New prostaglandin derivative for glaucoma treatment

Article abstract of DOI:10.1016/j.bmcl.2009.02.007

A hydrogen sulphide-releasing derivative of latanoprost acid (ACS 67) was synthesized and tested in vivo to evaluate its activity on reduction of intraocular pressure and tolerability. Glutathione (GSH) and cGMP content were also measured in the aqueous h

Full text of DOI:10.1016/j.bmcl.2009.02.007

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1639–1642
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New prostaglandin derivative for glaucoma treatment
a
b
b
c
b
a,*
Elena Perrino , Caterina Uliva , Cecilia Lanzi , Piero Del Soldato , Emanuela Masini , Anna Sparatore
a
Istituto di Chimica Farmaceutica e Tossicologica ‘Pietro Pratesi’, University of Milan, Via Mangiagalli 25, 20133 Milan, Italy
Department of Preclinical and Clinical Pharmacology, University of Florence, Viale G. Pieraccini n.6, 50139 Florence, Italy
CTG Pharma s.r.l., Viale Gran Sasso 17, 20131 Milan, Italy
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A hydrogen sulphide-releasing derivative of latanoprost acid (ACS 67) was synthesized and tested in vivo
to evaluate its activity on reduction of intraocular pressure and tolerability. Glutathione (GSH) and cGMP
content were also measured in the aqueous humour. The increased reduction of intraocular pressure,
with a marked increase of GSH and cGMP and the related potential neuroprotective properties, make this
compound interesting for the treatment of glaucoma. This is the first time that an application of a hydro-
gen sulphide-releasing molecule is reported for the treatment of ocular diseases.
Received 3 December 2008
Revised 2 February 2009
Accepted 2 February 2009
Available online 8 February 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Prostaglandin
Hydrogen sulphide
Glaucoma
Glaucoma is a group of diseases of the optic nerve involving
progressive degeneration of retinal ganglion cells resulting in a
pattern of irreversible loss of vision. Intraocular pressure (IOP)
represents a significant risk factor for developing glaucoma and
can be, at least partially, controlled by administering drugs which
either reduce the production of aqueous humour within the eye or
increase the fluid drainage.2
In recent years the physiological role of hydrogen sulphide
S), a compound that belongs to, along with nitric oxide (NO)
and carbon monoxide (CO), the family of labile biological media-
(H
2
1
8
,9
tors called gasotransmitters, has been elucidated. Several effects
are reported on biological targets, such as cytoprotection through
the increase of the intracellular glutathione (GSH) content, smooth
À
muscle relaxation and vasodilatatory effects through KATP or Cl
channels activation.
9
The use of prostaglandins for IOP reduction has been discussed
for the first time by Camras in 1981 when it was shown that PGF
2
a
Our research group has recently discovered that the class of
compounds known as dithiolethiones has the ability to release
3
effectively reduces IOP in monkeys. Since then there has been an
increasing interest in the research of new prostaglandin derivatives
to be used as drugs for glaucoma treatment.4 The first approach in
1
0
H
2
S in a controlled and long lasting way. We had previously ap-
plied the concept of preparing H S-releasing hybrids in other phar-
macological fields such as inflammation, with hybrid of
diclofenac, male erectile dysfunction, with a hybrid of sildena-
,5
2
modifying PGF
2a
included esterification of the carboxylic acid end
a
1
0–12
of the molecule to improve bioavailability and reduce the side ef-
fects.6 An extensive series of structure–activity studies led to the
fil, cardiovascular, with a hybrid of aspirin.
13
14
Ò
synthesis of latanoprost (Xalatan eye drop, Pfizer, Fig. 1) the most
It has also been reported that anethole dithiolethione (ADT) is
effective in in vitro lens cell protection against radiation injury.
7
prescribed prostaglandin derivative for treatment of glaucoma.
Ò 15
Anethole dithiolethione (Sulfarlem ) is a commercial drug used
as a hepatoprotective agent and for the treatment of drug- and
radiation-induced xerostomia, and 5-(4-hydroxyphenyl)-3H-1,2-
dithiole-3-thione (ADTOH, Fig. 2) is its main metabolite which
could be released from the former by in vivo demethylation.16
The working idea was to prepare a H S-releasing derivative of
2
latanoprost acid, potentially able to maintain or even increase
Figure 1. Structures of latanoprost acid and latanoprost.
*
Corresponding author. Tel.: +39 02 50319365; fax: +39 02 50319359.
E-mail address: anna.sparatore@unimi.it (A. Sparatore).
Figure 2. Structures of ADT and ADTOH.
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.007

1
640
E. Perrino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1639–1642
2
Scheme 1. Reagents and conditions: (a) EDAC, DMAP, anhydrous THF, N , rt, 3 h, 65%.
the anti-IOP activity and possessing significant neuroprotective
properties through the increase of anti-oxidant substances, such
as GSH. Thus a new compound (ACS 67) was designed by replacing
the isopropyl moiety of latanoprost with a phenyldithiolethione
group.
40
3
3
2
2
1
1
5
0
5
0
5
0
The route used for the synthesis of ACS 67 is reported in Scheme
1
0
1
and required firstly the preparation of the ADTOH intermediate
which was then esterified with latanoprost acid in the presence of
1
7
* *
EDAC and DMAP.
The effects of ACS 67 versus latanoprost on IOP in glaucomatous
pigmented rabbit over a four-hour time course are reported in Fig-
ure 3. The rabbits were made glaucomatous by injecting carbomer
into anterior chamber and, after stabilization, the IOP was mea-
sured after 30, 60, 120 and 240 min.18
Carbomer
Basal
0
30
60
120
240
The AUC (mm Hg h) was 75.2 ± 3.3 for ACS 67 and 108.8 ± 1.0
for latanoprost with a 30.8% reduction in IOP and the difference be-
tween the two groups was statistically significant already after
Time (min)
Figure 4. Effect on IOP in carbomer-induced glaucoma in New Zealand albino
rabbits after topical treatment with one drop (50 l) of 0.005% ACS 67 (-Ç-) versus
.005% latanoprost (-j-). p < 0.01 versus latanoprost.
3
0 min.
A similar experiment was conducted in albino rabbits (Fig. 4)
l
**
0
since it is reported that latanoprost failed to produce hypotensive
effects in albino rabbit and cat eyes. These differences may be
attributed to the different selectivity for prostaglandin receptors
and differences in prostaglandin receptor subtypes distribution in
1
9
the eye of these species. The increased IOP response in wild rab-
bits might be due to an increased affinity or an increased number
of prostaglandin receptors.
The AUC (mm Hg h) was 106.1 ± 4.2 for ACS 67 and 119.0 ± 5.0
for latanoprost and there was a statistical difference at fourth hour.
To exclude the fact that this response could only have been a
transient phenomenon, the effects of ACS 67 versus latanoprost
on IOP in glaucomatous pigmented rabbits were determined after
repeated administrations in a five-day time course,18 and the re-
sults reported in Figure 5.
The AUC (mm Hg h) was 2088.0 ± 31.0 for ACS 67 and
658.0 ± 59.1 for latanoprost with a 27% reduction versus latano-
prost (p < 0.001).
Figure 5. Effect on IOP in carbomer-induced glaucoma in pigmented rabbits after
repeated topical treatment with 0.005% ACS 67 (-Ç-) versus 0.005% latanoprost (-j-).
2
*
**
p < 0.05; p < 0.01 versus latanoprost.
We also measured the IOP after single administration (Table 1)
and after 5-day repeated administration (Table 2) of ADTOH, and of
the combination ADTOH + latanoprost at the concentration of
0
.005% in albino and pigmented rabbit. The data show the contri-
bution of the sulfurated moiety to IOP reduction in pigmented
rabbits.
The GSH content in the aqueous humour of normotensive rab-
bits at 3 different dose levels (Table 3) after single administration
and in glaucomatous pigmented rabbits after 5-day repeated
2
0
administration (Table 4) was determined.
ACS 67 significantly increases reduced glutathione levels in nor-
motensive pigmented rabbits after single administration in a dose
related manner, moreover ACS 67 also significantly increases, ver-
sus latanoprost, GSH levels in glaucomatous pigmented rabbits
after repeated 5-day administration (Table 4).
We also measured the GSH levels in normotensive pigmented
rabbits (basal 36.3 ± 2.9) after single administration of ADTOH
(58.3 ± 3.7), latanoprost (38.7 ± 3.5) and the combination
Figure 3. Effect on IOP in carbomer-induced glaucoma in pigmented rabbits after
topical treatment with one drop (50
l
l) of 0.005% ACS 67 (-Ç-) versus 0.005%
*
**
latanoprost (-j-). p < 0.05; p < 0.01 versus latanoprost.

E. Perrino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1639–1642
1641
Table 1
IOP (mm Hg) in albino and pigmented rabbits after single administration of ADTOH and latanoprost n = 4 (±sd)
Strain
Treatment
Basal
Carbomer
Time (min)
120
30
60
180
240
Albino rabbit
ADTOH
ADTOH + latanoprost
ADTOH
12.5 (±2.9)
13.2 (±2.5)
11.2 (±1.9)
12.5 (±1.3)
31.0# (±4.5)
29.5 (±1.9)
30.5 (±3.4)
30.0 (±5.2)
29.7 (±3.3)
28.0 (±2.3)
29.2 (±4.1)
29.0 (±4.2)
26.2 (±2.2)
27.7 (±4.0)
26.0 (±3.3)
25.0 (±1.4)
26.2 (±4.8)
24.5 (±2.1)
25.2 (±1.5)
27.2 (±4.6)
23.5 (±2.4)
#
32.2 (±3.9)
Pigmented rabbit
32.5# (±6.0)
32.3# (±3.1)
*
*
**
**
ADTOH + latanoprost
23.5 (±0.6)
19.2 (±2.2)
16.5 (±1.9)
16.2 (±2.1)
#
p < 0.01 carbomer versus basal.
p < 0.05 ADTOH versus ADTOH + latanoprost.
p < 0.01 ADTOH versus ADTOH + latanoprost.
*
*
*
Table 2
IOP (mm Hg) in pigmented rabbits after repeated 5-day administration of ADTOH and latanoprost n = 4 (±sd)
Strain
Treatment
Basal
Carbomer
Time (days)
3
1
2
4
5
Pigmented rabbit
ADTOH
ADTOH + latanoprost
11.2 (±1.9)
12.5 (±1.3)
32.5# (±6.0)
32.5# (±3.1)
26.7 (±3.4)
19.2 (±2.2)
26.0 (±1.6)
19.2 (±2.2)
23.0 (±2.6)
16.2 (±1.3)
22.2 (±1.3)
17.0 (±1.4)
22.0 (±1.4)
16.2 (±1.3)
*
**
**
**
**
#
p < 0.01 carbomer versus basal.
p < 0.05 ADTOH versus ADTOH + latanoprost.
p < 0.01 ADTOH versus ADTOH + latanoprost.
*
*
*
Table 3
Table 6
GSH levels in normotensive pigmented rabbits after single administration
Tolerability of ACS 67 in Draize test
GSH (pmol/ml)
Basal
Draize score
Vehicle
38.68 ± 2.15
0/5
0/5
0/5
0/5
ACS 67 0.005%
ACS 67 0.008%
ACS 67 0.01%
60.04 ± 11.03
69.83 ± 6.63
82.89 ± 5.69
ACS 67 0.005%
ACS 67 0.008%
ACS 67 0.01%
Table 4
data with the combination support the evidence that the increase
of cGMP is due to the sulfurated moiety of ACS 67.
GSH levels in glaucomatous pigmented rabbits after repeated 5-day administration
Finally the tolerability was measured in New Zealand albino
rabbits at three different concentrations of ACS 67 versus placebo
according to the Draize test and the results are reported in
Table 6.
GSH (pmol/ml)
Vehicle/carbomer
ACS 67 0.005%
20.92 ± 8.49
65.27 ± 10.97
36.44 ± 4.76
2
2
Latanoprost 0.005%
2 2a
This study shows that ACS 67, the new H S-donating PGF
derivative, was significantly more potent than latanoprost in
reducing IOP after either single or repeated topical administration
to glaucomatous pigmented rabbits. The compound was also very
well tolerated in Draize test.
ADTOH + latanosprost (61.2 ± 1.6) and in glaucomatous pigmented
rabbits (basal 36.3 ± 2.9) after 5-day repeated administration of
ADTOH (60.4 ± 3.1), latanoprost (33.7 ± 3.0), ADTOH + latanoprost
Furthermore the compound was found to be more potent than
latanoprost in reducing IOP also in the albino glaucomatous rabbits
particularly in consideration that the dose of ACS 67 is 28% lower
on a molar basis. This finding is of relevance since it is known that
(
63.9 ± 4.9) at the concentration of 0.005% .
These data support the evidence that the increase of GSH is due
to the sulfurated moiety of ACS 67.
The cGMP content in the aqueous humour of glaucomatous pig-
mented rabbits after 5-day repeated administration of latanoprost
and ACS 67 is reported (Table 5).21 Moreover the cGMP levels after
repeated 5-day administration in glaucomatous pigmented rabbits
19,23
in these latter animals latanoprost is only very poorly effective.
Thus both PG-dependent and independent effects in reducing IOP
can be identified for ACS 67. The PG-dependent effects could be
mediated by cAMP content similar in latanoprost and ACS 67
groups (unpublished data). The PG-independent effects are likely
to be due to the contribution of cGMP and sulfurated moiety via
(
basal 73.7 ± 2.8) of ADTOH (121.6 ± 17.5); latanoprost (69.9 ±
1
0
4.7); ADTOH + latanoprost (123.3 ± 13.6) at the concentration of
.005% were also measured.
its ability to release H
Recently, H S was found to possess relevant vasorelaxant ef-
fects in mice with depletion of cystathionine-
more H S was found to be neuroprotective through the GSH
formation.
2
S.
ACS 67 significantly increases the cGMP levels in glaucomatous
pigmented rabbits after 5-day repeated administration and the
2
24
c
-lyase. Further-
2
8
The increased anti-IOP response was accompanied by a marked
increase in GSH levels in aqueous humour of ACS 67 treated rabbits
as compared to the related values in latanoprost treated animals.
The observed GSH increase suggests a potential neuroprotective ef-
fect for ACS 67.
Table 5
Cyclic GMP levels in glaucomatous pigmented rabbits after repeated 5-day admin-
istration of latanoprost or compound ACS 67 at 0.005% concentration
cGMP (fmol/ml)
Vehicle/carbomer
ACS 67 0.005%
72.00 ± 3.76
118.16 ± 9.20
71.82 ± 4.34
This is the first time that an application of a hydrogen sulphide-
releasing molecule is reported for the treatment of ocular diseases.
Latanoprost 0.005%

1
642
E. Perrino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1639–1642
product (78 mg, yield 65%) has melting point (Büchi) 94–95 °C.
HRMS (Apex II ICR-FTMS Bruker Daltonics; ESI) m/z calcd for [C32
21.17736; found: 621.17694.
1
References and notes
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H S O
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.60 (m, 4H); 2.40–2.20 (m, 4H); 1.95–1.30 (m, 12H).
3
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8. High IOP levels were obtained injecting carbomer 0.25% (Siccafluid Farmila
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repeated dose experiment, and IOP was measured, using a Tono-Pen tonometer
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0. Determination of GSH levels in aqueous humour: aqueous humour was
centrifuged at 4 °C at 100.000g for 30 min and a fixed volume of supernatant
was diluted with phosphate-EDTA buffer to a final volume of 5 ml. For GSH
evaluation, 200
ll of 40 mM N-ethylmaleimide was added to 0.5 ml of the
supernatant, incubated for 30 min and diluted with 0.1 N NaOH to a final
volume of 5 ml. Phosphate-EDTA buffer and 0.1 N NaOH (1.8 and 0.1 ml,
respectively) and o-phthaldialdehyde solution (1 mg/ml, 0.1 ml) were added to
both mixtures. The samples were incubated at room temperature for 15 min
and then the fluorescence evaluated fluorometrically at 420 nm emission and
1
1
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7. Synthesis
of
(5Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(3R)-3-hydroxy-5-
phenylpentyl] cyclopentyl]-5-heptenoic acid 4-(3H-1,2-dithiole-3-thione-5-yl)-
350 nm excitation. Samples will be calibrated using a standard curve of GSH.
phenyl ester (ACS 67): 5-(4-Hydroxyphenyl)-3H-1,2-dithiol-3-thione, prepared
as previously described10 (91 mg, 0.40 mmol) and a catalytic amount of 4-
21. Determination of cyclic GMP (cGMP) levels in aqueous humour. The
concentration of cGMP was determined by means a radioimmunoassay kit
dimethylaminopyridine (DMAP) were added to
a
solution of (5Z)-7-
121
using [ I] labelled cGMP (Amersham, Bucks, UK). Five hundred
ll of 10%
[
(1R,2R,3R,5S)-3,5-dihydroxy-2-[(3R)-3-hydroxy-5-phenylpentyl]cyclopentyl]-
trichloroacetic acid (TCA) were added to the samples which were then
centrifuged and TCA extracted with 0.5 M tri-n-octylamine dissolved in 1,1,2-
trichloro-1,2,2-trifluoroethane. The samples were then acetylated in acetic
anhydrase and the amount of cGMP measured in aqueous phase. The values
were expressed as fmoles of cGMP per ml.
5
-heptenoic acid (latanoprost acid, 84 mg, 0.20 mmol) in 2 ml of anhydrous
tetrahydrofuran (THF), stirring under nitrogen at 0 °C. After few minutes 1-(3-
dimethylaminoisopropyl)-3-ethyl-carbodiimide hydrochloride (EDAC, 57 mg,
0
.30 mmol) was added and the reaction was stirred at room temperature for 3 h.
After evaporation of THF, the residue was dissolved in dichloromethane and
washed with cold water. The organic solution was dried on anhydrous sodium
sulphate, evaporated to dryness and the crude solid was purified by column
chromatography on silica gel (ethyl acetate/cyclohexane, 70:30) and then
washed with ethyl ether/ethyl acetate (70:30). The obtained red-coloured
2
2
2
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