Synthesis of water-Soluble prodrugs of BMS-191011: A maxi-K channel opener targeted for post-stroke neuroprotection

Article abstract of DOI:10.1016/S0960-894X(03)00296-8

A variety of water-soluble prodrugs of BMS-191011 was synthesized and evaluated for solution state stability and rate of conversion to BMS-191011 in rat and human plasma. The deoxycarnitine ester prodrug (11c) was selected for clinical evaluation based on

Full text of DOI:10.1016/S0960-894X(03)00296-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1695–1698
Synthesis of Water-Soluble Prodrugs of BMS-191011: A Maxi-K
Channel Opener Targeted for Post-stroke Neuroprotection
Piyasena Hewawasam,a,* Min Ding,a Nathan Chen,a Dalton King,a Jay Knipe,c
Lorraine Pajor,c Astrid Ortiz,b Valentin K. Gribkoffb and John Starretta
aDepartment of Chemistry, The Bristol-Myers Squibb Pharmaceutical Research Institute,
5 Research Parkway, Wallingford, CT 06492, USA
bDepartment of Neuroscience/Genitourinary Drug Discovery, The Bristol-Myers Squibb Pharmaceutical Research Institute,
5 Research Parkway, Wallingford, CT 06492, USA
cDepartment of Metabolism and Pharmacokinetics, The Bristol-Myers Squibb Pharmaceutical Research Institute,
5 Research Parkway, Wallingford, CT 06492, USA
Received 6 November 2002; accepted 6 March 2003
Abstract—A variety of water-soluble prodrugs of BMS-191011 was synthesized and evaluated for solution state stability and rate of
conversion to BMS-191011 in rat and human plasma. The deoxycarnitine ester prodrug (11c) was selected for clinical evaluation
based on its superior chemical stability, crystallinity and cleavage to BMS-191011 in human plasma.
# 2003 Elsevier Science Ltd. All rights reserved.
The incidence of stroke remains a major health concern
for an increasingly aging worldwide population.1
Strokes are classified into two types: hemorrhagic stroke
resulting from blood vessel rupture, and ischemic
stroke, which results from vessel occlusion accounts for
nearly 80% of all strokes. Proposed new post-stroke
therapies have focused on removal of the occluding
thrombus in ischemic stroke and neuroprotective agents
that protect neurons at risk. Thrombolytic agent, tissue
plasminogen activator (tPA) which restore blood flow
to ischemic neuronal tissue, is the only US FDA
approved therapy for acute stroke.2 Despite numerous
attempts to develop neuroprotective agents thus far
none have been approved due to lack of demonstrated
efficacy or because of poor side-effect profiles.3 We have
approached the problem of post-stroke neuroprotection
by developing potent and specific openers of large-con-
ductance calcium-activated (maxi-K) potassium chan-
nels. This concept has been validated preclinically in
animal models of stroke with BMS-204352 (Maxi-
PostTM)4 and BMS-191011.5
animal models of stroke including permanent MCAO in
the SHR rat and a normotensive model of focal stroke.5
However, clinical evaluation of BMS-191011 as an
intravenous neuroprotectant agent for the treatment of
acute ischemic stroke was hampered by its extremely
low aqueous solubility (<1 mg/mL of water).
Formation of water-soluble prodrugs has long been
recognized as an effective means of increasing the aqu-
eous solubility of drugs containing a hydroxyl group. In
general, sodium salts of phosphate ester prodrugs are
freely soluble in water and readily hydrolyzed by alka-
line phosphatases in vivo.6 Despite their good aqueous
solubility and solution stability, sulfate esters are highly
resistant to enzymatic hydrolysis in vivo.7,8 Dialkyl-
amino acid esters are readily cleaved by plasma esterases
but exhibit poor stability in aqueous solution.9,10 For
clinical evaluation the desired prodrug of BMS-191011
was required to have sufficient aqueous solubility (at
least 1 mg/mL), long duration of solution stability (up
to 24 h), and the ability to generate BMS-191011 upon
in-vivo administration.
The maxi-K channel opener, BMS-191011 (1) has been
identified as a potent neuroprotectant in two distinct
With the aim of improving aqueous solubility of
BMS-191011 (1), a series of potential prodrugs were syn-
thesized as shown in Scheme 1. Our approach was to
attach a water-solubilizing group directly or via a suitable
*Corresponding author. Tel.: +1-203-677-7815; fax: +1-203-677-
7702; e-mail: hewawasp@bms.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00296-8

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P. Hewawasam et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1695–1698
aqueous solubility, solution state stability and enzy-
matic cleavage in rat and human plasma.
While phosphate esters 9 and 14 displayed insufficient
solution state stability, the completely stable phosphate
prodrug 2 did not convert to the parent in rat or human
plasma. The dialkylamino ester derivatives 3a–c, 10a–c,
and 15 showed very poor solution state stability. In
order to overcome the instability of dialkylamino ester
prodrugs quaternization of the dialkylamino moiety was
attempted. Unlike dialkylamino ester prodrugs, the
corresponding quaternary ammonium salts were found
to be crystallizable solids with excellent aqueous solubi-
lity and exceptional solution state stability. In addition,
majority of the quaternary ammonium salts was imme-
diately cleaved to BMS-191011 in rat plasma (Table 1).
Based on its superior chemical stability, crystallinity and
formation of BMS-191011 in rat and human plasma,
the prodrug 11c (n=3) was selected for further eval-
uation. As a prelude to evaluating compound 11c in
animal models of stroke, more detailed in vitro conver-
sion studies (rat and human whole blood), as well as a
rat pharmacokinetic study, were performed.
Scheme 1. (a) R=PO(OH)2; (1) NaH, ((BnO)2P(O))2O, THF; (2)
PtO2, EtOAc, H2 (40 psi); (b) R=C(O)(CH2)nNR2; NaH,
The pharmacokinetics of 11c and its conversion to
BMS-191011 were studied following intravenous infu-
sion administration of 11c (1.7 mg/kg, equivalent to
1 mg/kg of BMS-191011) to male SD rats (N=3). To
prevent ex-vivo hydrolysis of 11c, blood samples imme-
diately after collection were added to an equal volume
of water and quenched with four volumes of acetonitrile
and stored at À20 ꢀC until analysis. Concentrations of
11c and BMS-191011 in the quenched blood samples
were determined by two separate LC/MS/MS assays.
The results are shown in Figure 1 and the pharmaco-
kinetic parameters are summarized in Table 2.
ꢀ
.
ClC(O)(CH2)nNMe2 HCl, ether; (c) MeSO3Me, EtOAc, 23 C; (d)
R=CH2Cl; (1) NaH, dry HMPA, ClCH2SMe; (2) SO2Cl2, DCM; (e)
R=CO2CH2Cl; (1) ClCO2CH2Cl, pyridine, DCM; (f) R=CO2CH2I;
NaI, acetone, reflux; (g) R=COCl; COCl2, cat. BnPh3PCl, PhCH3,
110–120 ꢀC, sealed tube; (h) (1) AgOP(O)(OBn)2, PhH, reflux; (2)
.
PtO2, EtOAc, H2 (40 psi); (i) HO2C(CH2)nNMe2 HCl, Cs2CO3, ace-
tone, 23 ꢀC; (j) HO(CH2)nNMe2, DCM, 0–23 ꢀC.
linker to the hydroxyl group of 1. Reaction of sodium
salt of 1 with tetrabenzyl pyrophosphate in THF gave
the corresponding dibenzylphosphate ester, which was
subjected to catalytic hydrogenation to provide the
phosphate ester 2. Acylation of the hydroxyl group of 1
11a,b
.
with ClCO(CH2)nNMe2 HCl
provided the desired
To determine the in vitro rate of conversion of 11c to
BMS-191011, incubations at 37 ꢀC were conducted in
dimethylamino esters 3a–c. Methylation of 3a–c with
methyl methanesulfonate in EtOAc afforded the tri-
methylammonium salts 4a–c. The chloromethyl ether 5
was prepared by chlorinating12 the corresponding
methylthiomethyl ether as indicated in Scheme 1. Reac-
tion of 5 with silver dibenzyl phosphate in refluxing
benzene gave the corresponding dibenzyl phosphate
ester which was debenzylated by catalytic hydrogena-
tion using PtO2 to afford the phosphate ester 9. Acyl-
oxylation of 5 with cesium salt of the dimethylamino
acids in acetone gave the esters 10a–c, which upon
methylation afforded the quaternary ammonium salts
11a–c. The chloromethyl carbonate 6, prepared by
acylation of 1 with chloromethyl chloroformate was
converted to iodomethyl carbonate 7 under Finkelstein
conditions. The iodomethyl carbonate 7 was converted
to the phosphate ester 14 under identical conditions
used for the preparation of 9. Chloroformylation of 1
with excess phosgene in the presence of catalytic
amount of BnPh3PCl at 110–120 ꢀC in a sealed tube
gave the chloroformate 8. Condensation of 8 with
HO(CH2)2NMe2 afforded the corresponding dimethyl-
aminoethyl carbonate 15, which upon methylation pro-
vided the trimethylammonium salt 16. The prodrugs
prepared by these methods were evaluated for their
Table 1. Chemical stability, aqueous solubility and rat plasma clea-
vage data of selected prodrugs of BMS-191011
Compd
% conversion to Solubility (@ pH) % of prodrug
BMS-191011a
in water mg/mL remains @ 24 h in
PEG400/water, 1:1
2
0
NT
NT
(5.5) 4.9
(7.2) 14.6
(7.0) 6.1
(8.4)>16
(2.4) 0.055
(7.5) 0.004
>4 mgb
ND
9
Not stable
Not stable
14
4a (n=1)
4b (n=2)
4c (n=3)
11a (n=1) Converted to an
unknown compound
100
100
95
>90
>90
>90
ND
>4 mgb
>4 mgb
(4.2) 4.5
(3.3) 4.5
(4.8) 3.7
>25 mgb
(3.0) 3.9
(4.1) 4.4
(5.5) 3.6
11c (n=3)
16
>80
100
>97
72
aAfter 30-min incubation in rat plasma at 37 ꢀC.
bSolubility of prodrug in deionized water.

P. Hewawasam et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1695–1698
1697
Figure 1. Mean (ÆSD) blood concentrations of 11c and BMS-191011
(1) in rats following administration of an IV dose (1.7 mg/kg) of 11c
(dosing vehicle: PEG-400/water, 1:1).
Figure 2. Disappearance of 11c as a function of time after its incu-
bation in human and rat whole blood and plasma.
Table 2. Summary of pharmacokinetics of 11c and BMS-191011 in
the rat following administration of 11c
Table 3. Percent reduction of neocortical infarct volume in rats after
IV administration of 11c (2 h post-MCA)a
Parameter
11c
BMS-191011
Dose (mg/kg)
AUC0-t (ng h/mL)
1258.6Æ153.8
2.7Æ0.2
52.4Æ4.3
1.2Æ0.5
—
0.00001 0.0001 0.001 0.1 1000
Apparent elimination t1=2 (h)
Clearance (mL/min kg)
Vdss (L/kg)
À16b À14b À12b À17b
22.9Æ0.8
2.6Æ0.3
% Reduction in infarct volume
Number of animals
À13
—
28
29
28
28
28
aCompared to vehicle-treated (2%DMSO/98% propylene glycol)
controls in the same study.
bp <0.05, ANOVA and Dunnett’s t-test.
rat and human plasma and whole blood. The results are
shown in Figure 2. Cleavage of 11c was not immediate
in either plasma or whole blood from rat or human; this
may, at least in part, be the cause of the apparent long
half-life of 11c observed in the rat. The identity of the
esterases responsible for the hydrolysis of 11c, the
quantitative expression and the inter-individual varia-
bility of those enzymes have not been determined. In the
absence of such information, quantitative extrapola-
tions of human pharmacokinetics cannot be made from
the human in-vitro data.
therapeutic window of 1.0 ng/kg–10 mg/kg for BMS-
191011.
In summary, we have successfully prepared a variety of
prodrug derivatives of BMS-191011 with significantly
enhanced aqueous solubility. Furthermore, we demon-
strated that several of these water-soluble prodrugs
possessed long duration of solution state stability and
undergo extensive conversion to the parent drug, BMS-
191011. Deoxycarnitine ester prodrug (11c) was selected
for clinical evaluation based on its superior chemical
stability, crystallinity and ability to generate BMS-
191011, in vitro and in vivo.
It was found that prodrug 11C crosses the rat blood
brain barrier but not as efficiently as BMS-191011,
resulting in brain/blood concentration ratios of 0.1
and 5.7 for 11c and BMS-191011, respectively. By
using whole-cell voltage clamp recordings and patch
clamp analysis it has been confirmed that prodrug 11c
is not a maxi-K opener prior to the cleavage to BMS-
191011.
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