Nitric oxide-donating carbonic anhydrase inhibitors for the treatment of open-angle glaucoma

Article abstract of DOI:10.1016/j.bmcl.2009.10.036

Novel bi-functional compounds with a nitric oxide (NO)-releasing moiety bound to a dorzolamide scaffold were investigated. Several compounds were synthesized and their activity as selective carbonic anhydrase inhibitors (CAI) evaluated in vitro on recombi

Full text of DOI:10.1016/j.bmcl.2009.10.036

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6565–6570
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Nitric oxide-donating carbonic anhydrase inhibitors for the treatment
of open-angle glaucoma q
a
a
a
a
Rebecca M. Steele a, , Francesca Benedini , Stefano Biondi , Valentina Borghi , Laura Carzaniga ,
*
Francesco Impagnatiello a, Daniela Miglietta a, Wesley K. M. Chong b, Ranjan Rajapakse b,
Alessandro Cecchi c, Claudia Temperini c, Claudiu T. Supuran c,
*
a NicOx Research Institute, Via Ariosto 21, 20091 Bresso, Milan, Italy
b Pfizer Global Research & Development-La Jolla, 10770 Science Center Drive, San Diego, CA 92121, USA
c Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel bi-functional compounds with a nitric oxide (NO)-releasing moiety bound to a dorzolamide scaf-
fold were investigated. Several compounds were synthesized and their activity as selective carbonic
anhydrase inhibitors (CAI) evaluated in vitro on recombinant hCA type I, II and IV enzyme isoforms where
they showed different degrees of potency and selectivity to hCA II. A high resolution X-ray crystal struc-
ture for the CA II adduct with 8 confirmed the high affinity of this class of compounds for the enzyme.
Compounds 4, 6, and 8 showed highly potent and efficacious NO-mediated properties as assessed by their
vascular relaxant effect on methoxamine-precontracted rabbit aortic rings. Finally, compounds 4 and 6
exerted potent intraocular pressure (IOP) lowering effects in vivo in normotensive rabbits thereby antic-
ipating their potential for the treatment of hypertensive glaucoma.
Received 21 September 2009
Revised 5 October 2009
Accepted 7 October 2009
Available online 13 October 2009
Keywords:
Carbonic anhydrase
NO-donating agent
Antiglaucoma drug
Sulfonamide
Ó 2009 Elsevier Ltd. All rights reserved.
Dorzolamide
X-ray crystallography
Open-angle glaucoma is a common multifactorial disease of
the eye which is often associated with elevated intraocular pres-
sure (IOP) leading to damage of the optic nerve and conse-
quently loss of vision.1,2 Indeed, targeting IOP with pressure
lowering agents still remains the main treatment strategy for
this disease.3
IOP depends on the delicate balance between production of
aqueous humor by the ciliary body and its drainage through
the conventional trabecular meshwork outflow facility and the
unconventional uveoscleral pathway.4 Selective activation of the
carbonic anhydrase type-II isozyme (CA II), which is expressed
in the ciliary bodies, or elicited via b-adrenergic stimulation,
facilitates aqueous formation and transport through cell mem-
branes, as well as its release into the posterior chamber and,
thereby unregulated CA can increase IOP.5–8 By contrast, allevia-
ventional outflow through the trabecular meshwork has recently
revealed therapeutic roles by a selective prostamide receptor
subtype10 and cytoskeletal-acting kinases.11 Interestingly, the
nitric oxide (NO)/soluble guanylyl cyclase (sGC) signaling path-
way12 is known to enhance local cyclic guanosine monophos-
phate (cGMP) levels, presumably beneficially for aqueous
humor homeostasis.13
Accordingly, topical carbonic anhydrase inhibitors (CAI’s), such
as dorzolamide (1; TrusoptÒ)14–17, or brinzolamide (2; AzoptÒ)18,19
together with b-blockers like timolol, (TimoptolÒ)20 and PGF2
a
analogues21 latanoprost (XalatanÒ) and travoprost (TravatanÒ),
are widely used drugs for controlling aqueous humor dynamics
and IOP in hypertensive glaucoma. Systemic CAI’s, such as aceta-
zolamide (3; Diamox)5 see limited use due to unpleasant side ef-
fects that arise, such as fatigue, paresthesias, and kidney stones
and emphasise the advantages of containing exposure via topical
dosing to the eye.6–8
tion of IOP by prostaglandins, specifically F2 (PGF2 ) receptors
a
a
modulate uveoscleral outflow.9 Emerging understanding of con-
O O
S
O O
O
S
O
S
S
N
S
q
S
O
O
NH2
H
The X-ray coordinates of the CA II—8 adduct are available in PDB with the ID
O
NH2
O
NH2
O
S
N
S
3K2F.
N N
* Corresponding authors. Tel.: +39 02 61036302; fax: +39 02 61036230 (R.M.S.);
tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.).
E-mail addresses: steele@nicox.it (R.M. Steele), claudiu.supuran@unifi.it
(C.T. Supuran).
O
HN
HN
1
2
3
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.036

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R. M. Steele et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6565–6570
Table 1
NO-donors are a relatively underexplored class in the field of ocular
therapeutics despite the ubiquitous presence of NO targets in all eye
compartments devoted to aqueous humor production and drain-
age.22 It has been observed that hypertensive glaucoma patients
have a decreased NO/cGMP content in the aqueous humor23 and
that some NO-donors have been shown to decrease IOP in normal
and pathological conditions.12,24,25
As part of our research activities, we decided to explore NO-
donating CAI, for topical administration, which would combine a
dual mechanism of action in a single molecule, and be capable of
enhancing NO/sGC/cGMP signaling while inhibiting CA enzymes.
These NO-donating CAI could represent an interesting new class
of drugs, with improved activity over existing treatments, to re-
duce IOP more efficiently in hypertensive glaucoma patients.
Inhibitory potency and selectivity of different compounds on recombinant human
carbonic anhydrase (CA) isoforms I, II and IV
NCX
Compound
Ki (nM)
Ki CAII/ Ki CAIV
hCA I
hCA II
hCA IV
Dorzolamide
ISMN
274
265
278
1
15
4
5
6
50,000
NE
2950
470
410
705
9
43
NE
4.7
NE
14
71
13
76
63
4360
46
181
339
3905
311.1
0.6
13.9
4.5
245
201
7
8
1520
62.0
Carbonic anhydrase (CA) activity was monitored using a CO2 hydrase stopped flow
assay.29
NE, not effective at concentrations up to 100 lM.
1
4
5
6
7
8
R = H (Dorzolamide)
R = CO(CH2)5ONO2 (NCX 274)
R = CO-p-(C6H4)-CH2ONO2 (NCX 265)
R = COO(CH2)3ONO2 (NCX 278)
R = COO(CH2)4ONO2 (NCX 245)
O
to be readily hydrolyzed to amines27 and were explored as a poten-
tial NO-donor connection moiety. However, the initial tests carried
out on compounds 4–8 to assess hydrolysis in rabbit corneal
homogenate21,28 documented that they are stable to eye esterases,
thus suggesting that these compounds are novel, stable NO-donat-
ing CAI that exert their activity as such.
O
S
O
S
S
O
NH2
RN
1-8
R = COOCH2CH(ONO2)CH2ONO2 (NCX 201)
As shown in Table 1, the new CAIs were initially assayed for
their inhibitory activity and selectivity on CA enzymes by exposing
an increasing concentration of test drug to the human recombinant
type I, II and IV isoforms.29 Dorzolamide (1) was used as the refer-
ence standard, with CA II Ki = 9 nM and Ki CAII/Ki CAIV = 4.7, as we
wanted to maintain a similar CA enzyme inhibition profile with
the novel NO-donating CAI.15 Compounds 4–8 inhibited CA II with
the following order of potency: 6 P 4 > 8 P 5 P 7 with Ki ranging
from 13 to 76 nM (Table 1). The alkyl nitrate linkers proved to be
more selective for CA II than the benzyl nitrate 5. Shortening the
carbamate chain from 4 to 3 carbon atoms (compounds 7 and 6),
increased significantly both potency on CA II and selectivity over
CA IV (Ki CAII = 13 nM and Ki CAII/Ki CAIV = 13.9 for compound 6).
Notably, compounds 4 and 6 (Ki = 14 and 13 nM, respectively) were
Our intended chemical strategy for attaining these new derivatives
foresaw attachment of an NO-donor linker (incorporating a nitrate
ester) to the amine functionality of dorzolamide. Thus, five new
NO-donating CAI 4–8 (see Scheme 1) were synthesized: amides26
4 and 5, and carbamates, 6, 7 and 8.
The NO-donating moieties included were mainly alkyl nitrates,
with the exception of the benzylic nitrate 5. The dinitrate ester 8
was included to eventually measure the effect of increasing the
theoretical deliverable amount of NO to eye tissues.
It was suspected that these derivatives would undergo hydroly-
sis by the enzymes present in the eye compartments to release
dorzolamide and the NO-donating moiety. Carbamates are known
O
O
O
O
O
O
S
O
S
S
S
O
S
S
O
NH2
S
O
S
(i)
(ii)
S
(iii), (iv)
O
N
O
N
4, 5
HN
NMe2
HN
NMe2
R''
N
O
1
9
10 R'' = (CH2)5Br
11 R'' = p-(C6H4)CH2Cl
(v)
O
S
O
O
S
S
(vi), (vii)
O
N
6, 7
NMe2
R'''
O
N
O
12 R''' = (CH2)3Cl
13 R''' = (CH2)2Cl
(viii), (ix)
14 R''' = CH=CH2
8
Scheme 1. Synthesis of amine derivatives 4–8.26 Reagents and conditions: (i) N,N-dimethylformamide dimethyl acetal, Et3N, DMF, rt, 3 h, 82%; (ii) for compound 10, 6-
bromocaproyl chloride, Et3N, DCM, rt, 22 h, 83%. For compound 11, 4-(chloromethyl)benzoyl chloride, Et3N, DCM, rt, 70 h, 86%; for compound 4: (iii) AgNO3, MeCN, 110 °C,
l
W, 20 min, 71%; (iv) 37% HCl, THF, 110 °C,
compound 12, 3-chloropropyl chloroformate, Et3N, DCM, rt, 66 h, 72%; For compound 13, 4-chlorobutyl chloroformate, Et3N, DCM, rt, 91 h, 60%; For compound 14, allyl
chloroformate, pyridine, DMAP, DCM, rt, 18 h, 33%. For compound 6: (vi) (a) NaI, MeCN, 150 °C, W, 30 min, (b) AgNO3, MeCN, 110 °C, W, 20 min, 75% over two steps; (vii)
37% HCl, THF, 110 °C, W, 28 min, 68%. For compound 7: (vi) 37% HCl, THF, 110 °C, W, 28 min, 94%; (vii) (a) NaI, MeCN, 150 °C, W, 30 min, (b) AgNO3, MeCN, 130 °C,
, 18 h, 69%; (ix) (a) I2, MeOH, À15 °C, 1 h, (b) AgNO3, MeCN, , 20 h, then AgNO3, MeCN, 120 °C,
lW, 28 min, 75%. For compound 5: (iii) 37% HCl, THF, 110 °C, lW, 28 min, 79%; (iv) AgNO3, MeCN, 110 °C, lW, 20 min, 77%. (v) For
l
l
l
l
l
l
l
W,
W,
4 min, 64% over two steps. For compound 8: (viii) 10% HCl, MeOH,
10 min, 21% from 14.
D
D

R. M. Steele et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6565–6570
6567
equipotent to dorzolamide (Ki = 9 nM) for CA II and comparably
selective inhibitors of CA II over CA IV (Ki CAII/Ki CAIV = 311 and
tive enzyme. Inspection of the electron density maps (Fig. 1)
showed the presence of one molecule of inhibitor 8 bound to the
active site (Fig. 2). The analysis of the structure showed that the
13.9, respectively, for compound 4 and 6) to dorzolamide (Ki CAII
/
Ki CAIV = 4.7).
To better understand the interaction of these new NO-donating
CAI with the CA active site, the X-ray crystal structure for the com-
plex of CA II with racemic dinitrate 8 was elucidated (Figs. 1–4, and
Table 2). The structure of the complex was similar to that of the na-
Figure 3. Superposition of the hCA II—8 adduct (inhibitor 8 in yellow, PDB file
3K2F) with the hCA II—dorzolamide (sky blue) adduct (PDB file 1cil) and hCA II—
brinzolamide (magenta) adduct (PDB file 1a42). The heterocyclic scaffolds of the
three inhibitors are superimposable whereas the tails (mainly the NO-donating one
of 8) adopt a different conformation and interact with various amino acid residues.
Figure 1. Omit electron density map of the inhibitor 8 (in yellow) bound within the
human CA contoured at 1.0
the binding.
r level and amino acid residues/Zn(II) ion involved in
Figure 2. Sulfonamide 8 (yellow) bound within the hCA II active site. The Zn(II) ion
(violet sphere), its three histidine ligands (His94, 96 and 119, in green) and proton
shuttle residue (His64, in violet) as observed. The inhibitor completely fills the
active site channel. The enzyme backbone is shown as green ribbon.
Figure 4. Interactions between sulfonamide 8 and amino acid residues/metal ion
within the hCA II active site.

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R. M. Steele et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6565–6570
geometry of the zinc+ binding site and the key hydrogen bonds be-
tween the sulfonamide moiety of the inhibitor 8 and the enzyme
active site were all retained with respect to other hCA II-sulfon-
amide complexes (Figs. 1 and 4). The ligand 8 was involved in mul-
tiple hydrogen bonds with several amino acid residues of the active
site: Asn62, His64, Asn67, Gln92, Thr199 and Pro201 (distances
provided in Table 3). Thus, ligand 8 makes three particularly inter-
esting and previously unobserved hydrogen bonds: secondary ni-
trate and ester oxygen with the Nd2 carboxamide hydrogens of
Asn62 and the primary nitrate oxygen with the imidazolic NH
moiety of His64. The thienothiopyran scaffold of 8 makes the same
type of interaction with hCA II as its ancestral CA inhibitors, dorzo-
lamide 1 or brinzolamide 2, as shown in the superposition of the
three hCA II—sulfonamide ligands as presented in Figure 3.
Compounds 4 and 6 and 8 were further characterized for NO-
donor capability by analyzing their cGMP-mediated vasorelaxant
properties on methoxamine-precontracted rabbit aortic rings.30
In this set of experiments, isosorbide mononitrate (ISMN, 15)
was used as a positive reference standard and dorzolamide 1 as
the comparative CA inhibitory compound (Table 4).30
H
O
HO
O
Table 2
ONO2
H
Crystallographic parameters and refinement statistics for the hCA II—8 adduct
Parameter
Value
15
Crystal parameter
Space group
P21
Cell Parameters
a = 41.63Å
b = 42.01 Å
c = 72.46 Å
b = 104.40°
The release of NO was clearly demonstrated for all three com-
pounds studied (see Fig. 5). More specifically, compounds 4, 6 and
8 were potent and effective stimulators of NO/sGC/cGMP signaling
Data collection statistics (20.0–2.0 Å)
No. of total reflections
No. of unique reflections
Completeness (%)a
F2/sig(F2)
with an EC50 of 4.25 0.80
0.05 M, respectively, compared to ISMN 15, which gave an EC50
of 10.8 2.4 M and dorzolamide which displayed little effect.
lM, 2.05 0.41 lM, and 0.37
54,133
16,290
98.2 (90.6)
20.9 (3.0)
14.4 (41.6)
l
l
Normotensive rabbit eyes have been shown to respond readily
to IOP lowering agents with CA inhibitory activity34 as well as to
standard NO-donors24, thus these were used in the model to ad-
dress the in vivo IOP lowering potentials of two CA II inhibitors
from our series, namely compounds 4 and 6 (Table 3). In this set
of experiments, ISMN and dorzolamide were also used as compar-
ators at clinically relevant doses. Both 4 and 6, despite their poor
solubility (0.25 and 15.1 mg/mL, respectively), performed as well
as an equimolar dose of dorzolamide 1 (solubility >20 mg/mL) in
lowering the IOP in normotensive rabbits (Table 3), reaching the
maximal response between 30 and 90 min post-dosing followed
by a slow decay over the next 180 min to reach basal IOP levels
at 360 min post-dosing. However, in this experiment, both 4 and
6 resulted in greater IOP lowering and over a longer duration than
the NO-donor ISMN 15 (Table 3), which attained a minimum of
2 mm below baseline at 30 min post-dosing and rapidly decayed
to basal level in a few hours.
R-sym (%)
Refinement statistics (20.0–2.0 Å)
R-factor (%)
22.3
27.0
0.012
1.54
R-free (%)b
Rmsd of bonds from ideality (Å)
Rmsd of angles from ideality (°)
a
Values in parenthesis relate to the highest resolution shell (2.1–2.0 Å).
Calculated using 5% of data.
b
Table 3
Interactions between various groups of the inhibitor 8 and amino acid residues within
the hCA II active site
Compound 8
hCA II residue
Distance (Å)
NAJ
Zn
1.96
3.08
3.78
2.86
3.05
3.05
2.97
3.34
3.70
OAL
OAT
OAL
CAS
CAS
OBB
OAV
OBG
Zn
O Pro201
N Thr199
N Asn67
N Gln92
Nd2 Asn62
Nd2 Asn62
Nd2 His64
In conclusion, a new class of NO-donating CAI were synthesized
and tested as potential IOP-lowering agents for the treatment of
open-angle glaucoma. These are new chemical entities with rele-
vant CA inhibitory properties via direct binding, as confirmed by
means of X-ray crystallographic work. Importantly 4 and 6 inhib-
ited CA II isozymes similarly to dorzolamide, and with selectivity
Table 4
Intraocular pressure (IOP) lowering effects of dorzolamide, ISMN and compounds 4 and 6 in normotensive New Zealand white rabbits
Compounda
IOP lowering effect in normotensive New Zealand white rabbits
Solubilityd (mg/mL)
b
c
Basal IOP (mmHg)
Post-treatment IOP (mmHg)
DDIOP (mmHg)
Tmax (min)
Vehicle
Dorzolamide
ISMN
NCX 274
NCX 278
20.1 0.6
19.7 0.7
19.2 0.5
19.8 0.7
19.2 0.5
19.9 0.7
16.7 0.8
16.6 0.5
15.9 0.7
16.5 0.4
0
0.6
0
60
30
60
120
1
15
4
À3.8 0.5**
À1.9 0.5*
À4.7 0.7**
À3.7 0.3**
>20
<20
0.25
6
15.1e
a
All drugs were administered at the final concentration of 55 mM in 50 lL using a positive displacer; IOP was recorded before treatment (pre-treatment) and at 30, 60, 90,
180 and 300 min thereafter using an applanation tonometer (Model 30 Classic, Medtronic, USA); Values are reported as mean SEM; N = 8/10 rabbits per group.
b
Post-treatment IOP is that reflecting the lowest measurement recorded over the observation period.
DDIOP reflects the maximal difference recorded in drug-treated versus vehicle (Cremophor EL 5%, DMSO 0.3%, BAK 0.02% in phosphate buffer, pH 6 at room temperature)
c
whereas Tmax is the time at which the maximal differences were recorded.
d
Solubility in Cremphor EL 5%, DMSO 0.3%, BAK 0.02%, pH 6 at room temperature.
Milky appearance even after microfiltration.
e
*
p <0.05 versus vehicle (Student t-test).
p <0.01 versus vehicle (Student t-test).
**

R. M. Steele et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6565–6570
6569
14. Ponticello, G. S.; Freedman, M. B.; Habecker, C. N.; Lyle, P. A.; Schwam, H.;
Varga, S. L.; Christy, M. E.; Randall, W. C.; Baldwin, J. J. J. Med. Chem. 1987, 30,
591.
15. Ponticello, G. S.; Sugrue, M. F.; Plazonnet, B.; Durand-Cavagna, G. In Integration
of Pharmaceutical Discovery and Development: Case Studies; Borchardt, R. T.,
Freidinger, R. M., Sawyer, T. K., Smith, P. L., Eds.; Pharmaceutical
Biotechnology; Plenum: New York, 1998; Vol. 11, pp 555–574.
16. Balfour, J. A.; Wilde, M. I. Drugs Aging 1997, 10, 384.
17. Sugrue, M. F. Prog. Ret. Eye Res. 2000, 19, 87.
18. Silver, L. H. Am. J. Ophthalmol. 1998, 126, 400.
19. Silver, L. H. Surv. Ophthalmol. 2000, 44, S147.
20. Cheng, J. W.; Cai, J. P.; Wei, R. L. Ophthalmology 2009, 116, 1243.
21. Perry, C. M.; McGavin, J. K.; Culy, C. R.; Ibbotson, T. Drugs Aging 2003, 20, 597.
22. Krauss, A.H., Toris, C.A.; Kallberg, M.E.; Gelatt, K.; Prasanna, G.; Impagnatiello,
F.; Chiroli, V.; Chong, W.K.M.; Carreiro, S.; Ongini, E. Abstract of Papers, The
Association for Research in Vision and Ophthalmology Annual Meeting, Fort
Lauderdale, FL, May 2009; Abstract 1471.
23. Galassi, F.; Renieri, G.; Sodi, A.; Ucci, F.; Vannozzi, L.; Masini, E. Br. J.
Ophthalmol. 2004, 88, 757.
24. Kotikoski, H.; Vapaatalo, H.; Oksala, O. Curr. Eye Res. 2003, 26, 119.
25. Becquet, F.; Courtois, Y.; Goureau, O. Surv. Ophthalmol. 1997, 42, 71.
26. Synthesis of compound
4 (NCX 274):To a solution of dorzolamide
hydrochloride (1; 5.00 g, 13.85 mmol; US Pharmacopeia) in dry N,N-
dimethylformamide (10 mL), under nitrogen, was added triethylamine
(2.12 mL, 15.23 mmol) followed by N,N-dimethylformamide dimethyl acetal
(2.21 mL, 16.62 mmol). The resulting solution was stirred at room temperature
for 3 h. The solution was cooled to 0 °C and water (25 mL) added slowly. The
mixture was extracted with ethyl acetate, the combined organic extracts were
washed with water, dried (Na2SO4) and the solvent was removed under
reduced pressure to give sulfonyl-amidine 9 as an off-white foam (4.35 g, 82%).
Figure 5. Vasorelaxant potency of vehicle (closed circle), dorzolamide (1, closed
square), ISMN (15, closed diamond) and compounds 4 (open triangle), 6 (open
diamond) and 8 (closed triangle).33
To
(2.00 mL), at 0 °C, under nitrogen, was added triethylamine (73
0.53 mmol) followed by dropwise addition of 6-bromocaproyl chloride
(79 L, 0.53 mmol). The solution was allowed to warm to ambient
temperature and stirred for 22 h. The solution was diluted with
dichloromethane and washed with water. The organic layer was dried
(Na2SO4) and the solvent was removed under reduced pressure. Purification
by flash chromatography, eluting with ethyl acetate gave the bromo-amide 10
as a white foam (122 mg, 83%).
a
solution of crude
9
(100 mg, 0.26 mmol) in dry dichloromethane
l
L,
towards the CA II isoform known to be consistent with beneficial
IOP lowering ability. Interestingly, compounds 4, 6 and 8 also re-
leased NO in vitro, showing higher potency than ISMN in the vaso-
relaxation of pre-contracted rabbit aorta rings. Compounds 4 and 6
proved to be promising in an in vivo IOP-lowering model, perform-
ing as well as an equimolar dose of dorzolamide, despite their low-
er solubility, and were superior to ISMN. Additional studies are
necessary to elucidate the detailed mechanism of NO donation
for this class of NO-donating CAI, as NO donation via hydrolysis
of the amide or carbamate bond by corneal homogenate esterases
is apparently not involved. The NO release of these and other such
bi-functional compounds (some of them in advanced clinical trials)
probably involves the reaction of the NO-releasing prodrug with
reducing agents, most probably thiol containing derivatives such
as cysteine residues from protein, or glutathione, a compound
found in high concentration in the eye tissues.35 Further studies
of novel NO-donating CAI in animal models of glaucoma are also
under consideration to further investigate this class.
l
To a solution of bromide 10 (370 mg, 0.66 mmol) in acetonitrile (16 mL), in a
microwave vial, was added silver nitrate (456 mg, 2.66 mmol). The mixture
was pre-stirred at ambient temperature in the microwave, for 2 min, then
heated at 110 °C with stirring, for 20 min. The salts were removed by filtration
over a pad of celite and the solvent was removed under reduced pressure. The
crude residue was dissolved in dichloromethane and washed with water. The
organic layer was dried (Na2SO4) and the solvent was removed under reduced
pressure. Purification by flash chromatography, eluting with ethyl acetate gave
the nitrate 10 as a white foam (253 mg, 71%).
To a solution of the amidine 10 (253 mg, 0.469 mmol) in THF at 0 °C, in a
microwave vial, was added 37% hydrochloric acid (1.873 mL) dropwise. The
solution was allowed to warm to ambient temperature, then heated in the
microwave at 110 °C, with stirring, for 28 min. The mixture was allowed to cool
to ambient temperature, then a saturated aqueous NaHCO3 solution added
slowly, until pH 8. The THF was removed under reduced pressure. The aqueous
residue was extracted with dichloromethane, the combined organics dried
(Na2SO4) and the solvent removed under reduced pressure. Purification by
flash chromatography, eluting with a gradient of 50% EtOAc/hexane to 100%
EtOAc gave sulfonamide 4 as a white solid (170 mg, 75%). 1H NMR (300 MHz,
DMSO-d6) d 7.99 (2H, s), 7.31 and 7.18 (1H, s), 5.40 and 5.00 (1H, s), 4.49 (2H, t,
J = 6.5 Hz), 3.92 (1H, m), 3.53–3.11 (2H, m), 2.75 (1H, m), 2.44–2.25 (3H, m),
1.80–1.30 (9H, m), 1.15 (3H, t, J = 7.2 Hz); 13C NMR (300 MHz, DMSO-d6) d
173.1, 150.0, 145.0, 129.1, 74.6, 56.2, 50.0, 42.8, 33.2, 26.8, 25.6, 25.1, 16.2,
12.4; ESI+ m/z = 484 (M+H+).
References and notes
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2. (a) Lutjen-Drecoll, E.; Futa, R.; Rohen, J. W. Invest. Ophthalmol. Vis. Sci. 1981, 21,
563; (b) Rohen, J. W. Ophthalmology 1983, 90, 758; (c) Alvarado, J.; Murphy, C.;
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a
l
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