Design, synthesis, and evaluation of no-donor containing carbonic anhydrase inhibitors to lower intraocular pressure

Article abstract of DOI:10.1021/acs.jmedchem.5b00043

The antiglaucoma drugs dorzolamide (1) and brinzolamide (2) lower intraocular pressure (IOP) by inhibiting the carbonic anhydrase (CA) enzyme to reduce aqueous humor production. The introduction of a nitric oxide (NO) donor into the alkyl side chain of do

Full text of DOI:10.1021/acs.jmedchem.5b00043

Article
pubs.acs.org/jmc
Design, Synthesis, and Evaluation of NO-Donor Containing Carbonic
Anhydrase Inhibitors To Lower Intraocular Pressure
Qinhua Huang,* Eugene Y. Rui,* Morena Cobbs, Dac M. Dinh, Hovhannes J. Gukasyan,
Jennifer A. Lafontaine, Saurabh Mehta, Brian D. Patterson, David A. Rewolinski, Paul F. Richardson,
and Martin P. Edwards
La Jolla Laboratories, Pfizer Worldwide Research and Development, 10770 Science Center Drive, San Diego, California 92121,
United States
ABSTRACT: The antiglaucoma drugs dorzolamide (1) and brinzolamide (2) lower intraocular pressure (IOP) by inhibiting the
carbonic anhydrase (CA) enzyme to reduce aqueous humor production. The introduction of a nitric oxide (NO) donor into the
alkyl side chain of dorzolamide (1) and brinzolamide (2) has led to the discovery of NO-dorzolamide 3a and NO-brinzolamide
4a, which could lower IOP through two mechanisms: CA inhibition to decrease aqueous humor secretion (reduce inflow) and
NO release to increase aqueous humor drainage (increase outflow). Compounds 3a and 4a have shown improved efficacy of
lowering IOP in both rabbits and monkeys compared to brinzolamide (2).
INTRODUCTION
■
Glaucoma is an eye disease characterized by a progressive loss
of vision due to irreversible damage to the optic nerve. The
chief pathophysiological feature of glaucoma is increased
intraocular pressure (IOP), and it has been demonstrated
that lowering IOP could control glaucoma and prevent vision
loss.1 IOP is determined primarily by the dynamic equilibrium
between the production of aqueous humor (inflow) in the
reported that nitric oxide (NO) is able to increase the aqueous
humor outflow rate, leading to IOP lowering.9 For example,
topical application of NO-donors, such as nitrovasodilators,10
nipradilol,11 sodium nitroprusside,12 SIN-1, and SNAP13 to
rabbit eyes reduces IOP through the conventional outflow
pathway. Furthermore, IOP lowering is correlated to the
intraocular NO production from NO-donors in the rabbit
eyes.13
It is common practice to use two or three different topical
antiglaucoma drugs in combination when a favorable response
of lowering IOP is not obtained with monotherapy, but the
research on lowering IOP through multiple mechanisms
achieved by a single small molecule remains an unexplored
arena.14 Herein, we report the design and synthesis of NO-
donor containing CAIs (NO-CAIs) to lower IOP through two
mechanisms: carbonic anhydrase inhibition to decrease
ciliary body and its efflux (outflow), mainly through the
trabecular meshwork and Schlemm’s canal.2
Aqueous humor is produced by the ciliary epithelial cells
located in the ciliary body, and its production is regulated by
the carbonic anhydrase (CA) enzymes.3 Inhibition of CA
enzymes decreases bicarbonate secretion and consequently
reduces aqueous humor secretion by the ciliary epithelial cells
into the posterior chamber, leading to a reduction of IOP.4
Therefore, systemic carbonic anhydrase inhibitors (CAIs), such
as acetazolamide, have been developed for the treatment of
glaucoma.5 However, systemic CAIs generally cause undesired
side effects because CA isoforms II, IV, XII are present in many
other tissues and organs.6 To avoid these side effects, topically
effective CAIs have been developed in the past 2 decades with
two drugs available on the market: dorzolamide (1)7 and
brinzolamide (2).8
While inhibiting the secretion of aqueous humor has become
an effective therapy for lowering IOP, another option is to
increase the drainage of aqueous humor out of the eye. It is
Received: January 9, 2015
Published: February 28, 2015
© 2015 American Chemical Society
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Scheme 1. Concept of Incorporating NO-Donor with Antiglaucoma Drugs
aqueous humor secretion (reduce inflow) and NO release to
increase aqueous humor drainage (increase outflow).
RESULTS AND DISCUSSION
■
Design of NO-Donor Containing Carbonic Anhydrase
Inhibitors. To design dual mechanism inhibitors to lower IOP,
we decided to take advantage of the IOP lowering efficacy
resulting from NO release.10−13 As shown in Scheme 1, when
an NO-donor is incorporated into an appropriate antiglaucoma
drug, the new molecule may provide two mechanisms to lower
IOP if NO can be intraocularly released. Obviously, a few
attributes are required of the chosen antiglaucoma drug in
order for the NO-donor containing antiglaucoma drug (NO-
antiglaucoma drug) to be effective. First, since NO increases
aqueous humor outflow, the NO-antiglaucoma drug may
Figure 1. Cocrystal structures of dorzolamide (1) (orange, PDB code
1c1l) and brinzolamide (2) (cyan, PDB code 3znc) with the CA
enzyme.
maximize the IOP lowering efficacy if the antiglaucoma drug
reduces aqueous humor inflow. Second, the NO-antiglaucoma
drug has to be topically administrated because of the in vivo
instability; therefore, a topical antiglaucoma drug is preferred.
Third, in order to achieve optimal levels of intraocular NO
production from the NO-antiglaucoma drug, the drug should
possess a relatively high formulated concentration. For example,
Xalatan contains only 0.005% formulated latanoprost,15 and the
corresponding NO-latanoprost is unlikely to achieve a high
level of in vivo NO release. Finally, both the parent NO-
antiglaucoma drug and its corresponding fragment resulting
from in vivo NO release should have sufficient potency to elicit
efficacy.
Of the approximately 20 antiglaucoma drugs on the market,
dorzolamide (1) and brinzolamide (2) distinguished them-
selves as ideal drugs to incorporate NO-donors.16 Both
dorzolamide (1) and brinzolamide (2) inhibit CA enzymes to
reduce aqueous humor production, utilize topical adminis-
tration, and possess relatively high concentrations (2% for
dorzolamide and 1% for brinzolamide) in the formulation.16 In
addition, the cocrystal structure analyses of dorzolamide (1)
and brinzolamide (2) with the CA enzyme reveal that
introduction of an NO-donor on the alkyl side chains, i.e.,
the methyl group from dorzolamide (1) and the methoxypropyl
group from brinzolamide (2), would have a minimum impact
on binding affinity (Figure 1). Those alkyl side chains reside on
the edge of the binding pocket and point toward the solvent,
while the sulfonamides interact with the protein in the deep
binding pocket.
NO-CAIs 3a−c and 4a bearing an NO-donor at the end of
the alkyl side chain were designed based on evaluation of
expected potency, stability, solubility, and synthetic accessibility
(Scheme 2). Compounds 3a−c were derived from dorzolamide
(1) with a three-carbon, two-carbon, and one-carbon linker,
while compound 4a was modified from brinzolamide (2) with a
three-carbon linker. A nitrate ester was chosen as a NO-donor
mainly because the alkyl nitrate group could intraocularly
decompose to release NO,9−13 and its relatively small size
would help NO-CAIs inherit topical druglike properties from
their parent CAIs for topical administration. As NO-CAIs 3a−c
and 4a penetrate into eye tissues after topical application, they
could gradually decompose and generate des-NO fragments
(5a−c and 6a) while releasing NO. Ideally, both NO-CAIs
(3a−c and 4a) and their fragments (5a−c and 6a) would be
potent.
Synthesis of NO-CAIs 3a−c and 4a. The synthesis of
NO-CAIs 3a−c is outlined in Schemes 3−5.7b The
thiopyranone 16 was efficiently constructed from commercially
available starting materials (Scheme 3). For the synthesis of
16a, PCC oxidation of 4-methoxybutan-1-ol followed by a
Wittig reaction afforded the trans alkene 8 in 50% yield over
two steps. The Michael addition of thiophene-2-thiol to alkene
8 proceeded very slowly to generate ester 9. This Michael
reaction was run neat with 10% Et3N and still required 2 days
to reach completion. When ester 9 was hydrolyzed in 6 M
aqueous HCl at 100 °C, it resulted in low conversion and
partially decomposed after being refluxed for an extended
period of time. The low conversion was due to the poor
solubility of ester 9 in 6 M aqueous HCl; the problem was
solved by adding glacial acetic acid as a cosolvent. Other mixed
solvents, such as aqueous THF or aqueous dioxane, failed to
improve the conversion. The crude hydrolyzed product was
then treated with 1.0 equiv of Tf2O in toluene at 0 °C to form
the desired thiopyranone 16a in 53% yield. For the synthesis of
16b, selective ketone reduction of β-ketoester 10 using NaBH4
at −78 °C, followed by a tosyl protection, afforded ester 11 in
∼40% yield over two steps. SN2 substitution by thiophene-2-
thiol generated ester 12, which was then treated with standard
hydrolysis and cyclization conditions (steps i and j, Scheme 3)
to afford thiopyranone 16b. For the synthesis of 16c,
commercially available ester 13 was reacted with Ag2O in
MeOH, followed by Michael addition of 1.0 equiv of
thiophene-2-thiol, generating ester 15 in 80% yield over two
steps. Ester 15 underwent our standard hydrolysis/cyclization
conditions (steps i and j, Scheme 3) to form thiopyranone 16c.
With thiopyranone 16 at hand, the subsequent sulfonamida-
tion using optimized reaction conditions afforded sulfonamide
17 in 49−68% yields (Scheme 4). In order to simplify
purification and obtain reasonable isolated yields, the
sulfonamidation steps required flash purified thiopyrane 16
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Scheme 2. Design of NO-CAIs 3a−c and 4a
a
Scheme 3. Synthesis of Thiopyranones 16a−c
a
Reagents and conditions: (a) 1.7 equiv of PCC, CH2Cl2, 25 °C, 3.5 h, 66%; (b) 1.8 equiv of Ph3PCO2Et, 0 °C, 30 min, CH2Cl2, 77%; (c) 1.15
equiv of thiophene-2-thiol, 1−10% Et3N, 25 °C, 2 d, 89%; (d) 1.1 equiv of NaBH4, MeOH, −78 °C 2 h, 82%; (e) 1.2 equiv of TsCl, pyridine, 25 °C,
30 h, 50%; (f) 1.05 equiv of thiophene-2-thiol, 1.2 equiv of Et3N, 25 °C, 2−3 h, 84%; (g) 0.75 equiv of Ag2O, MeOH, 25 °C, sonication, 12 h, (h)
1.0 equiv of thiophene-2-thiol, 0.1 equiv of Et3N, 25 °C, 12 h, 80% over two steps; (i) 6 M HCl/HOAc (v/v 7:1), 100 °C, 4 h; (j) 1.0 equiv of Tf2O,
toluene, 0 °C 30 min, 25 °C, 2 h, 28−67% over two steps.
because it was unstable even at 0 °C. Sulfonamide 17 was then
treated with 2.2 equiv of Oxone in MeOH/H2O to afford 18 in
71−100% yield. Interestingly, the reduction of 18 using NaBH4
in EtOH generated exclusively alcohol 19 as a cis
diastereoisomer in 77−95% yield. Alcohol 19 was exposed to
Ritter reaction conditions (4:1 CH3CN/conc H2SO4),7a
followed by amide reduction using BH3−THF to afford
amine 20 in a 5:1 trans/cis ratio. Chiral separation of amine
20 using supercritical fluid chromatography (SFC) produced
enantiomerically pure (−)-20 in 99% ee.
Interestingly, sulfonamides 20a−c showed different reactivity
toward 48% aq HBr (Scheme 4). When sulfonamide 20a
(three-carbon linker) was treated with 48% aq HBr at 80 °C,
the reaction reached completion within 2 days, generating a
quantitative yield of the brominated product 21 with complete
consumption of alcohol 5a. Sulfonamide 20b (two-carbon
linker) was also completely converted into the brominated
product 22 under 48% aq HBr at 85 °C without remaining
alcohol 5b; however, a much longer reaction time (7 days) was
required. Bromides 21 and 22 were then treated with excess
AgNO3 in CH3CN at 50 °C to form NO-CAIs 3a and 3b in
78−86% yield. On the other hand, treatment of sulfonamide
20c with 48% aq HBr failed to deliver the expected brominated
product 23. Instead, the corresponding alcohol 5c was obtained
in 100% yield, even at higher reaction temperatures (85−110
°C) and prolonged reaction times. The failure of direct
brominating sulfonamide 20c was likely due to the steric
hindrance of the reactive carbon. In addition the neighboring
electron-rich sulfone motif could further prevent the approach
of an electronegative nucleophile by electronic repulsion.
After numerous attempts, a successful route to access
compound 3c was outlined in Scheme 5, in which the nitro
group was introduced prior to the sulfone formation. As shown
in Scheme 5, reduction of sulfonamide 17c afforded the alcohol
24 in 81% yield as the cis diastereoisomer. Alcohol 24 was
exposed under strong Ritter amidation conditions (4:1
CH3CN/conc H2SO4) that were previously used for sulfone
19 (Scheme 4), but it decomposed immediately and gave no
desired amidation product 25. After numerous attempts, mild
Ritter reaction conditions were developed and successfully
converted alcohol 24 to amide 25 in 80% yield using catalytic
amounts of H2SO4 (0.7 equiv) at 25 °C. The reaction was also
performed under dilute conditions (in 0.035 M CH3CN) to
avoid dimer formation. Interestingly, this mild Ritter amidation
condition afforded a trans isomer exclusively. Reduction of
amide 25 with BH3−THF proceeded smoothly to afford amine
26 in 70% yield. When amine 26 was reacted with 48% aq HBr
at 80 °C for 12 h, bromide 27 was formed successfully in a
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a
Scheme 4. Complete Synthesis of NO-CAIs 3a and 3b
a
Reagents and conditions: (a) 1.05 equiv of ClSO3H, CH2Cl2, −5 °C for 30 min, 25 °C for 3 h; then 1.10 equiv of PCl5, 0−25 °C, 30 min; (b) 0.5
M NH3 in dioxane/7.0 M NH3 in MeOH (v/v 5:2), 0 °C, 2 h, 49−68% overall yield; (c) 2.2 equiv of Oxone, MeOH/water (v/v 5:2), 25 °C, 12 h,
71−100%; (d) 1.3 equiv of NaBH4, EtOH, 0 °C, 15 min; 2 N HCl, 25 °C, 77−95%; (e) CH3CN/conc H2SO4 (v/v 4:1), 25 °C, 48 h, 44−65%; (f)
3.4 equiv of BH3, THF, 25 °C, 48 h; 2.5 M H2SO4, 50 °C or 2 N HCl, 25 °C, 7 h; then chiral separation by SFC, 99% ee, 20−27% overall yield; (g)
48% aq HBr, reaction temperature and yield are indicated in the scheme; (h) 7.7 equiv of AgNO3, CH3CN, 50 °C, 24−38 h, 78−86%.
a
Scheme 5. Complete Synthesis of Racemic NO-CAI 3c
a
Reagents and conditions: (a) 1.3 equiv of NaBH4, EtOH, 0 °C, 15 min; 2 N HCl, 25 °C, 81%; (b) 0.7 equiv of conc H2SO4, CH3CN, 0.035 M, 25
°C, 2 d, 80%; (c) 3.4 equiv of BH3, THF, 25 °C, 12 h; 2.5 M H2SO4, 50 °C, 2 h, 70%; (d) 48% aq HBr, 80 °C, 12 h, quant; (e) 8.0 equiv of AgNO3,
CH3CN, 25 °C, 12 h; (f) 3.0 equiv of Oxone, MeOH/H2O (v/v 1:1), 25 °C, 12 h, 50% over two steps.
a
Scheme 6. Synthesis of NO-CAI 2a
a
Reagents and conditions: (a) 4.0 equiv of BBr3, CH2Cl2, −78 °C, 10 min, 66%; (b) 48% aq HBr, 80 °C, 18 h, 54%; (c) 5.0 equiv of AgNO3, conc
HNO3, CH3CN, 60 °C, 48 h, 28%.
quantitative yield. The nitration of bromide 27 proceeded at 25
°C to generate 28, and subsequent oxidation with Oxone
formed racemic NO-CAI 3c in 50% yield over two steps.
The synthesis of NO-CAI 4a is outlined in Scheme 6.8b
Commercially available brinzolamide (1) was reacted with BBr3
at −78 °C to afford alcohol 6a in 66% yield. The subsequent
bromination using 48% aq HBr at 80 °C generated bromide 29
in 54% yield. Bromide 29 was then reacted with an excess
amount of AgNO3 to form NO-CAI 4a in 28% yield.
In Vitro and in Vivo Efficacy of NO-CAIs 3a−c and 4a.
Compounds 3a−c and 4a were profiled together with
dorzolamide (1) and brinzolamide (2) as indicated in Table
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1. Compounds 3a−c and 4a indeed possessed similar CA2 and
suspension in Carbopol/Cremaphor,18 derived from the
commercial brinzolamide (2) formulation.19 Figure 2 summa-
rizes the rabbit PK for NO-CAIs 3a and 4a at the 4 h time
point after topical treatment with 2 drops (2 × 25 μL) of
formulated 3a or 4a. Both NO-CAIs 3a and 4a successfully
penetrated into the iris ciliary body and decomposed into their
corresponding des-NO fragments 5a and 6a as predicted. The
formation of des-NO fragments 5a or 6a was used as an
indicator for NO release, since direct detection of NO
CA4 enzymatic potency to their parent antiglaucoma drugs
Table 1. Enzymatic Potency, Lipophilicity, and Solubility of
Dorzolamide (1), Brinzolamide (2), 3a−c, and 4a
CA II Kd
(nM)
CA IV IC50
(nM)
solubility
(mg/mL)
a
b
compd
clogD
dorzolamide (1)
0.043
0.083
0.034
0.80
43
31
−0.5
0.3
>20
11.3
3a
formation in vivo has proved to be very challenging.20
A
3b
47
−0.2
−0.5
−0.9
−0.5
0.1
much higher concentration of 3a relative to 4a was measured in
the iris ciliary body, which suggested that 3a has superior in
vivo stability to 4a, resulting in less efficiency in NO release. In
aqueous humor, while a relatively high concentration of des-
NO fragments 5a and 6a was found, only trace amounts of
parent NO-CAIs 3a and 4a were detected.
3c
156
82
5a
0.170
0.089
0.090
0.136
brinzolamide (2)
45
0.5
2.2
4a
6a
31
165
−0.8
a
Compound 3c was racemic, and all other compounds were
b
The percent inhibition of CA activity was evaluated via a pH
STAT method.21 For NO-CAI 4a, its CA inhibition was
determined and compared to brinzolamide (2) at 2, 4, 6, 8 h
postdose after topical treatment with 2 drops (2 × 25 μL) of
formulated 4a (Figure 3). While nearly 90% CA inhibition was
attained at 2 h postdose with compound 4a, it gradually
decreased but still maintained about 45% inhibition after 8 h.
Apparently, the high CA affinity of des-NO fragment 6a also
contributed to the overall CA inhibition, since compound 4a
gradually decomposed to form 6a in the iris ciliary body and
only a trace amount of 4a was left after 4 h, as indicated in
Figure 2. Consistently, NO-CAI 4a possessed slightly improved
efficiency of CA inhibition compared to brinzolamide (2)
across all four measured time points. Note that, in our hands,
compound 4a could be formulated up to a 2% w/v suspension
in Carbopol/Cremaphor by which maximal CA inhibition was
achieved. On the other hand, 1%, 2%, 3% formulated
brinzolamide (2) resulted in similar CA inhibition and efficacy
enantiomerically pure. Calculated log D.
dorzolamide (1) and brinzolamide (2), respectively. The des-
NO fragments 5a and 6a were also potent but with a modest
loss in potency compared to their corresponding parent NO-
CAIs 3a and 4a, likely due to the decreased lipophlicity.17 The
calculated log D of NO-CAIs, dorzolamide (1), and
brinzolamide (2) demonstrated that the introduction of a
nitro group had a modest effect on the lipophilicity. Compound
3c had similar lipophilicity to dorzolamide (1). With the
introduction of more lipophilic CH2 groups, 3a and 3b became
slightly more lipophilic than dorzolamide (1). Compound 4a
possessed a little higher clogD than brinzolamide (2). The
solubility of 3a and 4a was also very comparable to 1 and 2,
respectively.
With the overall consideration of efficacy, formulation, and
compound supply, NO-CAIs 3a and 4a emerged as candidates
for rabbit pharmacokinetic (PK) studies using a formulated 2%
Figure 2. Concentration of NO-CAIs (3a and 4a) and their corresponding des-NO fragments (5a and 6a) in both iris ciliary body and aqueous
humor (N = 3 eye) in 4 h after topical treatment of 2 drops (2 × 25 μL) of 3a or 4a with a formulated 2% w/v suspension in Carbopol/Cremaphor.
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Figure 3. Percent inhibition of CA activities in the rabbit iris ciliary body (n = 3 eye) after topical treatment with 2 drops (2 × 25 μL) of formulated
NO-CAI 4a or brinzolamide (2) via pH Stat under the condition of 100 mL/min CO2.
Figure 4. Efficacy of lowering IOP under topical treatment with 2 drops (2 × 25 μL) of formulated 3a, 4a, or brinzolamide (2) in the rabbit
noncontact tonometry (N = 3 eye).
Figure 5. Efficacy of lowering IOP under topical treatment with 2 drops (2 × 25 μL) of formulated 3a or brinzolamide (2) in the OHT primate
tonometry (n = 3 eye).
of IOP lowering, indicating that 1% formulated brinzolamide
(2) has achieved its maximal CA inhibition.22 Therefore, in our
in vivo experiments, 1% commercial brinzolamide ophthalmic
suspension19 was used to compare with 2% formulated NO-
CAI 3a/4a, with each reaching its maximal CA inhibition and
efficacy of lowering IOP. For NO-CAI 3a, the CA inhibition
was only determined at the 4 h time point after topical
treatment with 2 drops (2 × 25 μL) of formulated 3a, resulting
in very similar CA activity inhibition (data not shown) to
compound 4a.
∼1−2 h under topical treatment with 2 drops (2 × 25 μL) of
formulated 3a or 4a. This drop was followed by a gradual IOP
increase beyond 2 h postdose. While exhibiting similar efficacy
in lowering IOP relative to each other, compounds 3a and 4a
displayed improved efficacy in lowering IOP compared to
brinzolamide (2) in rabbit tonometry. At trough, NO-CAIs 3a
and 4a lowered IOP to a maximum of 5 mmHg, which is
roughly twice the magnitude of effect shown by brinzolamide
(2). Although 2% formulated 3a or 4a possessed slightly higher
greater CA inhibition than 1% formulated brinzolamide (2), it
is likely that NO release from 3a and 4a in the rabbit tonometry
contributed to the overall efficacy of lowering IOP.
Interestingly, the NO effect on lowering IOP became much
more pronounced in the ocular hypertension (OHT) primate
The efficacy of lowering IOP for NO-CAIs 3a and 4a was
then evaluated in rabbits and monkeys in comparison to
brinzolamide (2). Using rabbit tonometry (Figure 4), IOP was
measured and was shown to decline and hit trough levels in
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C18 column at 80 °C, 4.6 mm × 150 mm, 5 μm, 5%−95% MeOH/
H2O buffered with 0.2% formic acid/0.4% ammonium formate, 3 min
run, flow rate 1.2 mL/min, UV detection (λ = 254, 224 nm).
Combustion analyses were performed by Atlantic Microlab, Inc.
(Norcross, Georgia).
Synthesis of Compound 3a. (E)-6-Methoxy-hex-2-enoic Acid
Ethyl Ester (8). To an orange suspension of PCC (60 g) in CH2Cl2
(700 mL) was added 4-methoxybutan-1-ol (compound 7, 17 g) slowly
at 25 °C. The resulting brown suspension was stirred at 25 °C for 3.5
h. The reaction mixture was diluted with 1.5 L of Et2O and violently
stirred at 25 °C for 10 min. The resulting gray suspension was filtered
through Celite. The reaction container was washed with 2 × 200 mL
of Et2O. The organic solutions were combined and passed through
Celite again. The filtrate was concentrated and the residue was
columned on silica gel using 1:1 hexane/EtOAc to afford 11.0 g of 4-
methoxybutyraldehyde in 66% yield as colorless oil.
tonometry. As shown in Figure 5, when monkeys were treated
with 2 drops (2 × 25 μL) of formulated 4a, a significant IOP
decrease of 10.5 mmHg was observed at 2 h postdose, and IOP
lowering was preserved for 24 h following treatment. In
contrast, treatment with 2 drops (2 × 25 μL) of brinzolamide
(2) lowered IOP by only about 2 mmHg. Given that
brinzolamide (2) shows close to maximal CA inhibition when
measured in the pH stat assay, the significant efficacy increase
from 4a is likely to involve in a second mechanism in addition
to just inhibiting CA. It appears that in vivo NO release from 4a
to increase aqueous humor drainage plays a key role in lowering
IOP in the OHT primate tonometry.
CONCLUSION
■
To a clear solution of Ph3PCO2Et (67.5 g, 194 mmol, 1.80 equiv)
in CH2Cl2 (400 mL) was added a solution of 4-methoxybutyraldehyde
(11.0 g) in CH2Cl2 (25.0 mL) at 0 °C under N2. The reaction was not
complete after being stirred at 0 °C for 30 min. Another 23 g (66
mmol) of Ph3PCO2Et was added, and the mixture was stirred at 0
°C for 30 min to reach completion. The solvent was removed under
reduced pressure, and the residue was diluted with 100 mL of hexane
to form a white suspension. The suspension was filtered to remove
phosphorus salts. The filtrate was concentrated under reduced
pressure and columned on silica gel using 4:1 hexane/EtOAc to
NO-CAIs 3a−c and 4a were designed to maximize efficacy of
IOP lowering by combining two mechanisms, reducing inflow
by CA inhibition to decrease aqueous humor secretion, and
increasing outflow by NO release to increase aqueous humor
drainage. A nitro group was utilized as an NO-donor and
incorporated in the side chains of two topically applied CAIs
dorzolamide (1) and brinzolamide (2) with appropriate carbon
linkers. While compound 4a was made from commercially
available brinzolamide (1) in three steps, the synthesis of
compounds 3a−c was completed in 12−13 linear steps which
featured a few key reactions, such as sulfonamidation, a Ritter
reaction, and bromination. The synthesized compounds 3a−c
and 4a displayed good CA enzymatic potency. In addition, the
rabbit PK illustrated that NO-CAIs 3a and 4a successfully
penetrated and decomposed in the iris ciliary body with a
slightly higher efficiency of CA inhibition. The efficacy of
lowering IOP from NO-CAIs 3a and 4a was evaluated in both
rabbits and monkeys in comparison to brinzolamide (2), and
improved efficacy was observed. In particular, the significant
improvement in efficacy from 4a in the OHT primate
tonometry revealed that the dual mechanism inhibitors have
great potential to be an effective therapy for lowering IOP.
1
afford 13.1 g of the desired product in 71% yield as colorless oil. H
NMR (400 MHz, CDCl3) δ ppm 6.97 (dt, J = 15.6, 7.0 Hz, 1H), 5.84
(dt, J = 15.6, 1.5 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.40 (t, J = 6.3 Hz,
2H), 3.34 (s, 3 H), 2.26−2.32 (m, 2H), 1.71−1.77 (m, 2H), 1.30 (t, J
= 7.1 Hz, 3H).
6-Methoxy-3-(thiophen-2-ylsulfanyl)hexanoic Acid Ethyl Ester
(9). To a mixture of compound 8 (13.1 g, 76.1 mmol, 1.0 equiv),
thiophene-2-thiol (7.06 mL, 76.1 mmol, 1.0 equiv), and THF (4 mL)
was added Et3N (0.2 mL, 1.47 mmol, 2 mol %) at 25 °C. The crude
1H NMR indicated that about 20% of starting material left after the
reaction was stirred at 25 °C for 16 h under N2. Therefore, another
1.06 mL of thiophene-2-thiol (11.4 mmol, 0.15 equiv) was added and
the resulting mixture was stirred at 25 °C for 12 h to reach completion.
The mixture was columned on silica gel using 10:1 heptane/EtOAc
(partially separated, three columns were run). In total, we obtained
1
19.5 g of the desired product in 89% yield as a pale yellow oil. H
EXPERIMENTAL METHODS
NMR (400 MHz, CDCl3) δ ppm 7.41 (dd, J = 1.3, 5.3 Hz, 1H), 7.17
(dd, J = 1.3, 3.8 Hz, 1H), 7.02 (dd, J = 3.5, 5.3 Hz, 1H), 4.10−4.22 (m,
2H), 3.39−3.42 (m, 2H), 3.34 (s, 3H), 3.19−3.26 (m, 1H), 2.62 (dd, J
= 7.6, 15.6 Hz, 1H), 2.49 (dd, J = 7.1, 15.8 Hz, 1 H), 1.86−1.97 (m,
1H), 1.50−1.80 (m, 3H), 1.28 (t, J = 7.0 Hz, 3H).
■
Starting materials and other reagents were purchased from commercial
suppliers and were used without further purification unless otherwise
indicated. All reactions were performed under a positive pressure of
nitrogen or argon or with a drying tube, at ambient temperature
(unless otherwise stated), in anhydrous solvents, unless otherwise
indicated. Analytical thin-layer chromatography was performed on
glass-backed silica gel 60_F 254 plates (Analtech (0.25 mm)) and
eluted with the appropriate solvent ratios (v/v). The reactions were
assayed by high-performance liquid chromatography (HPLC) or thin-
layer chromatography (TLC) and terminated as judged by the
consumption of starting material. The TLC plates were visualized by
UV, phosphomolybdic acid stain, or iodine stain. Microwave assisted
reactions were run in a Biotage Initiator. 1H NMR spectra were
recorded on a Bruker instrument operating at 400 MHz unless
6-(3-Methoxypropyl)-5,6-dihydrothieno[2,3-b]thiopyran-4-one
(16a). A mixture of compound 9 (17.0 g), 6.0 M aq HCl (350 mL),
and HOAc (50 mL) was warmed to 100 °C. After being refluxed at
100 °C for 4 h, the reaction was complete and was cooled to 25 °C.
The reaction mixture was concentrated, and the residue was diluted
with brine (300 mL) and toluene (200 mL). The organic layer was
separated, dried over Na2SO4, filtered, and concentrated to afford a
pale yellow oil. The resulting crude hydrolyzed product was dissolved
in dry toluene (250 mL), and Tf2O (10.9 mL, 64.8 mmol, 1.0 equiv)
was slowly added at 0 °C. A colorless solution was obtained. The
reaction mixture turned slowly from colorless to gray and eventually to
brown. The reaction was stirred at 0 °C for 30 min and at 25 °C for
2.0 h. Excess amounts of Tf2O were destroyed with water. The mixture
was diluted by EtOAc (500 mL), washed with brine (400 mL), dried
over Na2SO4, filtered, and concentrated. The resulting black residue
was columned on silica gel using 2:1 heptane/EtOAc to afford 7.5 g of
1
otherwise indicated. H NMR spectra are obtained as DMSO-d6 or
CDCl3 solutions as indicated (reported in ppm), using chloroform as
the reference standard (7.25 ppm) or DMSO-d6 (2.50 ppm). Other
NMR solvents were used as needed. When peak multiplicities are
reported, the following abbreviations are used: s = singlet, d = doublet,
t = triplet, m = multiplet, br = broadened, dd = doublet of doublets, dt
= doublet of triplets. Coupling constants, when given, are reported in
hertz. The mass spectra were obtained using liquid chromatography
mass spectrometry (LCMS) on an Agilent instrument using
atmospheric pressure chemical ionization (APCI) or electrospray
ionization (ESI). All test compounds showed >95% purity as
determined by combustion analysis or by high-performance liquid
chromatography (HPLC). HPLC conditions were as follows: XBridge
1
the desired product in 53% as a pale yellow oil. H NMR (400 MHz,
DMSO-d6) δ ppm 7.45 (d, J = 5.3 Hz, 1H), 7.02 (d, J = 5.5 Hz, 1H),
3.67−3.75 (m, 1H), 3.38−3.43 (m, 2H), 3.33 (s, 3H), 2.94 (dd, J =
16.6, 3.1, 1H), 2.74 (dd, J = 16.6, 11.1, 1H), 1.69−1.89 (m, 4H).
6-(3-Methoxypropyl)-4-oxo-5,6-dihydro-4H-thieno[2,3-b]-
thiopyran-2-sulfonic Acid Amide (17a). Chlorosulfuric acid (0.796
mL, 11.9 mmol, 1.05 equiv) was added into precooled DCM (20 mL).
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Article
The diluted colorless ClSO3H−DCM solution was added slowly into a
precooled solution of compound 16a (2.75 g, 11.35 mmol, 1.0 equiv)
in DCM (100 mL) at −10 °C (NaCl−ice bath). A green clear solution
was obtained. The mixture was stirred at −5 °C for 30 min and 25 °C
for 3.0 h to form a pale orange solution. PCl5 (2.60 g, 12.50 mmol,
1.10 equiv) was added portionwise at 0 °C, and an orange solution was
obtained. After being stirred at 25 °C for 30 min, the reaction mixture
turned from orange color into deep blue color. The reaction was
dumped into a funnel that contained ice. Water (200 mL) and DCM
(100 mL) were added. The organic layer became a blue color. The
organic solution was quickly separated, dried over Na2SO4, filtered,
and concentrated to a deep blue solution in a volume of around 15
mL. To the solution were added slowly 50 mL of 0.5 M NH3 in
dioxane and 20 mL of 7.0 M NH3 in MeOH at 0 °C. The reaction was
stirred at 0 °C until it reached completion. The mixture was
concentrated, and the residue was dissolved with EtOAc (200 mL) and
brine (200 mL). The organic layer was separated, dried over Na2SO4,
filtered, and concentrated to afford a yellow solid. The resulting yellow
solid was triturated twice with 20 mL of 1:1 EtOAc/hexane to afford
2.20 g of the pure desired product in 60% yield as a pale yellow solid.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.83 (s, 2H), 7.65 (s, 1H),
(1.45 mL, 15.3 mmol, 3.37 equiv) in 15 mL of THF at −10 °C. The
reaction was warmed to 25 °C, and a clear pale yellow solution was
obtained. It was stirred at 25 °C for 2 d to reach completion. The
reaction mixture was added into 20 mL of aq H2SO4 (2.5 M) at 0 °C.
After being stirred at 50 °C for 7 h, the mixture was cooled into 0 °C
and neutralized by using 10 and 1.0 M NaOH to tune pH to ∼8. The
neutralized mixture was extracted with 3 × 100 mL of 9:1 CHCl3/
MeOH. The organic solutions were combined, dried over MgSO4, and
concentrated to afford 1.74 g of the indicated compound as a mixture
of trans/cis isomers in a roughly 5:1 ratio. SFC technology was
employed for purification and chiral separation. The desired
enatiomerically pure compound (−)-20a was isolated in 27% yield
(470 mg) as a white solid with >99% ee. The obs is −0.011 and [α]D is
−29.86. LC/MS: (APCI) 383 (M+ + 1). 1H NMR (400 MHz, DMSO-
d6) δ ppm 7.96 (s, 2H), 7.55 (s, 1H), 3.90−3.95 (m, 1H), 3.81−3.88
(m, 1H), 3.37 (t, J = 6.0 Hz, 2H), 3.25 (s, 3H), 2.63−2.67 (m, 1H),
2.42−2.56 (m, 3H), 2.21−2.28 (m, 1H), 1.96−2.04 (m, 1H), 1.54−
1.80 (m, 3H), 1.03 (t, J = 7.0 Hz, 3H).
(4S,6S)-6-(3-Bromopropyl)-4-ethylamino-7,7-dioxo-4,5,6,7-tetra-
hydro-7λ6-thieno[2,3-b]thiopyran-2-sulfonic Acid Amide (21). At 0
°C, compound (−)-20a (white solid, 200 mg, 0.523 mmol) was
dissolved in 20 mL of HBr. A colorless solution was obtained. The
mixture was stirred 80 °C for 48 h to reach completion. It was cooled
to 25 °C and the solvent was removed under reduced pressure to
afford the desired product as a white solid in the form of HBr salts in
3.92−4.00 (m, 1H), 3.32 (t, J = 7.3 Hz, 2H), 3.21 (s, 3H), 2.94 (dd, J
= 3.3, 16.9 Hz, 1H), 2.77 (dd, J = 10.0, 16.9 Hz, 1H), 1.70−1.80 (m,
2H), 1.60−1.65 (m, 2H).
6-(3-Methoxypropyl)-4,7,7-trioxo-4,5,6,7-tetrahydro-7λ6-thieno-
[2,3-b]thiopyran-2-sulfonic Acid Amide (18a). A solution of Oxone
(8.42 g, 13.7 mmol, 2.20 equiv) in 40.0 mL of water was added slowly
into a solution of compound 17a (2.0 g, 6.22 mmol, 1.0 equiv) in 100
mL of MeOH at 0 °C. The reaction was stirred at 25 °C for 12 h to
reach completion. Methanol was removed under reduced pressure.
The resulting yellow suspension was diluted with 100 mL of water.
The solid was filtered and dried under reduced pressure at 60 °C to
1
quantitative yield. LCMS (APCI) m/z 431 (M+ + 1). H NMR (400
MHz, DMSO-d6) δ ppm 8.88 (br s, 2H), 8.18 (s, 2H), 7.86 (s, 1H),
4.71−4.74 (m, 1H), 4.02−4.06 (m, 1H), 3.63 (t, J = 5.8 Hz, 2H),
3.20−3.25 (m, 1H), 3.04−3.09 (m, 1H), 2.58−2.75 (m, 2H), 2.04−
2.13 (m, 3H), 1.74−1.81 (m, 1H), 1.24 (t, J = 7.3 Hz, 3H).
(4S,6S)-4-Ethylamino-6-(3-nitrooxy-propyl)-7,7-dioxo-4,5,6,7-tet-
rahydro-7λ6-thieno[2,3-b]thiopyran-2-sulfonic Acid Amide (3a). To
a pale yellow homogeneous solution of compound 21 (HBr salt, 268
mg, 0.523 mmol) in MeCN (15.0 mL) was added in one portion of
AgNO3 (444 mg, 2.61 mmol, 5.0 equiv) at 0 °C. The reaction was
1
afford 2.20 g of the desired product in 100% yield. H NMR (400
MHz, DMSO-d6) δ ppm 8.21 (s, 2H), 7.75 (s, 1H), 4.37−4.45 (m,
1H), 3.35 (t, J = 5.7 Hz, 2H), 3.23 (s, 3H), 3.19−3.23 (m, 2H), 2.03−
2.10 (m, 1H), 1.63−1.80 (m, 3H).
1
stirred at 50 °C for 20 h, and about /3 of SM was left. Another 240
cis-4-Hydroxy-6-(3-methoxypropyl)-7,7-dioxo-4,5,6,7-tetrahydro-
7λ6-thieno[2,3-b]thiopyran-2-sulfonic Acid Amide (19a). To a
solution of compound 18a (2.00 g, 5.66 mmol, 1.0 equiv) in EtOH
(100 mL) was added NaBH4 (0.278 g, 7.35 mmol, 1.30 equiv) slowly
at 0 °C. The reaction was stirred at 0 °C for 15 min to reach
completion. The solvent was removed under reduced pressure. The
oily residue was diluted with water (50 mL) and acidified with 2 N
HCl to pH = 2−3 to form lots of precipitate. The precipitate was
filtered, washed with small amounts of water, and dried to afford 1.7 g
of the desired compound in 77% yield as a pale yellow solid. The cis
stereochemistry was confirmed by NOSEY experiment. 1H NMR (400
MHz, DMSO-d6) δ ppm 8.05 (s, 2H), 7.56 (s, 1H), 6.08 (br s, 1H),
4.82−4.84 (m, 1H), 3.7−3.80 (m, 1 H), 3.37 (t, J = 5.9 Hz, 2H), 3.24
(s, 3H), 2.42−2.46 (m, 1H), 1.97−2.14 (m, 2H), 1.58−1.80 (m, 3H).
(4S,6S)-4-Ethylamino-6-(3-methoxy-propyl)-7,7-dioxo-4,5,6,7-tet-
rahydro-7λ6-thieno[2,3-b]thiopyran-2-sulfonic Acid Amide (20a).
To a suspension of compound 19a (3.00 g, 8.44 mmol, 1.0 equiv) in
anhydrous MeCN (100 mL) under ice−NaCl bath was added slowly
25 mL of conc H2SO4. A transparent pale orange solution was then
obtained. The reaction was stirred at 25 °C for 48 h to reach
completion. It was dumped into a mixture of water (200 mL) and
EtOAc (200 mL) at 0 °C and the organic layer was separated. The
aqueous solution was washed with 2 × 100 mL of EtOAc. Organic
solutions were combined, dried over MgSO4, and concentrated to
afford the crude product as brown sticky oil. It was columned on silica
gel using 15:1 and 10:1 DCM/MeOH to afford 2.20 g of an amide
intermediate in 65% yield as a white solid. The product is a mixture of
mg of AgNO3 (2.70 mmol, 1.41 equiv) was added, and the resulting
suspension was stirred at 58 °C for 18 h to reach completion. The
reaction was cooled to 25 °C, and the solvent was removed under
reduced pressure. The residue was dissolved in EtOAc (40 mL) and
passed through Celite. The filtrate was concentrated and purified by
HPLC (water/MeCN/HOAc) to afford the desired product in 78%
yield as a white solid. LCMS (APCI) m/z 414 (M+ + 1). 1H NMR for
the product in the form of HCl salt (400 MHz, DMSO-d6) δ ppm 9.39
(s, 1H), 9.16 (s, 1H), 8.19 (s, 2H), 7.91 (s, 1H), 4.71 (br s 1H), 4.62
(t, J = 6.0 Hz, 2H), 4.16−4.24 (m, 1H), 3.17−3.25 (m, 1H), 3.01−
3.09 (m, 1H), 2.75−2.79 (m, 1H), 2.56−2.67 (m, 1H), 1.91−2.10 (m,
3H), 1.72−1.81 (m, 1H), 1.26 (t, J = 7.1 Hz, 3H).
Synthesis of Compound 3b. Methyl 3-Hydroxy-5-methox-
ypentanoate. To a solution of methyl 5-methoxy-3-oxopentanoate
(compound 10, 36.0 g, 225 mmol, 1.0 equiv) in 500 mL of MeOH at
−78 °C was added portionwise NaBH4 (10.4 g, 247 mmol, 1.1 equiv).
During the addition, lots of bubbles were generated. Aftering being
stirred at −78 °C for 2 h, the reaction mixture was concentrated under
reduced pressure, diluted with 1 N HCl and extracted with EtOAc
twice. The combined organic layers were concentrated to afford 30 g
of the desired compound as yellow oil in 82% yield.
Methyl 5-Methoxy-3-{[(4-methylphenyl)sulfonyl]oxy}pentanoate
(11). To a solution of methyl 3-hydroxy-5-methoxypentanoate (30.0 g,
185 mmol, 1.0 equiv) in pyridine (200 mL) at 0 °C was added TsCl
(43.2 g, 222 mmol, 1.2 equiv). The resulting orange solution was
stirred at rt for 30 h, and the reaction turned into brown color. The
reaction mixture was diluted with 500 mL of EtOAc and neutralized
with 2 N HCl and 1 N HCl. The organic layer was separated, washed
with saturated NaHCO3 and brine, dried over Na2SO4, filtered, and
concentrated. The resulting residue was columned on silica gel using
4:1 and 2:1 heptane/EtOAc to afford 23 g of the desired compound in
50% yield. 1H NMR (400 MHz, CDCl3) δ ppm 7.81 (d, J = 8.06 Hz,
2H), 7.35 (d, J = 8.56 Hz, 2H), 5.03 (quin, J = 6.23 Hz, 1H), 3.61 (s,
1
trans/cis isomers in a 5:1 ratio. H NMR for trans isomer (400 MHz,
DMSO-d6) δ ppm 8.62 (d, J = 8.30 Hz, 1H), 8.06 (br s, 2H), 7.41 (s,
1H), 5.16−5.21 (m, 1H), 3.77−3.85 (m, 1H), 3.37 (t, J = 5.8 Hz, 2H),
3.23 (s, 3H), 2.41−2.50 (m, 1H), 2.32−2.38 (m, 1H), 1.96−2.05 (m,
1H), 1.87 (s, 3H), 1.57−1.77 (m, 3H).
The amide intermediate (a mixture of trans/cis isomers in a 5:1
ratio, 1.80 g, 4.53 mmol, 1.0 equiv) was added into a solution of BH3
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Article
3H), 3.33−3.40 (m, 1H), 3.23−3.32 (m, 1H), 3.17 (s, 3H), 2.65−2.81
(m, 2H), 2.45 (s, 3H).
into a solution of compound 17b (3.00 g, 9.80 mmol, 1.0 equiv) in 150
mL of MeOH at 0 °C. The resulting white suspension was then stirred
at 25 °C overnight. The solvent (MeOH) was removed under reduced
pressure. The residue was diluted with 100 mL of water and filtered.
The solid was collected and dried under reduced pressure at 60 °C for
3 h to afford 2.45 g of the desired product in 74% yield as a yellow
solid. LCMS (ACPI) m/z 338 (M+ − 1). 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.21 (s, 2H), 7.74 (s, 1H), 4.39−4.56 (m, 1H),
3.47−3.56 (m, 2H), 3.24 (s, 3H), 3.22−3.26 (m, 2H), 2.20−2.29 (m,
1H), 1.81−1.91 (m, 1H).
Methyl 5-Methoxy-3-(2-thienylthio)pentanoate (12). To a sol-
ution of compound 11 (21.70 g, 68.59 mmol, 1.0 equiv) in 6.0 mL of
THF (very small amounts) was added 2-thiophenethiol (7.05 mL, 72.0
mmol, 1.05 equiv). The resulting yellow solution was stirred at 25 °C
for 10 min, and Et3N (11.5 mL, 1.2 equiv) was added at 0 °C. Lots of
solids were generated. The suspension was stirred at 25 °C for 2−3 h
to reach completion. The mixture was diluted with EtOAc (400 mL)
and washed with brine (500 mL). The organic layer was collected,
dried over Na2SO4, filtered, and concentrated. The residue was
columned on silica gel using 10:1 and 4:1 heptane/EtOAc) to afford
cis-4-Hydroxy-6-(2-methoxyethyl)-5,6-dihydro-4H-thieno[2,3-b]-
thiopyran-2-sulfonamide 7,7-Dioxide (19b). To a suspension of
compound 18b (2.41 g, 7.10 mmol, 1.0 equiv) in 90 mL of EtOH at 0
°C was added slowly NaBH4 (0.322 g, 8.52 mmol, 1.20 equiv). The
resulting pale yellow solution was stirred at 0 °C for 15 min to reach
completion. EtOH was removed under reduced pressure. To the oily
residue was added 50 mL of water. The solution was acidified with 2 N
HCl to pH = 2−3 (small amounts of 2 N HCl were needed) to crush
out the product. The white solid was filtered, washed with 10 mL of
water, and dried under reduced pressure to give 2.0 g of the desired
product in 82% yield. The product is exclusively cis isomer. The cis
1
15.0 g of the desired compound in 84% yield as yellow oil. H NMR
(400 MHz, CDCl3) δ ppm 7.41 (dd, J = 5.41, 1.13 Hz, 1H), 7.17 (dd,
J = 3.53, 1.26 Hz, 1H), 7.00−7.04 (m, 1H), 3.71 (s, 3H), 3.48−3.67
(m, 2H), 3.36−3.43 (m, 1H), 3.35 (s, 3H), 2.53−2.68 (m, 2H), 1.75−
1.87 (m, 2H).
6-(2-Methoxyethyl)-5,6-dihydro-4H-thieno[2,3-b]thiopyran-4-
one (16b). A mixture of compound 12 (15.0 g, 57.6 mmol), 6.0 M
aqueous HCl (300 mL), and HOAc (50 mL) was heated to 100 °C.
After being refluxed at 100 °C for 4 h, the reaction was complete. The
mixture was cooled to 25 °C, and the mixture was concentrated under
reduced pressure. The orange sticky oily residue was diluted with
toluene (200 mL), dried over Na2SO4, filtered, and concentrated to
afford 14.0 g of the hydrolyzed intermediate as a pale yellow oil. The
crude hydrolyzed intermediate (14 g, 56.8 mmol) was dissolved in dry
toluene (300 mL), and Tf2O (10.5 mL, 62.5 mmol, 1.10 equiv) was
added slowly at 0 °C to give a colorless solution. The reaction mixture
turned slowly from colorless to gray and eventually to brown. After
being stirred at 0 °C for 30 min, the ice bath was removed and the
reaction was stirred at 25 °C for 1.5 h to reach completion. The excess
amounts of Tf2O were destroyed by 10 mL of water. The reaction
mixture was then diluted with 100 mL of EtOAc, washed with brine,
dried over Na2SO4, filtered, and concentrated. The black oily residue
was purified on silica gel using 4:1 and 2:1 heptane/EtOAc to afford
8.60 g of the desired product as yellow oil in 67% yield. 1H NMR (400
MHz, CDCl3) δ ppm 7.45 (d, J = 5.54 Hz, 1H), 7.02 (d, J = 5.29 Hz,
1H), 3.84−3.92 (m, 1H), 3.45−3.58 (m, 2H), 3.35 (s, 3H), 2.98 (dd, J
= 16.74, 3.40 Hz, 1H), 2.75 (dd, J = 16.87, 10.07 Hz, 1H), 1.98−2.07
(m, 2H).
1
stereochemistry was determined by NOSEY experiments. H NMR
(400 MHz, DMSO-d6) δ ppm 8.03 (s, 2H), 7.56 (s, 1H), 6.05 (d, J =
7.05 Hz, 1H), 4.85 (ddd, J = 10.45, 6.67, 5.54 Hz, 1H), 3.75−3.87 (m,
1H), 3.55 (t, J = 6.29 Hz, 2H), 3.27 (s, 3H), 2.48−2.54 (m, 1H),
2.05−2.26 (m, 2H), 1.81 (dd, J = 14.35, 7.81 Hz, 1H).
(−)-(4S,6R)-4-(Ethylamino)-6-(2-methoxyethyl)-5,6-dihydro-4H-
thieno[2,3-b]thiopyran-2-sulfonamide 7,7-Dioxide (20b). To a
suspension of compound 19b (2.00 g, 5.86 mmol, 1.0 equiv) in
anhydrous MeCN (36 mL) under ice−NaCl bath was added slowly 9
mL of conc H2SO4. The resulting transparent pale orange solution was
stirred at 25 °C for 48 h (CAUTION: excess reaction time will make
the reaction messier) to reach completion. The reaction mixture was
diluted with a mixture of water (150 mL) and EtOAc (150 mL) at 0
°C. The organic solution was separated, and the aqueous solution was
washed with 2 × 100 mL of EtOAc. Organic layers were combined,
dried over Na2SO4, and concentrated to form a brown sticky oil. The
oily compound was purified on silica gel using 10:1 DCM/MeOH to
afford 1.60 g (44% yield) of the desired amide intermediate as a trans/
cis mixture in 5:1 ratio. LCMS (APCI, negative) m/z 381 (M+ − 1).
1H NMR for trans isomer (400 MHz, DMSO-d6) δ ppm 8.63 (d, J =
6-(2-Methoxyethyl)-4-oxo-5,6-dihydro-4H-thieno[2,3-b]-
thiopyran-2-sulfonamide (17b). ClSO3H (1.59 mL, 23.8 mmol, 1.2
equiv) was added into a precooled DCM (20 mL). The resulting
diluted colorless ClSO3H−DCM solution was added slowly into a
precooled solution of compound 16b (4.53 g, 19.9 mmol) in 100 mL
of DCM at NaCl−ice bath. The resulting deep brown mixture was
stirred at rt for 6 h to reach completion. PCl5 (4.96 g, 23.8 mmol, 1.2
equiv) was then added into the reaction mixture portionwise at 0 °C.
The resulting orange solution was stirred at 25 °C for 0.5 h, during
which the reaction mixture turned from orange color to deep blue
color. The reaction mixture was poured into a separatory funnel that
contained ice. Another 200 mL of water and 100 mL of DCM were
added. The organic layer turned into a blue color, which was collected
quickly, dried over Na2SO4, filtered, and concentrated to be a 15 mL
blue solution. At 0 °C, to the 15 mL of blue solution were added
dioxane (100 mL), precooled 0.5 M NH3 in dioxane (50 mL), and 7.0
M NH3 in MeOH (50 mL). The resulting suspension was stirred at rt
for 30 min to reach completion. The solid was filtered, and the filtrate
was concentrated. The concentrated residue was diluted with EtOAc
(200 mL) and washed with brine (200 mL). The organic layer was
collected and concentrated under reduced pressure. The resulting
residue was kept in the freezer for solidification. The solid was
triturated with heptane/EtOH three times to afford 3.8 g of the
desired product in 60% yield. 1H NMR (400 MHz, DMSO-d6) δ ppm
7.82 (br s, 1H), 7.65 (s, 1 H), 3.95−4.05 (m, 1H), 3.39−3.50 (m, 2H),
3.24 (s, 3H), 2.91−3.00 (m, 1H), 2.75−2.84 (m, 1H), 1.85−2.06 (m,
2H).
8.31 Hz, 1H), 8.06 (br s, 2H), 7.41 (s, 1H), 5.16−5.23 (m, 1H), 3.81−
3.91 (m, 1H), 3.46−3.60 (m, 2H), 3.26 (s, 3H), 2.39−2.48 (m, 2H),
2.13−2.26 (m, 1H), 1.86 (s, 3H), 1.77−1.85 (m, 1H).
The obtained amide intermediate (trans/cis in 5:1 ratio, 1.60 g, 4.2
mmol) was added into a solution of BH3 (1.40 mL, 14.2 mmol, 3.37
equiv) in 12 mL of THF at 0 °C. After being stirred at rt for 48 h, the
reaction mixture was poured into 130 mL of aq H2SO4 (2.5 M) at 0
°C. Lots of white sticky solids were generated, which were slowly
redissolved. The mixture was then heated at 50 °C for 2 h to
completely decompose amine−borane complexes. The mixture was
cooled to 0 °C and basified by 10 M NaOH and 2.0 M NaOH to pH
≈ 8. The resulting mixture was then stirred at rt for another 1 h and
extracted with 3 × 100 mL of 9:1 CHCl3/MeOH. The organic
solutions were combined, dried over MgSO4, and concentrated to
1
afford 1.10 g of the desired product as a white solid. From H NMR,
the product consists of a mixture of trans/cis isomers in a 5:1 ratio.
The racemic product was separated by SFC to give 310 mg (20%
yield) of enatiomerically pure (−)-20b. LCMS (APCI) m/z 369.0 (M+
1
+ 1). H NMR (400 MHz, DMSO-d6) δ ppm 8.01 (s, 2H), 7.57 (s,
1H), 3.88−3.97 (m, 2H), 3.51−3.58 (m, 2H), 3.27 (s, 3H), 2.46−2.70
(m, 3H), 2.31−2.45 (m, 1H), 2.13−2.30 (m, 2H), 1.72−1.83 (m, 1H),
1.03 (t, J = 7.05 Hz, 3H).
(4S,6R)-6-(2-Bromoethyl)-4-(ethylamino)-5,6-dihydro-4H-thieno-
[2,3-b]thiopyran-2-sulfonamide 7,7-Dioxide (22). A solution of
compound (−)-20b (220 mg, 0.597 mmol) in 25 mL of 48% aqueous
HBr was stirred at 85 °C for 5 days. The reaction was cooled to 25 °C
and all the solvent was removed under reduced pressure to afford 298
mg of the desired product in a HBr salt form as a yellow solid in 100%
6-(2-Methoxyethyl)-4-oxo-5,6-dihydro-4H-thieno[2,3-b]-
thiopyran-2-sulfonamide 7,7-Dioxide (18b). A solution of Oxone
(13.20 g, 21.5 mmol, 2.2 equiv) in 60 mL of water was added slowly
1
yield. LCMS (APCI) m/z 416.8 (M+ + 1). H NMR (400 MHz,
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DMSO-d6) δ ppm 8.98 (br s, 2H), 8.20 (s, 2H), 7.87 (s, 1H), 4.73−
4.84 (m, 1H), 4.12−4.22 (m, 1H), 3.76 (t, J = 7.05 Hz, 2H), 3.16−
3.29 (m, 1H), 3.01−3.14 (m, 1H), 2.73−2.84 (m, 1H), 2.57−2.70 (m,
1H), 2.42−2.49 (m, 1H), 2.21−2.35 (m, 1H), 1.24 (t, J = 7.18 Hz,
3H).
the solid was filtered and the filtrate was concentrated. The residue
was diluted with 200 mL of EtOAc and washed with 200 mL of brine.
The organic layer was collected and the solvent was the removed to
afford a yellow residue, which was triturated twice with EtOAc/
1
heptane to afford 5.0 g of the pure desired product in 49% yield. H
(−)-2-[(4S,6R)-2-(Aminosulfonyl)-4-(ethylamino)-7,7-dioxido-5,6-
dihydro-4H-thieno[2,3-b]thiopyran-6-yl]ethyl Nitrate (3b). To a pale
yellow homogeneous solution of compound 22 (HBr salt, 290 mg,
0.582 mmol) in MeCN (20 mL) at 0 °C was added AgNO3 (770 mg,
4.5 mmol, 7.8 equiv). The resulting suspension was heated at 50 °C
for 24 h to reach completion. The reaction was diluted with CH3CN
and filtered through Celite. The filtrate was concentrated to give a
yellow solid, which was purified by HPLC to afford 200 mg of the
desired product in 86% yield. LCMS (APCI) m/z 400.0 (M+ + 1). 1H
NMR (400 MHz, DMSO-d6) δ ppm 8.88 (br s, 2H), 8.20 (s, 2H),
7.85 (s, 1H), 4.67−4.85 (m, 3H), 4.09−4.20 (m, 1H), 3.19−3.32 (m,
1H), 2.98−3.14 (m, 1H), 2.58−2.81 (m, 2H), 2.31−2.44 (m, 1H),
2.10−2.21 (m, 1H), 1.23 (t, J = 7.05 Hz, 3H).
NMR (400 MHz, DMSO-d6) δ ppm 7.83 (br s, 2H), 7.64 (s, 1H),
4.17−4.30 (m, 1H), 3.53−3.69 (m, 2H), 3.28 (s, 3H), 2.84−2.91 (m,
1H), 2.75−2.84 (m, 1H).
4-Hydroxy-6-(methoxymethyl)-5,6-dihydro-4H-thieno[2,3-b]-
thiopyran-2-sulfonamide (24). Compound 17c (1.50 g, 5.22 mmol,
1.0 equiv) was suspended in 20 mL of EtOH and cooled to 0 °C.
NaBH4 (243 mg, 6.78 mmol, 1.3 equiv) was added portionwise. The
pale yellow solution was stirred at 0 °C for 15 min and 25 °C for
another 1 h to reach completion. EtOH was removed under reduced
pressure. An amount of 20 mL of water was added into the oily
residue, and the mixture was acidified with 2 N HCl to pH 5−7. At
this point, white solids were precipitated out. The solid was filtered
and washed with 5 mL of water. The solid was collected and dried
under reduced pressure to give 1.25 g of the desired product in 81%
yield as a pale yellow solid. The cis isomer is the major product, which
contains <5% trans isomer. The cis stereochemistry was confirmed by
Synthesis of Compound 3c. Methyl 4-Methoxy-3-(2-
thienylthio)butanoate (15). To a solution of methyl trans-4-bromo-
2-butenoate (compound 13, 38.5 g, 215 mmol) in anhydrous MeOH
(100 mL) was added Ag2O (37.4 g, 161 mmol, 0.75 equiv). The
reaction mixture was sonicated overnight to reach completion. After
filtration to remove solid and rinsing solid with 50 mL of MeOH, the
filtrate was mixed with 2-thiophenethiol (25.0 g, 215 mmol, 1.0 equiv)
and Et3N (2.18 g, 21.5 mmol, 0.1 equiv). The reaction mixture was
stirred at room temperature overnight. After removal of solvent, the
residue was purified on silica gel column using hexane/EtOAc to afford
1
2D NMR. LCMS (APCI) m/z 294 (M+ − 1). H NMR (400 MHz,
DMSO-d6) δ ppm 7.59 (s, 2H), 7.44 (s, 1H), 5.65 (d, J = 6.04 Hz,
1H), 4.67 (ddd, J = 9.63, 5.29, 4.97 Hz, 1H), 3.89−3.99 (m, 1H), 3.67
(dd, J = 10.20, 4.66 Hz, 1H), 3.47 (t, J = 9.57 Hz, 1H), 3.28 (s, 3 H),
2.25 (ddd, J = 13.22, 5.16, 2.27 Hz, 1H), 1.70 (ddd, J = 13.22, 10.45,
10.32 Hz, 1H).
N-[2-(Aminosulfonyl)-6-(methoxymethyl)-5,6-dihydro-4H-thieno-
[2,3-b]thiopyran-4-yl]acetamide (25). H2SO4 (0.168 mL, 0.70 equiv)
was dissolved in anhydrous CH3CN (7.0 mL) at 0 °C. A solution of
H2SO4−CH3CN was added to a clear solution of compound 24 (1.00
g, 3.39 mmol, 1.0 equiv) in 90 mL of anhydrous CH3CN at 0 °C. After
being stirred at 0 °C for 2−3 h, the reaction was continued running at
25 °C for 2 days. The reaction mixture was then concentrated to give a
light green suspension. The suspension was placed still at least 3 h for
precipitation. The suspension was then filtered and washed with
EtOAc to afford 900 mg of the amide product in 80% yield. The trans
1
42.46 g of the desired product as oil in 80% yield over two steps. H
NMR (400 MHz, CDCl3) δ ppm 7.68−7.77 (dd, J = 1.2, 5.3 Hz, 1H),
7.18−7.27 (dd, J = 1.3, 3.2 Hz, 1H), 7.05−7.16 (dd, J = 5.3, 3.5 Hz,
1H), 3.62 (s, 3H), 3.41−3.51 (m, 1H), 3.30−3.41 (m, 2 H), 3.25 (s,
3H), 2.62−2.75 (m, 1H), 2.39−2.49 (m, 1H).
6-(Methoxymethyl)-5,6-dihydro-4H-thieno[2,3-b]thiopyran-4-
one (16c). To a mixture of compound 15 (41.3 g, 168 mmol) in 1,4-
dioxane (660 mL) was added 6 N aqueous HCl. After being stirred at
80 °C for 16 h, the reaction mixture was allowed to cool to room
temperature, diluted with water (600 mL), and extracted with EtOAc
(3 × 400 mL). The organic layers were combined, dried over MgSO4,
filtered, and concentrated. The residue was passed through a large
silica gel plug, eluted with 2 L of 40% EtAOc/hexanes, and
concentrated to dryness. The resulting 42.4 g of acid product was
diluted with toluene (600 mL) and treated with trifluoroacetic
anhydride (151 mL, 1.1 mol, 6.0 equiv). After being stirred at 25 °C
for 3 days, the mixture was passed through a silica gel plug, eluted with
500 mL of 25% EtOAc/hexane, and concentrated to dryness. Crude
material was purified via normal phase chromatography, eluting with
5−25% EtOAc/hexane to afford 13.79 g of the desired compound as a
1
stereochemistry was confirmed by 2D NMR. H NMR (400 MHz,
DMSO-d6) δ ppm 8.37 (d, J = 8.31 Hz, 1H), 7.63 (br s, 2H), 7.50−
7.76 (m, 2H), 7.27 (s, 1H), 5.04 (ddd, J = 8.50, 4.53, 4.34 Hz, 1H),
3.78−3.88 (m, 1H), 3.67 (dd, J = 10.07, 5.29 Hz, 1H), 3.44−3.54 (m,
1H), 3.30 (s, 3H), 2.02−2.11 (m, 1H), 1.85−1.93 (m, 1H), 1.84 (s,
3H).
4-(Ethylamino)-6-(methoxymethyl)-5,6-dihydro-4H-thieno[2,3-
b]thiopyran-2-sulfonamide (26). Neat solid compound 25 (900 mg,
2.68 mmol, 1.0 equiv) was added into a solution of BH3−DMS (0.863
mL, 9.10 mmol, 3.40 equiv) in THF (12.0 mL) at 0 °C. The reaction
mixture was stirred at 0 °C for 5 min, and a yellow transparent
solution was obtained. The reaction was then stirred at 50 °C for 12 h
to reach completion. The reaction was cooled to 0 °C and then poured
into 20 mL of aq H2SO4 (2.5 M). After being stirred at 25 °C for 20
min and at 50 °C for 2 h, all amine−borane complexes were converted
into the desired product as indicated by LCMS. The mixture was
cooled to 0 °C and was neutralized by using 10 M NaOH and 2.0 M
NaOH to tune pH to ∼8. The neutralized mixture was then stirred at
25 °C for another 1 h. The reaction mixture was then extracted with 3
× 30 mL of 7:1 CHCl3/MeOH. The organic solutions were combined,
dried over Na2SO4, concentrated, and purified by HPLC to afford 600
mg of the desired product in 70% yield as a white solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 8.20 (s, 1H), 7.58 (br s, 2H), 7.40 (s, 1H),
3.88−3.97 (m, 1H), 3.84 (t, J = 3.78 Hz, 1H), 3.68 (dd, J = 10.07, 5.04
Hz, 1H), 3.48 (dd, J = 9.82, 8.81 Hz, 1H), 3.30 (s, 3H), 2.64−2.73 (m,
1H), 2.53−2.61 (m, 1H), 2.17−2.24 (m, 1H), 1.68 (ddd, J = 14.23,
10.95, 3.78 Hz, 1H), 1.04 (t, J = 7.05 Hz, 3H).
1
brown oil. H NMR (400 MHz, CDCl3) δ ppm 7.84 (d, J = 5.4 Hz,
1H), 7.05 (d, J = 5.3 Hz, 1H), 3.89−4.03 (m, 1H), 3.60−3.69 (m,
2H), 3.43 (s, 3H), 2.83−2.94 (m, 2H).
6-(Methoxymethyl)-4-oxo-5,6-dihydro-4H-thieno[2,3-b]-
thiopyran-2-sulfonamide (17c). ClSO3H (2.40 mL, 35.3 mmol, 1.05
equiv) was added into a precooled DCM (20 mL). The diluted
colorless ClSO3H−DCM solution was added slowly into a precooled
solution of compound 16c (7.20 g, 34.9 mmol) in DCM (200 mL) at
NaCl−ice bath. A deep brown solution was obtained. After being
stirred at −5 to 0 °C for 10 min and at 25 °C for 3 h, a brown solution
was obtained. The LCMS indicated that the reaction was complete.
PCl5 (7.35 g, 35.3 mmol, 1.05 equiv) was added portionwise at 0 °C,
and an orange solution was obtained. The ice bath was removed after
the completion of addition. After being stirred at 25 °C for 0.5 h, the
reaction mixture turned form orange color to deep blue color. The
reaction was dumped into a separatory funnel that contained ice.
Another 200 mL of water and 100 mL of DCM were added. The
organic layer became a blue color, which was collected quickly and
dried over Na2SO4, filtered, and concentrated to be about 15 mL of
deep blue solution. The solution was placed in 0 °C, and 100 mL of
precooled (0 °C) 0.5 M NH3 in dioxane was added, followed by added
150 mL of 7.0 M NH3 in MeOH. After being stirred at 0 °C for 2 h,
6-(Bromomethyl)-4-(ethylamino)-5,6-dihydro-4H-thieno[2,3-b]-
thiopyran-2-sulfonamide (27). A solution of compound 26 (100 mg,
0.31 mmol) in 5.0 mL of 48% aqueous HBr was stirred at 80 °C for 24
h to reach completion. The resulting pale green solution was
concentrated to afford 141 mg of the desired product as a HBr salt
in 100% yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.72 (br s, 2H),
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7.76 (s, 2H), 7.65 (s, 1H), 4.62 (br s, 1H), 3.96−4.10 (m, 2H), 3.81−
3.89 (m, 1H), 3.19 (br s, 1H), 2.99 (br s, 1H), 2.63 (d, J = 2.27 Hz,
1H), 2.10 (ddd, J = 15.11, 11.33, 4.28 Hz, 1H), 1.23 (t, J = 7.18 Hz, 3
H).
concentration of 1 mM, 50 μM, and then 0.5 μM and transferred to a
96-well polypropylene plate for further serial dilutions (1:3 dilutions,
11 points) in duplicate. Final compound concentration in CA II Kd FP
tight binding assay is 0.01 μM. Assays were conducted in a final
volume of 100 μL in 50 mM Tris/HCl (pH 7.6), 100 mM Na2SO4,
and 0.005% Tween-20 in a 96-well Microfluor 1, black assay plate.
Labeled BODIPY558/568-acetazolamide was used as the tracer.
Binding inhibition was determined by pipetting 8 μL of human CA
II (1.5 nM) into assay plate containing 2 μL of compound and 2 μL of
tracer (0.5 nM) in 88 μL of assay buffer. The assay plate was incubated
at room temperature for 1 h and read in the fluorescence polarization
reader (Molecular Devices, Analyst) at 524/45 nm (excitation) and
595/60 nm (emission). The Kd binding was calculated using
GraphPad Prism and Morrison tight binding ligand equation.
Determination of CA IV IC50 Using Fluorescence Polarization
Assay. Compounds were dissolved in DMSO at a concentration of 1
mM, then to 250 μM in DMSO and transferred to a 96-well plate for
further dilutions (1:3 dilutions, 11 points) in duplicate. Final
compound highest concentration in CA IV FP assay is 5 μM. Assays
were conducted in a final volume of 100 μL in 50 mM Tris/HCl (pH
7.6), 100 mM Na2SO4, and 0.005% Tween-20 in a 96-well black assay
plate. BODIPY558/568-acetazolamide (catalog no. B-12270 from
Invitrogen-Molecular Probes, discontinued item) was used as the
tracer. Binding inhibition was determined by pipetting 8 μL of human
CA IV (25 nM) into assay plate contained 2 μL of compound and 2
μL of tracer (2 nM) in 88 μL of assay buffer. The assay plate was
incubated at ambient temperature for 30 min and read in the
fluorescence polarization reader (Molecular Devices, Analyst) at 524/
45 nm (excitation), 595/60 nm (emission), and 561 nm (beam
splitter). The IC50, the inhibitor concentration resulting in 50%
inhibition of the enzyme activity, was calculated using GraphPad Prism
or similar in-house software with the IC50 curve fitting using the four-
parameter logistic equation.
Formulation Methods. Compounds 3a and 4a have physical
properties similar to those of brinzolamide (2) and are poorly soluble
in water, making them suitable for a topical ophthalmic suspension
formulation. The compounds were formulated at up to 2% w/v final
concentration as sterile, aqueous suspensions to be readily suspended
and slow settled, following shaking. The final pH was set between 5.5
and 7.5 and the osmolality at 300 mOsm/kg. Each milliliter of
formulation contained up to 20 mg of the respective active ingredient
and was preserved with benzalkonium chloride (0.2 mg). The vehicle
was composed of aqueous 4.5% w/v mannitol, 0.35% w/v carbomer
974P, 1−5% w/v polyethoxylated castor oil, 0.01% w/v edetate
disodium, with hydrochloric acid and/or sodium hydroxide to adjust
pH if needed. The active ingredients were homogenized and
suspended in the aforementioned vehicle to achieve dosing
concentrations (0.1−20 mg/mL) required for preclinical evaluation
using a magnetic stir bar and magnetic stir plate with 700 rpm mixing
speed for 24 h. All the excipients (except for Cremophor EL) used to
formulate compounds 3a and 4a were USP/NF grade, obtained from
Sigma-Aldrich (St. Louis, MO).
[trans-2-(Aminosulfonyl)-4-(ethylamino)-7,7-dioxido-5,6-dihy-
dro-4H-thieno[2,3-b]thiopyran-6-yl]methyl Nitrate (3c). A mixture
of compound 27 (HBr salt, 141 mg, 0.31 mmol), silver nitrate (421
mg, 2.47 mmol, 8.0 equiv), and CH3CN (5.0 mL) was stirred at 25 °C
for 12 h to reach completion. The resulting suspension was filtered to
afford a crude compound 28, which was subjected for the next Oxone
oxidation without further purification. To a solution of crude
compound 28 in 3 mL of MeOH was added slowly a solution of
Oxone (0.78 mmol, 2.5 equiv) in water (3.0 mL) at 0 °C. A white
suspension was obtained. The resulting suspension was stirred at 25
°C for 12 h to reach completion. The reaction mixture was carefully
diluted with 20 mL of water and extracted with 2 × 30 mL of DCM.
The organic layers were combined, dried over Na2SO4, concentrated,
and purified by HPLC to give 60 mg of the desired product as a yellow
1
solid in 50% yield. LCMS (APCI) 385.9 (M+ + 1). H NMR (400
MHz, DMSO-d6) δ ppm 9.47 (br s, 1H), 9.34 (br s, 1H), 8.22 (s, 2H),
7.97 (s, 1H), 4.98−5.14 (m, 2H), 4.70−4.82 (m, 2H), 3.16−3.33 (m,
1H), 3.00−3.12 (m, 1H), 2.80−2.90 (m, 1H), 2.65−2.76 (m, 1H),
1.27 (t, J = 6.7 Hz, 3H).
Synthesis of Compound 4a. (R)-4-(Ethylamino)-2-(3-hydrox-
ypropyl)-3,4-dihydro-2H-thieno[3,2-e][1,2]thiazine-6-sulfonamide
1,1-Dioxide (6a). To a slurry of compound 1 (397 mg, 1.04 mmol, 1.0
equiv) in 20 mL of DCM under N2 at −78 °C was added a solution of
BBr3 (1.0 M, 4.1 mL, 4.10 mmol, 3.94 equiv) dropwise. The slurry was
allowed to stir for 10 min at −78 °C and gradually warmed to 25 °C.
After being stirred at 25 °C for 24 h, it was carefully quenched by 20
mL of MeOH at 0 °C, and the resulting colorless homogeneous
mixture was then stirred at 25 °C for 30 min. The solution was then
concentrated to provide an oil that was azeotroped with 3 × 10 mL of
MeOH. The crude oil was purified by reverse HPLC (1−30%,
CH3CN/H2O, 0.1% AcOH) to afford 255 mg of the desired product
1
in 66% yield as a white solid. LCMS (ESI) m/z 370.0 (M+ + 1). H
NMR (300 MHz, D2O) δ ppm 7.82 (s, 1H), 4.76−4.80 (m, 1H), 4.23
(dd, J = 15.6, 4.3 Hz, 1H), 4.09 (dd, J = 15.6, 5.8 Hz, 1H), 3.68 (t, J =
6.0 Hz, 2H), 3.53 (dt, J = 14.3, 7.3 Hz, 1H), 3.38 (dt, J = 13.8, 6.8 Hz,
1H), 3.14 (q J = 7.3 Hz, 2H), 1.82−2.01 (m, 2H), 1.28 (t, J = 7.2 Hz,
3H).
(R)-2-(3-Bromopropyl)-4-(ethylamino)-3,4-dihydro-2H-thieno-
[3,2-e][1,2]thiazine-6-sulfonamide 1,1-Dioxide (29). A solution of
compound 6a (1.05 g, 2.74 mmol) in 10 mL of HBr (48%) was stirred
at 80 °C for 18 h to reach completion. The resulting brown
homogeneous solution was concentrated under vacuum and the
residue was purified by reverse HPLC (1−30%, CH3CN/H2O, 0.1%
AcOH) to afford 738 mg the desired product in 54% yield as a white
solid. LCMS (ESI) m/z 432.0 (M+ + 1). 1H NMR (300 MHz, DMSO-
d6) δ ppm 8.21 (s, 2H), 7.99 (s, 1H), 4.97 (S, 1H), 4.05−4.23 (m,
2H), 3.40−3.71 (m, 4H), 3.23−3.39 (m, 1H), 2.98−3.21 (m, 2H),
2.17 (dt, J = 13.2, 6.6 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H).
(R)-3-(4-(Ethylamino)-1,1-dioxido-6-sulfamoyl-3,4-dihydro-2H-
thieno[3,2-e][1,2]thiazin-2-yl)propyl Nitrate (4a). To a solution of
compound 29 (738 mg, 1.49 mmol) in 25 mL of CH3CN were added
nitric acid (1.0 mL) and AgNO3 (726 mg, 3.0 equiv). The mixture was
stirred at 60 °C for 24 h, at which point additional AgNO3 (508 mg,
2.0 equiv) was added in one portion. The resulting mixture was then
stirred at 60 °C for additional 24 h to reach completion. The mixture
was cooled to 25 °C and diluted with EtOAc (50 mL) and brine (20
mL). The organic layer was collected, dried over MgSO4, filtered, and
concentrated. The residue was purified by reverse HPLC (1−30%,
CH3CN/H2O, 0.1% AcOH) to afford 174 mg of the desired product
Ocular Pharmacokinetic Assessments in Rabbits. Pigmented
male Dutch-belted (DB) rabbits (n ≥ 3 rabbits/time point) weighing
1.5−2.0 kg were used. All animals received a 2 × 25 μL topical dose of
formulated 3a or 4a in each eye. At 4 h time point, the rabbits were
euthanized and enucleated and ocular tissues were dissected.
Compounds 3a and 5a, or 4a and 6b, were isolated from the
homogenized rabbit ocular tissue (cornea, iris ciliary body, and
aqueous humor) by protein precipitation and quantitated using high
performance liquid chromatography/mass spectrometry (LC/MS/
MS). The LC/MS/MS system used consisted of two Shimadzu LC-
10AD HPLC pumps (Columbia, MD), a CTC HTS PAL autosampler
(LEAP, Carbrboro, NC), and a Sciex API 4000 triple quadrupole mass
spectrometer (Life Technologies, Foster City, CA). Peak area
determination, calculation of the ratio between the analyte to internal
standard peak area, and determination of sample concentrations were
done using Analyst software (Analyst 1.4.1, Life Technologies, Foster
City, CA).
1
in 28% yield. LMCS (ESI) m/z 415.0 (M+ + 1). H NMR(300 MHz,
DMSO-d6) δ ppm 8.05 (br s, 2H), 7.69 (s, 1H), 4.59 (t, J = 6.1 Hz,
2H), 4.14 (br s, 1H), 3.84 (s, 2H), 3.45 (dt, J = 14.1, 7.3 Hz, 1H),
3.21−3.32 (m, 1H), 2.56−2.67 (m, 2H), 2.02 (dt, J = 12.8, 6.4 Hz,
2H), 1.04 (t, J = 6.4 Hz, 3H).
Determination of CA II Kd Using Fluorescence Polarization
Tight Binding Assay. Compounds were diluted in DMSO at a
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Inhibition of CA Activity by Using a PH-STAT Method.
Pigmented male Dutch-belted (DB) rabbits (n ≥ 3 rabbits/time point)
weighing 1.5−2.0 kg were used. All animals received a 2 × 25 μL
topical dose of formulated 3a, 4a, or brinzolamide (2) in each eye. At
the certain time points, the rabbits were euthanized and enucleated
and ocular tissues were dissected. The ciliary bodies were rinsed with
water and mechanically homogenized with small glass beads in buffer
containing 4-(hydroxylethyl)-1-piperazineethanesulfonic acid
(HEPES) 0.01 M and tris[hydroxylmethyl]aminomethane hydro-
chloride (TRIZMA hydrochloride), 0.01 M, with Na2SO4, 0.1 M,
adjusted to pH 7.5. The sample was agitated for 2 h and then
centrifuged at 12 000 rpm for 15 min. The supernatant was used for
CA activity determination, which was assessed using the pH stat
assay.21 This assay measures the rate of hydration of CO2 by
determining the rate at which a standard solution of NaOH has to be
added to Tris-HCl buffer, pH 8.6, to maintain a constant pH as CO2 is
bubbled into the buffer. Enzymatic activity is proportional to the
volume of 0.025 N NaOH that is required to maintain the pH at a
value of 8.3.
Intraocular Pressure Measurements. IOP was measured in
conscious normotensive Dutch-belted (DB) rabbits or unilaterally
lasered ocular hypertensive cynomolgus monkeys following topical
ocular dosing of test articles (vehicle and active compound containing
formulations). Each animal was dosed topically in the right eye (OD)
with 2 × 25 μL drops of freshly formulated NO-CAI (3a or 4a) or
brinzolamide (2). As controls, contralateral eyes were treated with
placebo vehicle only. All test and control articles were administered in
the morning (9:00 a.m.) in a masked fashion. IOP was measured using
a model 30 classic pneumatonometer (Medtronic, Minneapolis, MN)
before administration of test articles (0 h) and then at predetermined
time points after test article administration. Animals received 30 μL of
topical 0.25% tetracaine HCl (anesthetic, Henry Schein, USA) prior to
each IOP measurement. An average of three IOP measurements was
taken at each time point for both eyes of an animal. All animal-related
procedures were conducted in accordance with the Association for
Research in Vision and Ophthalmology (ARVO) statement for the use
of animals in ophthalmic research and were approved by the
appropriate animal care and use committees of each institute.
(3) Krupin, T.; Wax, M.; Moolchandani, J. Aqueous production.
Trans. Ophthalmol. Soc. U. K. 1986, 105, 156−161.
(4) Mincione, F.; Scozzafava, A.; Supuran, C. T. The development of
topically carbonic anhydrase inhibitors as antiglaucoma agents. Curr.
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AUTHOR INFORMATION
Corresponding Authors
*Q.H.: phone, 858-622-3165; email, qinhua.huang@pfizer.com.
*E.Y.R.: phone, 858-526-4666; email, eugene.rui@pfizer.com.
■
Notes
The authors declare no competing financial interest.
(13) Behar-Cohen, F. F.; Goureau, O.; D’Hermies, F.; Courtois, Y.
Decreased intraocular pressure induced by nitric oxide donors is
correlated to nitrite production in the rabbit eyes. Invest. Ophthalmol.
Visual Sci. 1996, 37, 1711−1715.
ACKNOWLEDGMENTS
We are grateful to Dr. Ted W. Johnson at Pfizer for helpful
discussions.
■
(14) (a) Patterson, B. D.; Rui, E. Y. Pfizer Inc. Thieno [3,2-E][1,2]
thiazine derivative as inhibitor of carbonic anhydrase. WO2008120099,
October 9, 2008. (b) Huang, Q.; Rui, E. Y. Preparation of thieno[2,3-
b]thiopyrans as therapeutic inhibitors of carbonic anhydrase for
treating eye disease. WO2009007814, January 15, 2009 (c) Steele, R.
M.; Benedini, F.; Biondi, S.; Borghi, V.; Carzaniga, L.; Impagnatiello,
F.; Miglietta, D.; Chong, W. K.; Rajapakse, R.; Cecchi, A.; Temperini,
C.; Supuran, C. T. Nitric oxide-donating carbonic anhydrase inhibitors
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Cecchi, A.; Temperini, C.; Formicola, G.; Pacileo, I.; Scozzafava, A.;
Masini, E.; Supuran, C. T. Synthesis and crystallographic analysis of
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(e) Chen, J.; Runyan, S. A.; Robinson, M. R. Novel ocular
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2011, 5, 667−677.
ABBREVIATIONS USED
■
IOP, intraocular pressure; CA, carbonic anhydrase; CAI,
carbonic anhydrase inhibitor; NO, nitric oxide; NO-CAI,
NO-donor containing carbonic anhydrase inhibitor; NO-
antiglaucoma drug, NO-donor containing antiglaucoma drug
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