G. Cynkowska et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3524–3527
3527
Table 1. The concentrations of acetonitrile in the mobile phase, the
detection wavelength, and the retention times (RT) for the selected
drug-conjugates and for the parent drugsa
promise efficient binding at the active site of the plasma
esterase enzyme; also, this same steric hindrance may
shield the ester moiety from general base hydrolysis in
phosphate buffer.
Compound
Mobile phase
RT (min)
ECA–ATL, 5
ECA–PEG, 1a
ECA–PEG4, 1b
ECA–PEG6, 1c
ECA
A/45% MeCN
A/45% MeCN
A/45% MeCN
A/45% MeCN
B/30% MeCN
B/10% MeCN
14.3
12.9
12.3
11.8
10.5
8.5
For the treatment of glaucoma, both the ECA–ATL co-
drug and the ECA–PEG mono-ester prodrugs represent
less toxic and more efficacious forms for the delivery of
both ECA and/or ATL. Furthermore, these novel drug
conjugates could provide a longer duration of action
for the pharmacological effect of these compounds
(i.e., reducing the IOP) compared to the parent drug(s)
alone. Incorporation of a diuretic drug with a b-adren-
ergic receptor antagonist into a codrug structure may
also provide better efficacy in the treatment of a number
of other therapeutic conditions, such as ischemic heart
disease, certain arrhythmias, and airway diseases such
as bronchitis.
ATL
a Wavelength was 254 nm for all except for ATL, it was 225 nm.
Enzymatic hydrolysis was determined in a similar way
using human serum, and the samples were deproteinized
with acetonitrile before HPLC analysis.
A number of prodrugs containing ECA linked to a short
chain PEG moieties consisting of 2, 4, or 6 oxyethylene
units, and two codrugs of ECA covalently linked to the
b-adrenergic receptor antagonists ATL or TML, have
been synthesized. The parent drugs were linked via ester
moieties to permit facile non-enzymatic hydrolytic
cleavage at physiological pH or via enzyme-mediated
catalysis in plasma. The main goal of this approach
was to improve drug delivery of the parent drugs by
overcoming solubility problems and to enhance perme-
ation through topical membranes, as well as to afford
a reduction in toxicity.
The ECA–ATL codrug and several of the ECA–PEG
mono-ester prodrugs described above are currently
undergoing further evaluation in in vivo studies.
Acknowledgment
This research was supported by a grant from Control
Delivery Systems, Inc.
References and notes
The reaction of the acid chloride of ECA with a variety
of short chain PEGs yielded both the desired mono-ester
prodrug and di-ester side products, which were easily
separated. All the ECA–PEG monoesters studied under-
went facile hydrolysis in both phosphate buffer at pH 7.4
(half-lives 110–130 min) and in human serum (half-life
<2 min for all ECA–PEG monoesters studied), to gener-
ate ECA and the corresponding PEG. Esterfication of
ECA with ATL afforded the mono-substituted codrug
(half-life in serum = 30 min, and in buffer, pH
7.4 = 14 h). The hydrolysis of the ECA–ATL codrug
and the ECA–PEG mono-esters prodrugs exhibited first
order kinetics under the conditions used to generate the
corresponding drugs, that is, ECA and ATL, or ECA
and PEG, respectively. The half-lives of the ECA pro-
drugs and the ECA–ATL codrug in pH 7.4 buffer and
in serum are listed in Table 2.
1. Koeehel, D. A.; Rankin, G. O. J. Med. Chem. 1978, 21,
764.
2. Griffiths, N. M.; Simmons, N. L. J. Exp. Physiol. 1987, 72,
313.
3. Shimazaki, A.; Ichikawa, M.; Vasantha Rao, P.; Kirihara,
T.; Konomi, K.; Epstein, D.; Hara, H. Biol. Pharm. Bull.
2004, 27, 1019.
4. Tingey, D. P.; Schroeder, A.; Epstein, M. P. M.; Epstein,
D. L. Arch. Ophthalmol. 1992, 110, 699.
5. Epstein, D. I.; Roberts, B. C.; Skinner, L. L. Invest.
Ophthalmol. Vis. Sci. 1997, 38, 1526.
6. Melamed, S.; Kotas-Neumann, R.; Barak, A.; Epstein,
D. L. Am. J. Ophthalmol. 1992, 113, 508.
7. Liang, L. L. et al. Arch. Ophthalmol. 1992, 33, 2640.
8. Erickson-Lamy, K.; Schroeder, A.; Epstein, D. L. Invest.
Ophthalmol. Vis. Sci. 1992, 33, 2631.
9. Epstein, D. L.; Anderson, P. Appl. Pharmacol. 1984,
135–150.
10. Gluchowski, C.; Cheng Bennett, A.; Epstein D.L. PCT
Int. Appl. WO 9508990, 1995; Chem. Abs. 1995, 122,
306569g.
11. Peczon, J. D.; Grant, W. M. Am. J. Ophthalmol. 1968, 66,
680.
The ECA–ATL codrug exhibited a longer half-life, both
in buffer and in serum, compared to the ECA mono-
esters prodrugs; this might be due to the steric hindrance
around the ester linkage in the codrug that could com-
12. Shargel, L.; Mutnick, A.; Souney, P.; Swanson, L.; Block,
L.; Comprehensive Pharmacy Review: Baltimore: Mary-
land, 1997.
13. Palkama, A.; Uusitalo, H.; Raij, K.; Uusitalo, R. Acta
Ophthalmol. 1985, 63, 9.
Table 2. Hydrolysis of the ECA–ATL codrug and the ECA–PEG
prodrugs
Compound
Half-life (time)
In buffer, pH 7.4 In serum (min)
14. Safran, A. B. et al. Int. Ophthalmol. 1993, 17, 109.
15. All ester prodrugs of ECA and the ECA–ATL, ECA–
1
ECA–ATL, 5
14 h
30
TML codrugs were fully characterized utilizing H NMR
and 13C NMR spectroscopy, CHN combustion analysis.
In every case, the spectral and analytical data were
consistent with the proposed structures.
ECA–PEG2, 1a (n = 2)
ECA–PEG4, 1b (n = 4)
ECA–PEG6, 1c (n = 6)
110 min
130 min
120 min
<2
<2
<2