Controlled drug release: New water-soluble prodrugs of an HIV protease inhibitor

Article abstract of DOI:10.1016/S0960-894X(01)00007-5

We designed and synthesized a series of highly water-soluble prodrugs of an HIV protease inhibitor, KNI-727 (1), containing tandem-linked two auxiliary units, a solubilizing moiety and a self-cleavable spacer. Prodrugs with an ionized amino group at the solubilizing moiety exhibited a remarkable increase of water-solubility (>10⁴ fold) compared to the parent drug 1. These prodrugs released 1 not enzymatically, but chemically via an intramolecular cyclization-elimination reaction through an imide formation in physiological conditions. Diversified rates of parent drug release were observed when the chemical structure of both the solubilizing and the spacer moieties were modified. This new approach for water-soluble prodrugs will enable to control chemically the release of parent drug as well as to maintain high water-solubility.

Full text of DOI:10.1016/S0960-894X(01)00007-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 605±609
Controlled Drug Release:
New Water-Soluble Prodrugs of an HIV Protease Inhibitor
Hikaru Matsumoto, Youhei Sohma, Tooru Kimura, Yoshio Hayashi
and Yoshiaki Kiso*
Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University,
Yamashina-ku, Kyoto 607-8412, Japan
Received 22 November 2000; accepted 6January 2001
AbstractÐWe designed and synthesized a series of highly water-soluble prodrugs of an HIV protease inhibitor, KNI-727 (1), con-
taining tandem-linked two auxiliary units, a solubilizing moiety and a self-cleavable spacer. Prodrugs with an ionized amino group
at the solubilizing moiety exhibited a remarkable increase of water-solubility (>104 fold) compared to the parent drug 1. These
prodrugs released 1 not enzymatically, but chemically via an intramolecular cyclization-elimination reaction through an imide for-
mation in physiological conditions. Diversi®ed rates of parent drug release were observed when the chemical structure of both the
solubilizing and the spacer moieties were modi®ed. This new approach for water-soluble prodrugs will enable to control chemically
the release of parent drug as well as to maintain high water-solubility. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
HIV PR inhibitor conjugated with a reverse tran-
scriptase inhibitor by spontaneously cleavable linkers
such as glutarylglycine and succinylglycine.12 In
physiological conditions, these linkers can release the
parent compounds spontaneously via intramolecular
cyclization through an imide formation.12À15 Therefore,
we considered these linkers to be useful as the solubiliz-
ing moiety, which could be attached to the parent drug.
The sparing water-solubility of drugs often cause unde-
sirable pharmaceutical properties such as erratic oral
absorption pro®le, poor oral bioavailability and the
limitation in the availability of both oral and parenteral
dosage forms.1 To date, there are many drugs posses-
sing low aqueous solubility due to the inherent high
lipophilicity, which ultimately reduces their therapeutic
ecacy.2À6 This undesirable limitation often occurs in
many of the peptidomimetic drugs such as HIV protease
(PR) inhibitors and renin inhibitors.7À10 Thaisrivongs
and co-workers9,10 reported highly water-soluble phos-
phate prodrugs of HIV PR inhibitors with improved
biological activity. Therefore, increasing the water-
solubility of these drugs is of more concern.
The objective of the present study is to design water-
soluble prodrugs by utilizing this spontaneously clea-
vable linker strategy. As a model compound, we selec-
ted a dipeptide-based HIV PR inhibitor with poor
water-solubility, KNI-727 (1) (Fig. 1).16,17 For the
design of prodrugs, two auxiliary units, a solubilizing
moiety and a self-cleavable spacer, were tandemly
One of the e€ective strategies to improve the solubility
is to convert the water-insoluble compounds into pro-
drugs by covalently attaching an appropriate solubiliz-
ing moiety.11 The critical requirement for the prodrug is
to attach an solubilizing moiety to the parent drug in a
bioreversible manner.11 In our previous studies, we had
reported `double-drug' strategy, a new prodrug form of
*Corresponding author. Tel.: +81-75-595-4635; fax: +81-75-591-
9900; e-mail: kiso@mb.kyoto-phu.ac.jp
Figure 1. Structure of water-insoluble HIV protease inhibitor, 1
(KNI-727).
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00007-5

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H. Matsumoto et al. / Bioorg. Med. Chem. Lett. 11 (2001) 605±609
linked to the parent drug 1 (Fig. 2). As a spacer, we used
substituted succinamide or glutaramide to link the par-
ent drug and the solubilizing moiety. In physiological
conditions (pH 7.4), we expect the spacer to sponta-
neously cyclize to the imide derivative and thereby
release the parent drug.12 The advantages of water-
soluble prodrugs utilizing this type of spacers are as
follows: (a) As prodrugs can release the parent drug by
chemical reaction that is dependent on the cyclization
tendency of the spacer, the rate of the parent drug
release can be controlled by the modi®cation of the
structure of the spacer units. The controlled release
prodrug strategy will be widely applicable to various
kinds of drugs and dosage forms. (b) Various solubiliz-
ing moieties (e.g. acidic, basic or nonionizable) can be
introduced on demand.
Based on these premises, we synthesized water-soluble
prodrugs containing various solubilizing moieties and
spontaneously cleavable spacers. These prodrugs were
examined for their water-solubility and the rate of drug
release. The prodrug strategy employed in this paper
allowed us to control the drug release rate by altering
the chemical structure of the auxiliary as well as to
increase the water-solubility.
Results and Discussion
Chemistry
Figure 2. General structure of water-soluble prodrug, and its intra-
molecular cyclization-elimination reaction releasing the parent drug
and an imide fragment.
The synthesis of these prodrugs is summarized in
Schemes 1 and 2. KNI-727 (1) was coupled with
Scheme 1. Reagents and conditions: (a) succinic anhydride or glutaric anhydride, DCHA, THF±ether (1:2); (b) NH2COCH2CH2COOH, EDCꢀ HCl,
DMAP, DMF; (c) HClꢀ H-Gly-OBut or HClꢀ H-b-Ala-OBut, Et3N, BOP, HOBt, DMF; (d) TFA; (e) HClꢀ H-Gly-NH2, EDCꢀ HCl, Et3N, HOBt,
DMF; (f) Boc-NH(CH2)2NH2, EDCꢀ HCl, HOBt, DMF; (g) 4 N HCl±dioxane; (h) (Me)2NCH2CH2NH2, EDCꢀ HCl, HOBt, DMF.
Scheme 2. Reagents and conditions: (i) 2,2-dimethylsuccinic anhydride, DCHA, THF±ether (1:2); (j) Boc-NH(CH2)2NH2, EDCꢀ HCl, HOBt, DMF;
(k) 4 N HCl±dioxane.

H. Matsumoto et al. / Bioorg. Med. Chem. Lett. 11 (2001) 605±609
607
succinic anhydride in THF±ether in the presence of
dicyclohexylamine (DCHA) to yield the half-ester 2a.12
Compound 2b was prepared from glutaric anhydride in
a similar manner. Compound 3 was prepared by cou-
pling of 1 with succinamic acid using 1-ethyl-3-(3-
On the other hand, prodrugs 7±10 containing the amine
structures, which were hydrochloride salts, drastically
increased water-solubility more than 5100-fold. Among
these prodrugs, 10 showed the highest solubility with
the value of 15450-fold (171.5 mg/mL) compared to 1
(0.011 mg/mL).
(dimethylamino)propyl)carbodiimide
hydrochloride
(EDCꢀ HCl) in the presence of dimethylaminopyridine
(DMAP). Condensation of 2a and tert-butyl ester of
glycine or b-alanine by use of benzotriazole-1-yloxy-tris
(dimethylamino) phosphonium hexa¯uorophosphate/1-
hydroxybenzotriazole (BOP/HOBt) in the presence of
triethylamine, and following deprotection by tri-
¯uoroacetic acid (TFA) a€orded 4 and 5. Compound 6
was obtained by coupling 2a with glycine amide using
EDC/HOBt. Compounds 7 and 8 were prepared by
coupling 2a or 2b and N-Boc-ethylenediamine by the
use of EDC/HOBt followed by the deprotection of the
Boc group under acidic conditions.
Disintegration rate of prodrugs
Disintegration pro®le of the prodrugs in phosphate
bu€ered saline (PBS, pH 7.4) at 37 ꢁC was determined
by HPLC analysis.19 In all the cases, prodrugs com-
pletely released the parent drug. A typical HPLC chro-
matogram (using 7 as an example) and a kinetic pro®le
for the disappearance of the prodrug and the appear-
ance of parent drug 1 are shown in Figure 3 and 4,
respectively. Prodrug
7
(Rt=12 min) released
1
(Rt=18 min) distinctly with t1/2 of 21 min. The imide
derivative that is the other disintegration product from
7 was invisible, because it was undetectable at 230 nm in
HPLC analysis. Kinetic analysis of the disintegration
showed that 1 was released following pseudo-®rst-order
kinetics, consistent with an intramolecular reaction as
shown in Figure 2.
Compound 9 was prepared as shown in Scheme 2. The
treatment of 1 with 2,2-dimethylsuccinic anhydride in
the presence of DCHA a€orded the half ester 2c. Con-
densation of 2c and N-Boc-ethylenediamine followed by
the deprotection of Boc group a€orded 9. Compound
10 was prepared by coupling 2a with N,N-dimethyl-
ethylenediamine by the use of EDC/HOBt. All the
crude products were puri®ed and converted to corre-
sponding hydrochloride salts by HPLC using binary
solvent system (linear gradient of CH3CN in 12 mM
aqueous HCl), and after lyophilization the obtained
amorphous products were used for the evaluation.
In order to con®rm the disintegration mechanism of the
prodrugs, compound 11 containing succinylbenzyl-
amide was synthesized, and the cleavage products of 11
in PBS (pH 7.4, 37 ꢁC) were analyzed by HPLC and
mass spectrometry. Two new peaks appeared, which
were assigned as parent drug 1 and succinimide deriva-
tive. In the same condition, hemisuccinate 2a was stable
for at least 48 h. These results suggested that the regen-
eration of the parent compound was not due to the
simple hydrolysis of the ester bond but intramolecular
cyclization-elimination reaction through the imide for-
mation. The stability of the prodrugs to the enzymatic
Water-solubility of prodrugs
Water-solubility of the prodrugs was determined and
compared to the parent drug, 1 (Table 1).18 Compounds
3, 4 and 6 showed a moderate increase in water-solubility.
Table 1. Water-solubility and disintegration rate of prodrugs
Compound
X
R
Water-solubility
mg/mL
t1/2a (min)
Relative
1
3
4
5
6
7
8
9
Ð
Ð
H
11.1Â10À3
89.5Â10À3
73.5Â10À3
N.D.b
1.0
8.1
6.6
N.D.b
18.9
10,400
5,130
12,320
15,450
Ð
Ð
960
1122
1008
20
ÀCH2CH2À
ÀCH2CH2À
ÀCH2CH2À
ÀCH2CH2À
ÀCH2CH2À
ÀCH2CH2CH2À
ÀCH2C(Me)2À
ÀCH2CH2À
ÀCH2CH2À
ÀCH2COOH
À(CH2)2COOH
ÀCH2CONH2
À(CH2)2NH2ꢀ HCl
À(CH2)2NH2ꢀ HCl
À(CH2)2NH2ꢀ HCl
À(CH2)2N(CH3)2ꢀ HCl
ÀCH2C6H5
210Â10À3
115.4
56.9
136.7
171.5
21
1680
<1
11
10
11
<11.1Â10À3
528
at1/2 is the time required for 50% release of parent drug (1) at 37 ꢁC in phosphate bu€ered saline (pH 7.4).
bNot determined.

608
H. Matsumoto et al. / Bioorg. Med. Chem. Lett. 11 (2001) 605±609
hydrolysis was examined using carboxyesterase (porcine
liver esterase, E.C. 3.1.1.1.). Consequently, the prodrugs
exhibited resistance towards esterase hydrolysis, and the
disintegration pro®le was almost the same as seen in the
case of PBS (pH 7.4).
Interestingly, the disintegration rates of prodrugs were
a€ected by the structure of both spacer and solubilizing
moiety. Prodrug 7 containing succinyl moiety as the
spacer released 1 much faster (t1/2=21 min) than 8 con-
taining glutaryl group (t1/2=28 h). This is attributed to
the lower energy for cyclization reaction of 7 in the for-
mation of the ®ve-membered cyclic imide.15 The
geminal methyl substitution of the succinyl moiety
drastically increased the disintegration rate of the pro-
drug. The prodrug 9 released 1 completely within 5 min.
This acceleration of the intramolecular cyclization rate
is attributed to a unique interlocking of the geminal
methyl group, which produces a severe conformation
restriction (gem e€ect).20À25
Prodrug 4 and 5 containing the carboxyl group as a
solubilizing moiety (R) released 1 much slower (t1/
2=18.7 h and 16.8 h, respectively) than 7. When the
carboxyl group of 4 was converted to carboxamide,
resulting prodrug 6 disintegrated at an almost similar
rate (t1/2=20 min) as 7. The stability of 4 and 5 is
probably due to the presence of a negative charge in the
same part of the molecule, which may reduce the
nucleophilicity of amide nitrogen and inhibit the cycli-
zation reaction.14,15 Prodrug 10 containing N,N-dime-
thylethylenediamide disintegrated about two times
faster (t1/2=11 min) than 7, whereas 3 containing succi-
namide disintegrated more slowly (t1/2=16h).
Figure 3. Typical HPLC chromatograms of the disintegration of pro-
drug 7 in PBS (pH 7.4, 37 ꢁC), at 0, 5, 15, 30 and 45 min. The retention
times of 1 (KNI-727) and prodrug 7 were 18 and 12 min, respectively.
The observed di€erences of the drug release rate suggest
that there are at least three factors a€ecting the kinetics
of cyclization reaction of the auxiliary, that is: (1)
energy for cyclic imide formation; (2) conformational
restriction caused by `gem e€ect'; and (3) the presence of
negative charge in the same residue. Consequently, this
type of prodrug enables to control the rate of drug
release through chemical modi®cation of the auxiliary.
The disintegration rate of 7 was examined by HPLC at
various pH (at 37 ꢁC). The rate was pH dependent. A
typical kinetic pro®le showing the disintegration of 7 at
various pH is shown in Figure 5. At pH 5.5, prodrug 7
released 1 slowly compared to the case of pH 7.4. At pH
2.0, prodrug 7 was stable for at least 48 h. Even prodrug
9, which possessed the shortest half-life (<1.0 min) at
pH 7.4, was stable for at least 2 weeks at pH 2.0. The
good water-solubility and smooth conversion of pro-
drug to the parent drug in physiological conditions (pH
7.4) would expect to be suitable for intravenous admin-
istration, and the sucient chemical stability at low pH
will allow the routine handling and storage.26
Figure 4. Typical kinetic pro®le showing the disappearance of prodrug
7 and the appearance of parent drug 1 in PBS, pH 7.4 at 37 ꢁC.
Conclusion
We designed and synthesized new water-soluble pro-
drugs with controlled release of the parent drug. All the
prodrugs had increased water-solubility, and among
them, prodrug 10 containing an amino group had a
drastic increase of the water-solubility, that is more than
15000-fold compared to that of the parent drug. These
prodrugs released the parent drug not enzymatically but
chemically via intramolecular cyclization through imide
Figure 5. Typical kinetic pro®le showing the disintegration of prodrug
7 in bu€er at various pH (at 37 ꢁC). The bu€ers used were as follows:
pH 2.0, glycine±HCl bu€er; pH 5.5 and 7.4, phosphate bu€ered saline.

H. Matsumoto et al. / Bioorg. Med. Chem. Lett. 11 (2001) 605±609
609
formation, and the rate of parent drug release can be
controlled by: (1) chemical modi®cation of the spacer
and the solubilizing moiety; and (2) altering the pH of
the media. We suggested that this prodrug strategy
would be useful to increase the water-solubility as well
as to control the rate of parent drug release, and be
applicable to various water-insoluble drugs containing a
hydroxyl group.
12. Matsumoto, H.; Hamawaki, T.; Ota, H.; Kimura, T.;
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13. Tamamura, H.; Ishihara, T.; Oyake, H.; Imai, M.; Otaka,
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HPLC.
Acknowledgements
This research was supported in part by the Frontier
Research Program of the Ministry of Education,
Science and Culture of Japan, and grants from the
Ministry of Education, Science and Culture of Japan
and Japan Health Science Foundation. We thank Dr. C.
V. Ramesh for his help in preparing the manuscript.
References and Notes
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by HPLC. HPLC was performed using a C18 reverse phase
column with a linear gradient of 40±100% acetonitrile in 0.1%
aqueous TFA over 30 min, detected at UV 230 nm.
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