissue levels
at every time point. This ocular pharmacokinetic profile is
consistent with clinical data, particularly the rapid clearance of
drug from the iris and ciliary body. Therefore, this tissue was
used as a baseline to test prodrugs for hydrolysis and improved
exposure of 1 in the eye.
In Vivo PK Testing Identified Two Prodrugs of 1 with
Improved Properties. On the basis of prioritization of in
vitro stability and solubility data shown above, 10 compounds
were promoted for further testing in vivo (bolded compounds
in Table 2). Because of the composite, labor-intensive, nature
of ocular pharmacokinetic studies, an abbreviated design was
adopted for prodrug screening. In this case, the dose was
increased to a 0.1% solution, as all chosen compounds
displayed improved solubility compared to 1. Each animal
was given a single dose of 4 μL of formulation to each eye, and
each study consisted of time points at 15, 60, and 180 min
(0.25, 1, and 3 h), with three animals per group. Plasma and iris
E
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The development of novel glaucoma drug classes has been
challenged by a combination of ocular barriers and delivery
hurdles, including rapid loss through drainage of tear film and
lipophilic corneal and scleral epithelia.39 For CB1/2 agonists, an
additional challenge is the maintenance of very low systemic
and brain absorption. Alternative strategies to improve anterior
segment exposure include formulation of sustained delivery
depots, extensive reformulation, and addition of permeability
enhancers. In the case of 1, reformulation would have been
challenging due to poor aqueous solubility and corneal
permeability, yet the compound was clinically efficacious and
displayed a critical low brain:plasma ratio. The present prodrug
strategy simultaneously addressed solubility and permeability
without changing the active compound.
One important benefit to this approach is to minimize the
initial characterization necessary for each new derivative, as the
desired active compound remains identical. The molecules
identified from this work can now be focused on for
formulation at higher concentrations than 1 and promoted
into advanced safety and efficacy studies such as administration
to normotensive and ocular hypertensive animal models. Also,
as CB1/2 agonists have been noted for potent neuroprotective
effects, it will be of great interest to assess distribution of these
prodrugs to the posterior of the eye as well.
EXPERIMENTAL SECTION
■
General Information. All reagents and solvents were used as
supplied. All carboxylic ester formation reactions (apart from 45 and
46) were performed in a high-throughput lab in a combinatorial
chemistry fashion using commercially available carboxylic acid starting
material. The isolated yields were in the range of 10−90%. Only data
of the 10 compounds selected for in vivo pk studies are reported
Figure 4. Identification of two prodrugs of 1 with improved exposures
in iris and ciliary body. Results are shown for 0.1% 1 and two prodrugs,
24 and 36, which were dosed as part of an abbreviated ocular PK study
over 3 h and analyzed for 1. (A) Concentrations of 1 were significantly
increased in the iris and ciliary body when 24 and 36 were
administered, as compared to dosing of 1 alone. Of particular note
is the 3 h time point, where levels of 1 where below detection after
dosing parent compound alone. (B) In contrast, plasma levels of 1
remained low after administration of all three compounds (n = 3
animals/time point, bars represent SE).
1
below. H NMR spectra were recorded using an internal deuterium
lock at ambient temperature on a Varian 400 MHz spectrometer. Data
are presented as follows: chemical shift (in ppm on the δ scale
relatively to δTMS = 0), multiplicity (s = singlet, d = doublet, t =
triplet, q = quartet, quin = quintuplet, m = multiplet, br = broad, dd =
doublet of doublet, dt = doublet of triplet, dq = doublet of quartet),
coupling constant (J/Hz), and integration. Mass spectra were recorded
using an Agilent 6220 mass spectrometer with electrospray ionization
source and Agilent 1200 liquid chromatograph. The resolution of the
HRMS system was approximately 11000 (fwhm definition). The
semipreparative HPLC used in purification adopted a C18 column and
prodrug candidates has been time and labor intensive. In this
regard, development of accurate and consistent in vitro ocular
permeability models remains a key goal.35,36 Yet, no such assay
has been established and fully validated. In its absence, many
investigations rely on corneal or scleral permeability modeling,
using excised rabbit, bovine, or human tissues (e.g., refs
32,37,38). While providing a powerful experimental tool, these
ex vivo models can be difficult to assemble, are dependent on
tissue availability, and are largely restricted to passive processes.
Likewise, in vivo pharmacokinetic studies tend to be costly and
involve composite data sets from numerous animals.
We have presented here a novel strategy for rapid screening
of prodrugs for improvement of ocular delivery of compounds
to the anterior segment. To accomplish this, we capitalized on a
combination of in vitro and abbreviated in vivo modeling and
have subsequently shown that plasma T1/2, aqueous solubility,
and chemical stability provide a successful testing cascade to
prioritize compounds for in vivo prodrug studies. We have
further developed a rat ocular PK screening model that can be
modified to evaluate compounds for any anterior segment
target or disease. This methodology has potential to become a
great asset for current and future efforts in topical delivery of
small molecules to the eye.
1
a mobile phase of CH3CN/H20 (0.01% TFA). H NMR and mass
spectra (LC and HRMS) were used to determine purity of the
compounds. All compounds used for in vivo studies had purity ≥95%.
Experimental Procedures. Ethyl 5,7-Dimethyl-3-(2-(N-
methylsulfamoyl)phenyl)-4-oxo-2-((((2-(phosphonooxy)ethyl)-
carbamoyl)oxy)methyl)-3,4-dihydroquinazoline-6-carboxylate (2).
To a solution of dibenzyl diisopropylphosphoramidite (973 mg, 2.82
mmol, 5 equiv) in DCM (10 mL), 2H-tetrazole (197 mg, 2.82 mmol, 5
equiv) was added and the reaction allowed to stir for 10 min at rt. 1
(300 mg, 0.563 mmol) was then added, and the reaction allowed to
stir overnight. At this point, m-CPBA (694 mg, 2.82 mmol, 5 equiv)
was added and the reaction stirred for 4 h at room temperature. The
reaction was evaporated, and the crude product was purified by
semipreparative HPLC. The collected fractions were evaporated, and
the product (300 mg, 0.378 mmol) was dissolved in t-butanol (6 mL)/
water (1 mL). NaHCO3 (127 mg, 1.514 mmol, 4 equiv) and palladium
black (500 mg, 4.70 mmol, 160% w/w) were added and the flask
flushed 3 times with a balloon of H2 and then stirred for 4 h under the
same atmosphere of H2. The organic layer was removed by filtration,
the dark residue washed with water (20 mL), and the collected
aqueous layer lyophilized to give the pure product as a white solid
(200 mg, 0.326 mmol, 58%). 1H NMR gave extremely broad and
noninterpretable peaks (see Supporting Information). MS (ESI) m/z
613.1 (M + 1).
F
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Ethyl 2-((2-(2-Aminoacetoxy)ethylcarbamoyloxy)methyl)-5,7-di-
methyl-3-(2-(N-methylsulfamoyl)phenyl)-4-oxo-3,4-dihydroquina-
zoline-6-carboxylate (9). 1 (0.25 g, 0.478 mmol), 2-(tert-
butoxycarbonylamino)acetic acid (0.135 g, 0.777 mmol, 1.6 equiv),
and DMAP (0.005 g, 0.041 mmol, 0.1 equiv) were placed in DCM (10
mL) and cooled to 0 °C, EDC (0.174 g, 0.91 mmol, 1.9 equiv) was
added, and the reaction was stirred at room temperature overnight.
The reaction mixture was then washed with 1 N HCl, dried, and
concentrated to an oil.
Ethyl 5,7-Dimethyl-3-(2-(N-methylsulfamoyl)phenyl)-2-((2-
(nicotinoyloxy)ethylcarbamoyloxy)methyl)-4-oxo-3,4-dihydroqui-
nazoline-6-carboxylate (36). The title compound was synthesized
using the same procedure as for prodrug (32). White solid (110 mg,
0.172 mmol, 30% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.11
(d, J = 2.02 Hz, 1 H) 8.75−8.77 (m, 1H) 8.26 (dd, J = 7.83, 2.02 Hz, 1
H) 8.00 (d, J = 7.83 Hz, 1 H) 7.61−7.83 (m, 3 H) 7.48−7.51 (m, 1 H)
7.29 (s, 1 H) 4.68 (d, J = 14 Hz, 1 H) 4.35−4.44 (m, 3 H) 4.27 (br s, 2
H) 3.37 (br s, 2 H) 2.59 (s, 3 H) 2.42 (d, J = 3.28 Hz, 3 H) 2.30 (s, 3
H) 1.33 (t, J = 7.07 Hz, 3 H). MS (ESI) m/z 638.0 (M + 1).
(R)-Ethyl 2-((2-(2-Amino-2-(2-chlorophenyl)acetoxy)-
e t h y l c a r b a m o y l o x y ) m e t h y l ) - 5 , 7 - d i m e t h y l - 3 - ( 2 - ( N -
methylsulfamoyl)phenyl)-4-oxo-3,4-dihydroquinazoline-6-carboxy-
late (40). The title compound was synthesized using the same
procedure as for prodrug (9). White solid (60 mg, 0.08 mmol, 15%
yield). MS (ESI) m/z 700.14, 703.1 (M + 1).
The oil was dissolved in 5 mL of DCM and treated with 2 mL of
TFA at 0 °C. The reaction was complete after 2 h. The reaction was
concentrated and then dissolved in 2 mL of MeOH and purified by
preparative HPLC to obtain the title compound as a white solid (49
1
mg, 0.083 mmol, 17%). H NMR (400 MHz, DMSO-d6) δ ppm 8.19
(br s, 2 H) 8.02 (dd, J = 7.83, 1.52 Hz, 1 H) 7.77−7.87 (m, 2 H)
7.66−7.68 (m, 1 H) 7.61 (d, J = 7.33 Hz, 1 H) 7.54 (s, 1 H) 7.43 (s, 1
H) 4.69 (d, J = 14 Hz, 1 H) 4.36−4.47 (m, 3 H) 4.14−4.16 (s, 2 H)
3.80 (br s, 2 H) 3.25 (br s, 2 H) 2.62 (s, 3 H) 2.43 (d, J = 4.80 Hz, 3
H) 2.37 (s, 3 H) 1.33 (t, J = 7.07 Hz, 3 H). MS (ESI) m/z 590.0 (M +
1).
Ethyl 2-((2-(3-Aminopropanoyloxy)ethylcarbamoyloxy)methyl)-
5,7-dimethyl-3-(2-(N-methylsulfamoyl)phenyl)-4-oxo-3,4-dihydro-
quinazoline-6-carboxylate (12). The title compound was synthesized
using the same procedure as for prodrug (9). White solid (110 mg,
0.182 mmol, 50% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.02
(dd, J = 7.83, 1.52 Hz, 1 H) 7.76−7.86 (m, 2 H) 7.65−7.73 (m, 3 H)
7.61 (dd, J = 7.58, 1.26 Hz, 1 H) 7.55 (m, 1 H) 7.42 (s, 1 H) 4.68 (d, J
= 14 Hz, 1 H) 4.36−4.47 (m, 3 H) 4.02−4.08 (m, 2 H) 3.21−3.23 (m,
2 H) 3.01−3.04 (m, 2 H) 2.61−2.68 (m, 5 H) 2.43 (d, J = 4.80 Hz, 3
H) 2.37 (s, 3 H) 1.33 (t, J = 7.07 Hz, 3 H). MS (ESI) m/z 604.1 (M +
1).
Ethyl 5,7-Dimethyl-3-(2-(N-methylsulfamoyl)phenyl)-4-oxo-2-((2-
(piperidine-2-carbonyloxy)ethylcarbamoyloxy)methyl)-3,4-dihydro-
quinazoline-6-carboxylate (16). The title compound was synthesized
using the same procedure as for prodrug (9). White solid (150 mg,
0.23 mmol, 58% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.01−
9.03 (br s, 1 H), 8.87−8.89 (br s, 1 H), 8.02 (dd, J = 7.71, 1.64 Hz, 1
H) 7.77−7.87 (m, 2 H) 7.65−7.67 (m, 1 H) 7.60−7.62 (m, 1 H)
7.54−7.58 (m, 1 H) 7.42 (s, 1 H) 4.67 (d, J = 14 Hz, 1 H) 4.36−4.48
(m, 3 H) 4.18−4.25 (m, 1 H) 4.0−4.15 (m, 2 H) 3.24−3.26 (m, 3 H)
3.28−3.32 (m, 1 H) 2.61 (s, 3 H) 2.42 (d, J = 4.80 Hz, 3 H) 2.37 (s, 3
H) 2.01−2.04 (m, 1 H) 1.31−1.68 (m, 5 H) 1.33 (t, J = 7.20 Hz, 3 H).
MS (ESI) m/z 644.1 (M + 1).
(S)-Ethyl 2-((((2-((2-Amino-3-methylbutanoyl)oxy)ethyl)-
carbamoyl)oxy)methyl)-5,7-dimethyl-3-(2-(N-methylsulfamoyl)-
phenyl)-4-oxo-3,4-dihydroquinazoline-6-carboxylate (44). The title
compound was synthesized using the same procedure as for prodrug
(9). White solid (120 mg, 0.19 mmol, 20% yield). HRMS calcd for
C29H37N5O9S M+ 632.2390, found 632.2383.
(9R,12S)-Ethyl 2-(12-Amino-9-isopropyl-13-methyl-3,8,11-trioxo-
2,7-dioxa-4,10-diazatetradecyl)-5,7-dimethyl-3-(2-(N-
methylsulfamoyl)phenyl)-4-oxo-3,4-dihydroquinazoline-6-carboxy-
late (46). To a solution of 1 (300 mg, 0.563 mmol) in DCM (5 mL),
EDC (162 mg, 0.845 mmol, 1.5 equiv), D-Boc-valine (184 mg, 0.845
mmol, 1.5 equiv), and DMAP (13.76 mg, 0.113 mmol, 0.2 equiv) were
added at 0 °C. The reaction was allowed to reach rt and stirred for 2 h.
LCMS showed complete conversion so a solution of 1 N HCl was
added, and the product was extracted with EtOAc. The crude product
was dried and evaporated and then dissolved in DCM (10 mL), and
then TFA (10 mL, 130 mmol) was added. After 30 min, the organic
solvents were evaporated and the compound dried in vacuo. At this
point, the crude product was dissolved in DMF (5 mL), DIPEA (0.197
mL, 1.127 mmol, 2 equiv), and L-Boc-valine (147 mg, 0.676 mmol, 1.2
equiv), HATU (257 mg, 0.676 mmol, 1.2 equiv), and HOAt (92 mg,
0.676 mmol, 1.2 equiv) were added. After 2 h, the reaction was
complete and the mixture was diluted with EtOAc, washed with 1N
HCl, and extracted with EtOAc to give the crude product. It was then
again dissolved in DCM/TFA and stirred for 30 min, the solvent
mixture was then evaporated, and the crude product was purified by
semipreparative HPLC to give the title compound as a white solid
(0.252 g, 0.345 mmol, 61%). 1H NMR (400 MHz, DMSO-d6) δ ppm
8.72−8.74 (m, 1 H) 8.10−8.14 (m, 1 H) 8.02 (dd, J = 7.71, 1.39 Hz, 1
H) 7.77−7.87 (m, 2 H) 7.56−7.69 (m, 3 H) 7.43 (s, 1 H) 4.67 (dd, J
= 14.27, 3.16 Hz, 1 H) 4.36−4.47 (m, 3 H) 4.30−4.32 (m, 1 H) 3.85−
4.07 (s, 2 H− under water peak) 3.76 (br s, 1 H) 3.22 (br s, 2 H) 2.61
(s, 3 H) 2.42 (d, J = 1.26 Hz, 3 H) 2.37 (s, 3 H) 2.09−2.20 (m, 2 H)
1.33 (t, J = 7.07 Hz, 3 H) 0.84−0.97 (m, 12 H). MS (ESI) m/z 731.2
(M + 1).
Prodrugs 3, 4, 5, 6, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 34,
and 35 were synthesized using the same procedures as outlined for 32.
Prodrugs 7, 8, 10, 11, 13, 14, 15, 17, 18, 19, 37, 38, 39, 41, 42, and
43 were synthesized using the same procedures as outlined for 9.
In Vitro Plasma Stability Assay. First, 10 μL of a prodrug was
added to 990 μL of pooled plasma to a 1 μM final concentration in 2
mL microcentrifuge tubes. Tubes were immediately placed in a water
bath at 37 °C with samples removed at 0, 0.083, 0.25, 0.5, 1, and 1.5 h
(n = 3/time point). Then 25 μL aliquots were removed at each
stability time points, and they were immediately protein precipitated
using cold acetonitrile having 100 ng/mL of internal standard
(glyburide). The amount of 1 formed was determined by quantitating
the unknown levels using the compound 1 plasma calibration standard
curve. The analysis was performed on API-4000 LC/MS/MS using
multiple reaction monitoring (MRM) mode. Prodrug T1/2 was
determined in the same analytical run using the peak area ratio of
prodrug vs internal standard and normalizing the values against the T0
hour time point.
Ethyl 5,7-Dimethyl-3-(2-(N-methylsulfamoyl)phenyl)-4-oxo-2-
((((2-(2-(piperidin-1-yl)acetoxy)ethyl)carbamoyl)oxy)methyl)-3,4-di-
hydroquinazoline-6-carboxylate (24). The title compound was
synthesized using the same procedure as for prodrug (32). White
1
solid (90 mg, 0.137 mmol, 15% yield). H NMR (400 MHz, DMSO-
d6) δ ppm 8.02 (dd, J = 7.8, 1.5 Hz, 1 H) 7.78−7.87 (m, 2 H) 7.57−
7.72 (m, 3 H) 7.43 (s, 1 H) 4.67 (d, J = 14 Hz, 1 H) 4.47 (d, J = 14
Hz, 1 H) 4.36−4.40 (m, 2 H) 4.10−4.40 (m, 4 H) 3.23−3.42 (m, 4
H) 2.85−2.93 (m, 2 H) 2.62 (s, 3 H) 2.42 (d, J = 5 Hz, 3 H) 2.38 (s, 3
H) 1.71−1.80 (m, 6 H) 1.33 (t, J = 7.20 Hz, 3 H). MS (ESI) m/z
658.2 (M + 1).
Ethyl 2-((2-(Imidazo[1,2-a]pyridine-2-carbonyloxy)-
e t h y l c a r b a m o y l o x y ) m e t hy l ) - 5 , 7 - d i m e t h yl - 3 - ( 2 - ( N -
methylsulfamoyl)phenyl)-4-oxo-3,4-dihydroquinazoline-6-carboxy-
late (32). 1 (250 mg, 0.469 mmol), imidazo[1,2-a]pyridine-2-
carboxylic acid (112.1 mg, 0.691 mmol, 1.5 equiv), and DMAP
(5.73 mg, 0.047 mmol, 0.1 equiv) were placed in a vial along with
DCM (5 mL) and cooled to 0 °C. EDC (122 mg, 0.636 mmol, 1.35
equiv) was added, and the reaction was allowed to stir at rt overnight.
The reaction was concentrated and then dissolved in 2 mL of MeOH
and purified by preparative HPLC to obtain the title compound as a
white solid (93 mg, 0.137 mmol, 30%). 1H NMR (400 MHz, DMSO-
d6) δ ppm 8.50−8.53 (m, 1 H) 8.01 (dd, J = 7.8, 1.5 Hz, 1 H) 7.73−
7.87 (m, 2 H) 7.62−7.67 (m, 3 H) 7.42−7.44 (m, 1 H) 7.27 (s, 1 H)
7.03−7.08 (m, 1 H) 4.70 (d, J = 14 Hz, 1 H) 4.37 (d, J = 14 Hz, 1 H)
4.36−4.38 (m, 2H) 4.27 (br s, 2 H) 3.36 (m, 2 H) 2.57 (s, 3 H) 2.43
(d, J = 5 Hz, 3 H) 2.27 (s, 3 H) 1.33 (t, J = 7.07 Hz, 3 H). MS (ESI)
m/z 677.1 (M + 1).
Figure 5 below demonstrates a time course in vitro plasma stability
study for prodrug 24 in rat plasma at 37 °C.
G
dx.doi.org/10.1021/jm4004939 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
ACKNOWLEDGMENTS
■
Many thanks to Vinayak Hosagrahara, Peter Tarsa, and Jean-
Claude Bizec for their input and Michael Dechantsreiter and
Jonathan Grob for their technical assistance in the high-
throughput chemistry lab. J. Sivak holds the Toronto Western
Hospital Glaucoma Research Chair.
ABBREVIATIONS USED
■
CB1/2, cannabinoid receptor 1 and 2; AUC, area under the
curve; IOP, intraocular pressure; POAG, primary open angle
glaucoma; THC, Δ-9 tetrahydrocannabinol; EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; DCM, dichloromethane;
EtOAc, ethyl acetate; HATU, O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; HOAt,
1-hydroxy-7-azabenzotriazole; m-CPBA, meta-chloroperoxyben-
zoic acid; HPLC, high performace liquid chromatography
Figure 5. In vitro rat plasma stability of prodrug. Peak area ratio
(prodrug/internal standard and 1/internal standard) demonstrate
rapid conversion of prodrug to 1 in rat plasma. The concentration of 1
in rat plasma was determined using the plasma calibration standard
curve.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR of the prodrugs tested in vivo. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*For N.M.: phone, 1-617-871-7394; E-mail, nello.mainolfi@
novartis.com. For J.M.S.: phone, 1-416 581-8171; E-mail,
jsivak@uhnres.utoronto.ca.
■
Present Address
∥For J.M.S.: Department of Ophthalmology and Vision Science,
University of Toronto, and Department of Vision Sciences,
Toronto Western Research Institute, 399 Bathurst Street,
Toronto, Ontario M5T 2S8, Canada.
Notes
The authors declare no competing financial interest.
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