Water soluble prodrug of a COX-2 selective inhibitor suitable for intravenous administration in models of cerebral ischemia

Article abstract of DOI:10.1016/j.bmcl.2005.05.002

A water soluble choline prodrug (17) of a COX-2 selective inhibitor (16) suitable for intravenous dosing in models of cerebral ischemia has been developed. Constant infusion studies using 17 demonstrate that extrapolated brain levels of 16 may be maintained for over 24 h in rats.

Full text of DOI:10.1016/j.bmcl.2005.05.002

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3197–3200
Water soluble prodrug of a COX-2 selective inhibitor suitable
for intravenous administration in models of cerebral ischemia
Nicholas D. Smith,a,* Thomas S. Reger,a Joseph Payne,a Jasmine Zunic,a Dan Lorrain,b
Lucie Correa,b Nicholas Stock,a Merryl Cramer,a Weichao Chen,a Jennifer Yang,a
Peppi Prasita and Benito Munoza
aDepartment of Medicinal Chemistry, Merck Research Laboratories, MRLSDB2, 3535 General Atomics Court,
San Diego, CA 92121, USA
bDepartment of Pharmacology, Merck Research Laboratories, MRLSDB1, 3535 General Atomics Court,
San Diego, CA 92121, USA
Received 2 April 2005; revised 28 April 2005; accepted 3 May 2005
Abstract—A water soluble choline prodrug (17) of a COX-2 selective inhibitor (16) suitable for intravenous dosing in models of cere-
bral ischemia has been developed. Constant infusion studies using 17 demonstrate that extrapolated brain levels of 16 may be main-
tained for over 24 h in rats.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Stroke, resulting from an interruption of cerebral blood
flow, is one of the leading causes of death and neurolog-
ical disability worldwide.1 A multitude of mechanisms
are involved in the brain damage that accompanies
stroke2 and previous work has implicated the cyclooxy-
genase-2 (COX-2) enzyme as a contributor to this dam-
age.3–5 However, previous work aimed at inhibiting the
COX mechanism in stroke models using small molecule
inhibitors has led to conflicting results.3,6–10 Further,
those inhibitors reported to be effective were dosed via
intra-peritoneal (ip) or per oral (po) routes. As stroke pa-
Figure 1. General prodrug strategy.
tients are often unconscious, intravenous (iv) administra-
tion is the preferred dosing route and so we sought to
approach was to open the lactone moiety of 1 and to
append water solubilizing groups X and/or Y on the
resulting acid and alcohol moieties, respectively, to give
prodrug 2. Ideally, X and Y would be endogenous to the
identify an effective prodrug of a COX-2 inhibitor suit-
able for intravenous administration.
We initially selected COX-2 inhibitor 1 to test in our
body (minimizing any possible adverse side effects) and
stroke model due to its good potency against COX-2
result in a water soluble and stable prodrug 2 that upon
in Human Whole Blood (HWB) IC50 = 0.9 lM
(Fig. 1).11
iv dosing would rapidly release the COX-2 inhibitor 1.
An example of the synthesis of prodrugs of general
structure 2 is exemplified below for the synthesis of 14
(Scheme 1).
As 1 is insoluble in water and unsuitable for iv dosing,
we sought to develop a water soluble prodrug. Our
Briefly, the parent COX-2 inhibitor 1 was prepared by
reacting bromoketone 3 with the appropriately substi-
tuted phenyl acetic acid in the presence of base. Lactone
Keyword: Stroke cerebral ischemia COX-2 prodrug infusion PGE2.
*
Corresponding author. Tel.: +1 858 202 5248; fax: +1 858 202
5752; e-mail: Nicholas_Smith@merck.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.002

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N. D. Smith et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3197–3200
Table 1. Prodrugs of 1
Compound
Water Plasma
stablea concen-
tration
of 1b
X
Y
H
7
8
H
NAc
No
NAc
NAc
H
Scheme 1. Synthesis of prodrug 14. Reagents and conditions: (a) 3,4-
difluorophenylacetic acid, DIEA, DMF, 70 ꢁC. (b) DIBALH, CH2Cl2,
À78 ꢁC to RT. (c) Acetyl chloride, Et3N, DMAP, 0 ꢁC. (d) (i) Dess–
Martin Periodinane (ii) NaClO2. (e) (2-bromoethyl)trimethylammo-
nium bromide, K2CO3, DMF.
9
H
H
No
NAc
10
NAc,d
NAc
1 was then reduced using DIBALH to give the diol 4,
which was acylated with 1 equivalent of acetyl chloride
to give a mixture of products. The desired regio-isomer
5 was readily isolated by silica chromatography from
this mixture and then oxidized to acid 6. Alkylation of
acid 6 with a choline equivalent gave the desired pro-
drug 14.
11
12
Et
Et
No
No
NAc
Table 1 shows a selection of compounds that were syn-
thesized in an analogous manor to this route and that
were evaluated as potential prodrugs of COX-2 inhibi-
tor 1.
NAc
Thus, ring opened lactone 7 with a free acid and alcohol
(X = H, Y = H) could not be isolated presumably due to
immediate ring closure to the parent lactone 1. To pre-
vent spontaneous ring closure, the alcohol of 7 was
capped while maintaining the free acid (X = H) as a po-
tential water-solubilizing group. Thus, capping the alco-
hol with acetate (8), arginine (9) or nicotinic acid (10)
led, in each case, to a compound that could be isolated.
Unfortunately, nicotinate 10 was found to be insoluble
in water (2 mg/mL) as either the hydrochloride or so-
dium salt. Conversely, although 8 and 9 were soluble
in water at 4 mg/mL, the solutions formed were found
to decompose over 24 h making them unsuitable as pro-
drugs. It was hypothesized that the aqueous instability
of 8 and 9 might be due to the presence of a free acid
in these prodrugs and so the acid was capped as the
ethyl ester as in 11, 12, and 13 (X = Et). Unfortunately,
11 (Y = aspartic acid) and 12 (Y = serine), although
both soluble, were also found to decompose over 24 h
as an aqueous solution. However, 13, where Y = lysine,
was found to be stable as a solution in water. To
test whether 13 would function as a prodrug of 1, a
2 mg/kg iv dose was administered to rats and the plasma
was monitored for the formation of 1. Gratifyingly at
the 30 min time point, 0.4 lM of 1 was detected, indicat-
ing the conversion of 13 to 1 in vivo.
13
14
15
Et
Yes
Yes
Yes
0.4 lM
0.9 lM
1.4 lM
a 4 mg/mL solution in water. Stability judged at 24 h by LCMS.
b Concentration of 1 following a 2 mg/kg iv dose of prodrug in Spra-
gue–Dawley rats (n = 2); measuring at 5, 15, 30, and 60 min. Value
quoted is at 30 min.
c Not applicable.
d Compound not soluble in water at 2 mg/mL as HCl salt.
the solubilizing group can be appended to the acid of 2
(i.e., group X). Thus, while keeping the alcohol capped
with an acetate group, 14 (X = choline) and 15 (X = eth-
yl-serine) were synthesized. Both 14 and 15 were stable
as solutions in water, and more importantly, generated
1 when dosed in the rat iv screen. Both 14 and 15 dis-
played an increase in Ôprodrug efficiencyÕ over 13, with
In 11, 12, and 13, the water-solubilizing moiety is ap-
pended to the alcohol of 2 (i.e., group Y). Alternatively,

N. D. Smith et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3197–3200
3199
Figure 3. 17—a choline prodrug of COX-2 inhibitor 16.
Figure 2. Plasma levels of 14 and 1 following a 2 mg/kg iv dose of 14.
Thus, we synthesized 17, the choline prodrug of 16
(Fig. 3). Gratifyingly, as with 14, 17 was stable as a solu-
tion in water, released the parent inhibitor 16 upon iv
administration in rats, and had good brain penetration
of approximately 50% (vide supra).
0.9 and 1.4 lM of 1 being detected at 30 min,
respectively.
Figure 2 shows the rat plasma levels of the prodrug
14 and the parent COX-2 inhibitor 1 following the
2 mg/kg iv dose of 14. As can be seen, not only are
there high levels of 1 detected at the earliest time
point (5 min), there is also a rapid decrease in the lev-
els of circulating prodrug 14.
The animal model we intended to use for cerebral ische-
mia proof-of-concept experiments using prodrug 17 is
the transient middle cerebral artery model (tMCAO).17
In this model, blood flow to the middle cerebral artery
(MCA) of rats is occluded by the use of a filament. After
a 90-min occlusion period, the filament is withdrawn to
allow blood to reperfuse into the MCA. To determine
the time course of COX-2 up-regulation in this model
(and hence the required time course of COX-2 inhibi-
tion), we carried out a microdialysis experiment to
examine the levels of PGE2 (a product of COX-2 enzy-
matic activity) in the brain (Fig. 4).18
Although higher levels of 1 were detected in vivo with
15, compound 14 was selected for further profiling as
it utilizes choline as a water-solubilizing group. This is
because citicholine, a compound shown to be beneficial
in human clinical trials of cerebral ischemia,12,13 is
metabolized to uridine and choline in vivo.14 In this
way, we hoped to obtain higher levels of neuroprotec-
tion due to the potential additive effect of a COX-2
inhibitor and choline.
Figure 4 illustrates that PGE2 levels in the stroked hemi-
sphere of the rat brain increase compared to those in
sham operated animals starting at 6 h after occlusion,
peaking at 10 h, and with high levels maintained out
to 24 h and beyond.19
As we wanted to inhibit COX-2 in the brain, we next
determined the brain levels of 1 after dosing with 14.
Thus following a 10 mg/kg iv dose of 14 in rats, at the
3 h time point, the brain and plasma levels of 1 were
found to be 0.7 and 1.4 lM, respectively—a brain pene-
tration of 50%. For our in vivo stroke studies, we
wanted levels of 1 in the rat brain of 10· its COX-2
IC50 (i.e., 9 lM).15 With rat brain penetration of 50%,
this would mean plasma levels of 1 of 18 lM. However,
previous publications have suggested that COX-1 inhibi-
tion is detrimental to stroke outcome,16 indicating that
COX-2 inhibition in the brain is desired, while inhibition
of COX-1 in the brain and periphery is not. At 18 lM,
the plasma concentration of 1 would, indeed, result in
undesired COX-1 inhibition in the periphery (COX-1
IC50 = 13 lM).
Since dosing 17 via a single iv bolus would provoke
transient inhibition of the COX-2 enzyme in the brain
(rat t1/2 of 16 = 2 h), we wished to establish a protocol
to enable continuous COX-2 inhibition over the course
of the experiment. For this reason, we next carried out
infusion studies with 17 aimed at maintaining steady-
600
500
400
300
200
100
0
tMCAO Rat Brain
Sham Operated Rat Brain
(n = 8)
Due to this undesired COX-1 inhibition with 1, we iden-
tified an alternative COX-2 selective inhibitor 16
(Fig. 3).11 Although 16 has less absolute potency against
COX-2 compared to 1 (1.3 lM vs 0.9 lM), it has greater
selectivity for COX-2 (50-fold versus 15-fold). This
would mean that at 10· the COX-2 IC50 concentration
of 16 in the brain (i.e., 13 lM), the concentration in
the plasma would be 26 lM (the brain penetration of
16 is also approximately 50%—vide supra). This plasma
level is significantly less than the COX-1 IC50 of 16 of
65 lM, so minimizing any undesired peripheral COX-1
inhibition.
0
6
12
Time (Hr)
18
24
Figure 4. Microdialysis levels of PGE2.

3200
N. D. Smith et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3197–3200
Table 2. Infusion studies with 17a
Dose of prodrug 17
Concentration of 16 (lM)b
Brain/plasma 24 h (%)
Plasma 6 h
Plasma 24 h
Brain 24 h
10 mg/kg Bolus + 5 mg/kg/h infusion
5 mg/kg Bolus + 3 mg/kg/h infusion
16
20
11
55
59
6.0
9.2
5.5
a Sprague–Dawley rats (n = 3).
b Plasma concentration of 16 measured at 0.08, 0.25, 0.5, 1, 2, 4, 6, and 24 h.
2. Lee, J. M.; Grabb, M. C.; Zipfel, G. J.; Choi, D. W. J.
Clin. Invest. 2000, 206, 723.
3. For a review see: Hurley, S. D.; Olschowka, J. A.;
OÕBanion, M. K. J. Neurotrauma 2002, 19, 1.
4. Nogawa, S.; Zhang, F.; Ross, M. E.; Iadecola, C. J.
Neurosci. 1997, 17, 2746.
state concentrations of the parent compound 16 in the
plasma (and hence brain) of rats over 24 h. We targeted
brain concentrations of 16 in the rat at two levels—13
and 6.5 lM (10· and 5· the COX-2 IC50, respectively,
Table 2).
5. Iadecola, C.; Niwa, K.; Nogawa, S.; Zhao, X.; Nagayama,
M.; Araki, E.; Morham, S.; Ross, M. E. Proc. Natl. Acad.
Sci. USA 2001, 98, 1294.
6. Govoni, S.; Masoero, E.; Favalli, L.; Rozza, A.; Scelsi, R.;
Viappiani, S.; Buccellati, C.; Sala, A.; Folco, G. Neurosci.
Lett. 2001, 303, 91.
7. Candelario-Jalil, E.; Alvarez, D.; Castaneda, J. M.; Al-
Dalain, S. M.; Martinez-Sanchez, G.; Merino, N.; Leon,
O. S. Brain Res. 2002, 927, 212.
8. Sugimoto, K.; Iadecola, C. Brain Res. 2003, 960, 273.
9. Cole, D. J.; Piyush, P. M.; Lowell, R.; Drummond, J. C.;
Marcantonio, S. J. Pharmacol. Exp. Ther. 1993, 266, 1713.
10. Joshita, H.; Asano, T.; Hanamura, T.; Takakura, K.
Stroke 1989, 20, 788.
11. Prasit, P.; Wang, Z.; Brideau, C.; Chan, C. C.; Charlsen,
S.; Cromlish, W.; Ethier, D.; Evans, J. F.; Ford-Hutch-
inson, A. W.; Gauthier, J. Y.; Gordon, R.; Guay, J.;
Gresser, M.; Kargman, S.; Kennedy, B.; Leblanc, Y.;
Leger, S.; Mancini, J.; OÕNeil, G. P.; Ouellet, M.; Percival,
M. D.; Perrier, D.; Riendeau, D.; Rodger, I.; Tagari, P.;
Therien, M.; Vickers, P.; Wong, E.; Xu, L. J.; Young, R.
N.; Zamboni, R. Bioorg. Med. Chem. Lett. 1999, 9, 1773.
12. Adibhatla, R. M.; Hatcher, J. F. J. Neurosci. Res. 2002,
70, 133.
13. Krupinski, J.; Ferrer, I.; Barrachina, M.; Secades, J. J.;
Mercadal, J.; Lozano, R. Neuropharmacology 2002, 42,
846; Adibhatla, R. M.; Hatcher, J. F.; Dempsey, R. J.
J. Neurochem. 2002, 80, 12.
14. Wurtman, R. J.; Regan, M.; Ulus, I.; Yu, L. Biochem.
Pharm. 2000, 60, 989.
Table 2 shows that a 10 mg/kg bolus dose followed by a
5 mg/kg/h infusion of 17 results in levels of 16 of 11 lM
in the brain and 20 lM in the plasma at 24 h (55% brain
penetration). Gratifyingly, this is close to the targeted
brain levels of 13 lM (10· COX-2 IC50) and importantly
at 20 lM, the plasma levels of 16 are significantly below
the COX-1 IC50 of 65 lM. Further, plasma levels of 16
were maintained at 16–20 lM during the course of the
experiment, meaning COX-1 inhibition in the periphery
is minimal (COX-1 IC50 = 65 lM).
These infusion studies with prodrug 17 demonstrate that
high levels of 16 may be sustained in the rat brain for the
extended period of time required to inhibit the COX-2
enzyme activity indicated by the PGE2 time-course in
Figure 4. In vivo proof-of-concept experiments using
prodrug 17 in animal models of cerebral ischemia are
currently underway.
In summary, a water soluble and stable prodrug of a
COX-2 selective inhibitor 17 has been designed using
choline as a solubilizing group. Microdialysis experi-
ments measuring PGE2 levels in the stroked hemisphere
of rat brain indicate COX-2 activity over a prolonged
period from 6 to 24 h following occlusion. Infusion stud-
ies in rats with 17 demonstrate that rapid conversion to
the parent inhibitor 16 occurs and that steady state
levels of 16 may be achieved in the plasma (and by
extrapolation the brain) over the desired time period
of COX-2 up-regulation indicated by the microdialysis
experiment. In vivo proof-of-concept experiments using
17 are currently underway.
15. As no PD assay was available, we targeted the brain
concentration of drug at high levels of 10· COX-2 IC50 to
ensure full inhibition of the COX-2 enzyme.
16. Iadecola, C.; Sugimoto, K.; Niwa, K.; Kazama, K.; Ross,
M. E. J. Cereb. Blood Flow Metab. 2001, 21, 1426.
17. Longa, E. Z.; Weinstein, P. R.; Carlson, S.; Cummins, R.
Stroke 1989, 20, 84.
18. In this experiment, PGE2 levels are measured in the
stroked hemisphere of the rat brain following a 90 min
MCA occlusion and compared to the PGE2 levels of sham
operated animals.
References and notes
1. Lo, E. H.; Dalkara, T.; Moskowitz, M. A. Nat. Rev.
Neurosci. 2003, 4, 399.
19. A similar time-course in the up-regulation of COX-2
mRNA has been reported: See Ref. 4.