Novel prodrugs with a spontaneous cleavable guanidine moiety

Article abstract of DOI:10.1016/j.bmcl.2016.02.060

Water-soluble prodrug strategy is a practical alternative for improving the drug bioavailability of sparingly-soluble drugs with reduced drug efficacy. Many water-soluble prodrugs of sparingly-soluble drugs, such as the phosphate ester of a drug, have been reported. Recently, we described a novel water-soluble prodrug based on O–N intramolecular acyl migration. However, these prodrug approaches require a hydroxy group in the structure of their drugs, and other prodrug approaches are often restricted by the structure of the parent drugs. To develop prodrugs with no restriction in the structure, we focused on a decomposition reaction of arginine methyl ester. This reaction proceeds at room temperature under neutral conditions, and we applied this reaction to the prodrug strategy for drugs with an amino group. We designed and synthesized novel prodrugs of representative sparingly soluble drugs phenytoin and sulfathiazole. Phenytoin and sulfathiazole were obtained as stable salt that were converted to parent drugs under physiological conditions. Phenytoin prodrug 3 showed a short half-life (t_1/2) of 13?min, whereas sulfathiazole prodrug 7 had a moderate t_1/2of 40?min. Prodrugs 3 and 7 appear to be suitable for use as an injectable formulation and orally administered drug, respectively.

Full text of DOI:10.1016/j.bmcl.2016.02.060

Accepted Manuscript
Novel prodrugs with a spontaneous cleavable guanidine moiety
Yoshio Hamada
PII:
S0960-894X(16)30171-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.060
BMCL 23614
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
25 December 2015
5 February 2016
19 February 2016
Please cite this article as: Hamada, Y., Novel prodrugs with a spontaneous cleavable guanidine moiety, Bioorganic
& Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.060
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Bioorganic & Medicinal Chemistry Letters
Novel prodrugs with a spontaneous cleavable guanidine moiety
Yoshio Hamada
Medicinal Chemistry Laboratory, Kobe Pharmaceutical University, Motoyamakita, Higashinada, Kobe 658-8558, Japan,
A RT ICL E INFO
A BST RA CT
Article history:
Received
Revised
Accepted
Available online
Water-soluble prodrug strategy is a practical alternative for improving the drug bioavailability of
sparingly-soluble drugs with reduced drug efficacy. Many water-soluble prodrugs of sparingly-
soluble drugs, such as the phosphate ester of a drug, have been reported. Recently, we described
a novel water-soluble prodrug based on O-N intramolecular acyl migration. However, these
prodrug approaches require a hydroxy group in the structure of their drugs, and other prodrug
approaches are often restricted by the structure of the parent drugs. To develop prodrugs with no
restriction in the structure, we focused on a decomposition reaction of arginine methyl ester.
This reaction proceeds at room temperature under neutral conditions, and we applied this
reaction to the prodrug strategy for drugs with an amino group. We designed and synthesized
novel prodrugs of representative sparingly soluble drugs phenytoin and sulfathiazole. Phenytoin
and sulfathiazole were obtained as stable salt that were converted to parent drugs under
physiological conditions. Phenytoin prodrug 3 showed a short half-life (t1/2) of 13 min, whereas
sulfathiazole prodrug 7 had a moderate t1/2 of 40 min. Prodrugs 3 and 7 appear to be suitable for
use as an injectable formulation and orally administered drug, respectively.
Keywords:
guanidino acid
phenytoin
prodrug
quinine
sulfa drug
2015 Elsevier Ltd. All rights reserved.
Prodrug strategy is a practical alternative for improving drug
bioavailability, since it overcomes various barriers that reduce
drug efficacy, including poor solubility. There are many well-
known sparingly-soluble drugs, and they often exhibit reduced
bioavailability and have undesirable pharmaceutical properties.1-3
For example, most human immunodeficiency virus type 1 (HIV-
1) protease inhibitors are poorly soluble in aqueous solutions,
such as gastric juice. HIV-1 protease is a dimer, and the pockets
around its active site are hydrophobic. Thus, potent inhibitors
that are optimized for the pockets of HIV-1 protease show poor
water-solubility. The formulations of HIV-1 protease inhibitors,
such as ritonavir, contain some solubilizers, such as
polyoxyhydrogenated caster oils, which lead to some side effects.
The antiepileptic phenytoin is a representative sparingly-soluble
drug. Because phenytoin is only dissolved in strongly-alkaline
aqueous solutions, the injectable formulations of phenytoin are
prepared as an aqueous solution at pH 12, often resulting in
strong irritant properties. Recently, we reported a series of β-
secretase (β-site amyloid precursor protein cleaving enzyme 1;
BACE1) inhibitors as anti-Alzheimer’s disease drugs.4-16 Because
BACE1 also has some hydrophobic pockets around its active site,
BACE1 inhibitors optimized for the hydrophobic pockets were
often sparingly-soluble. To improve the poor solubility, one
effective strategy is to convert the poor-soluble drugs into
hydrophilic prodrugs by covalently attaching the appropriate
solubilizing moieties, such as phosphates,17,18 sugars,19,20 and
or chemically under physiological conditions. The hydroxy
groups in drugs are often modified for the application to prodrugs
because they are easily cleaved. The phosphate-type water-
soluble prodrugs of HIV-1 protease inhibitors were reported by
Taisrivongs et al.23 These prodrugs can release the parent drugs
by cleaving a phosphate ester bond through the help of an
alkaline phosphatase. Thus, this type of prodrug requires a
hydroxy group in the structure of the parent drug. Previously, we
reported novel water-soluble prodrugs based on O-N
intramolecular acyl migration reaction.24-28 These prodrugs,
wherein the amide bond of the parent drug is converted to an
ester bond, could be rapidly converted to the corresponding
parent drugs under physiological conditions. However, there is a
structural restriction for the application of these prodrugs, in
particular, as the parent drugs must have a hydroxy group in their
structure. Moreover, our water-soluble ritonavir prodrug
exhibited a drug release rate that was too slow (t1/2 = 42 h).24 The
prodrugs with a steric hindered hydroxy group, such as ritonavir,
might have slow drug release rates because O-N intramolecular
acyl migration requires a conformational change for the
formation of
a
transition state that accompanies the
intramolecular migration reaction. As seen above, there are
various structural restrictions for the design of water-soluble
prodrugs. New prodrug strategies are required with no structural
restriction and with a more rapid drug release rate and spread
wide modifiable functional groups,such as an amino group,other
than hydroxy group.
amines,21,22 which can eliminate fromthe prodrugs enzymatically
———
 Corresponding author. Tel.: +81-78-441-7555; e-mail: pynden@gmail.com

Figure 1. (A) Decomposition reaction of arginine methyl ester. (B) Design of two types of prodrugs based on the decomposition reaction of
arginine methyl ester.
To overcome these issues, we focused on a decomposition
reaction of arginine methyl ester, which was reported by Photaki
et al.,29 as shown in Figure 1A. In this reaction, the guanidino
group of an arginine methyl ester attacks the ester’s carbonyl
carbon of another arginine methyl ester, forming the arginine
dimer. Next, N-terminal amino group of the dimer attack the
guanidine’s carbon within the molecule, forming a heterocyclic
compound and ornithine methyl ester. Surprisingly, this
decomposition reaction proceeds at room temperature under
neutral conditions. Hence, we believe that the second reaction in
the decomposition of arginine methyl ester can be applied to
prodrug design. We focused on the aminoacylguanidine structure
of the arginine dimer and designed a novel prodrug (Type A), as
shown in Figure 1B. This prodrug is thought to release a
heterocyclic compound—glycocyamidine—forming the parent
drug. We assumed that a driving force of this reaction was the
release of the heterocyclic compound that was stabilized with a
conjugate structure. Because a compound in which the order of
the acyl and guanidino group of the prodrug (Type A) is reversed
is thought to release the same heterocyclic compound to the
prodrug (Type A), we speculated that the release of the
heterocyclic compound might become a driving force for the
cleavage reaction of an amide bond and designed another novel
prodrug (Type B), as shown in Figure 1B.
First, we elected 2-aminobenzimidazole as a model compound
for evaluating our prodrug approach, because many sparingly-
soluble drugs have a/some amino group(s) on a heterocyclic ring
or aromatic ring, whereas drugs with an aliphatic amino group
are easily-soluble as a salt in aqueous solutions.We designed and
synthesized two types of prodrugs (1 and 2) of 2-
aminobenzimidazole, as shown in Figure 2. Subsequently, we
synthesized the prodrugs 2-7 of the representative sparingly-
soluble drugs, phenytoin and sulfathiazole, and also the prodrugs
8 and 9 of the anti-malarial quinine with a hydroxy group.
Compounds 1-9 were synthesized using the common solution-
phase synthesis method as shown in Scheme 1. Amide and ester
bond formation for preparing 10 and 11a-e was performed using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide•HCl (EDC) in
the presence of 4-dimethylaminopyridine (DMAP) as a coupling
reagent. Guanidination reaction for preparing 13a-e was
performed using N,N’-Bis(tert-butoxycarbonyl)1H-pyrazole-1-
carboxamidine in acetonitrile or acetonitrile containing a small
amount of dimethylformamide (DMF). The guanidination
reaction generally proceeded more rapidly in low polar solvents.
Because most materials are poorly soluble in low polar solvents,
we used acetonitrile with/without DMF for the guanidination
reaction. Boc-deprotection of compounds 11a-e was performed
using anisole and 4N HCl/dioxane, whereas Boc-protection for
preparing 1-9 was performed using trifluoroacetic acid (TFA).
Prodrugs 1-9 were obtained as TFA salts and stored in a
refrigerator.
Figure 2. Design of two kinds of 2-aminobenzimidazole
prodrugs.

S cheme 1. Reagents and condition: ( a) Boc-Gly -OH, EDC, DMAP/DMF, room temp erature, 1 day ; (b) TFA, room temp erature, 2 h; (c)
Boc-Gly -OH, Boc-Ala-OH, or Boc-Sar-OH, EDC, DMAP/DMF, room temp erature, 1 day ; (d) anisole, 4 M HCl/dioxane, room
temp erature, 2h.; (e) N,N-Bis(tert-butoxy carbonl)1H-py razole-1-carboxamidin e, acetonitrile, room temp erature, 1day .
Prodrugs 1-9 were evaluated by high-performance liquid
chromatography (HPLC) analysis using a reverse phase C18
column and a linear gradient system of acetonitrile and 0.1%
aqueous TFA. These prodrugs and peptide were incubated under
physiological condition with pH 7.4 phosphate buffered saline
(PBS) at 37°C and measured by HPLC. Prodrug 1 was stable as a
TFA salt, but 1 was labile in polar solvents,such as methanol and
aqueous solvents, and released quickly the parent compound 2-
aminobenzimidazole. Thus, Type A prodrugs appeared to be not
appropriate for the prodrug strategy. Prodrug 2 (Type B) was
obtained as a TFA salt that was stable in acidic media, methanol,
and unbuffered water. Prodrug 2 (Type B) appeared to be present
as a TFA salt in methanol and unbuffered water. HPLC profile
and time course of 2 in pH 7.4 PBS at 37°C are shown in Figure
3A and B, respectively. Prodrug 2 released time-dependently (t1/2
value: 38 min, Table 1) its parent drug 2-aminobenzimidazole
with no byproduct.
Figure 3. (A) HPLC profile of prodrug 2 in pH 7.4 PBS at 37°C. (B) Formation rate of 2-aminobenzimidazole from prodrug 2 in pH 7.4
PBS at 37°C.

Figure 4. Formation of parent drugs from phenytoin prodrugs (3-5), sulfathiazole prodrug (7), and quinine prodrugs (8 and 9) in pH 7.4
PBS at 37°C.
Based on these results, we synthesized a series of Type B
prodrugs (2-9) of phenytoin,sulfathiazole, and quinine.Although
the NH group of phenytoin is not an amino group but an imide
group, the prodrugs (3-5) of representative sparingly-soluble
drug, phenytoin, were synthesized first. Prodrug 3 rapidly
converted to the parent drug, phenytoin, in a time-dependent
manner with no byproduct. The phenytoin prodrug Fostoin
(fosphenytoin), which possesses a phosphate ester bond, was
approved by the US Food and Drug Administration in 2004 for
injection. Fostoin releases the parent drug, phenytoin, and
formaldehyde in the presence of an alkaline phosphatase, and
shows a t1/2 value of 8min in human plasma, which is close to
that of prodrug 3 (t1/2 = 13 min).30 Prodrug 3 appears to be
interchangeable with Fostoin for clinical use and is therefore
suitable as an injectable formulation. Prodrug 4 and 5, which
possess an α-methyl or N-methyl group in the amino acid moiety,
were converted more rapidly to the parent drug than prodrug 3.
As it is well-known that a/some methyl group(s) on a leaving
group accelerate the cyclization reaction via nucleophile attack,31
this result seemes predictable. The sulfa drug sulfathiazole is a
representative of a sparingly-soluble drug and has a typical
aromatic amino group. Prodrug
6 of sulfathiazole was
synthesized, but 6 and its parent drug sulfathiazole had the same
retention time on HPLC, and the drug release rate of 6 could not
be evaluated. Therefore, we synthesized prodrug 7, which
possesses an α-methyl group in the amino acid moiety. Prodrug 7
was converted to the parent drug in a time dependent manner,
and it showed a moderate drug release rate (t1/2 value; 40 min)
and appeared to be suitable as an orally-administered drug.
Table 1. Drug release rate and solubility of prodrugs
t1/2 value*1
solubility (mg/mL)
parent drug*3 prodrug
ratio
of solubility*2
prodrug
parent drug
R1
R2
n
(min)
2
3
4
5
7
8
9
2-aminobenzimidazole
phenytoin
-H
-H
-H
-H
0
0
0
0
0
0
1
38
13
—
6.3 × 10-3
—
—
10.2
—
—
1620
—
phenytoin
-CH3
-H
-H
1.1
0.78
40
phenytoin
-CH3
-H
—
—
—
sulfathiazole
quinine
-CH3
-H
1.4 × 10-3
—
7.1
5071
—
-H
<1
—
quinine
-H
-H
1.1
2.1 × 10-1
>10
>48
*1t1/2 is the time required for 50% release of parent drugs in pH 7.4 PBS at 37°C.
*2prodrug/parent drug
*3The parent drugs, sulfathiazole and quinine, were used as a TFA salt and an HCl salt, respectively, for measuring the solubilities.

Scheme 2. Reagents and condition: (a) Boc-NH(CH2)3COOH,
EDC, DMAP/DMF, room temperature, 1 day; (b) TFA, room
temperature, 2 h.
Figure 5. Conventional ester-typeprodrug of quinine.
To demonstrate the wide application of our prodrug strategy,
prodrug 8 of quinine with a hydroxy group was synthesized. As
an ester bond is more cleavable than an amide bond, the drug
release rate of prodrug 8 was more rapid than that of amide-type
prodrugs 2 –7. We previously reported that ester-type prodrugs
based on O-N intramolecular acyl migration via formation of a
six-membered ring showed slower drug release rates than the
same type prodrugs that formed a five-membered ring.25 Because
the drug release rate of prodrug 8 was extremely rapid (t1/2 value;
<1 min, Table 1), prodrug 9 was synthesized via formation of a
six-membered ring to delay the drug release rate. Prodrug 9 was
rapidly converted to the parent drug in a time-dependent manner
(t1/2 value; 1.1 min, Table 1) with no byproduct (see Supporting
Information) as shown in Figure 4.
14 was synthesized using a similar method as that of prodrugs 2-
9, as shown in Scheme 2. Prodrug 14 with a γ-aminobutyric
group was obtained as a TFA salt, and seems to exist as a TFA
salt in unbuffered aqueous solutions, such as water. Prodrug 14
releases the parent drug quinine and
a five-membered
heterocyclic compound derived fromthe γ-aminobutyric group as
shown in Figure 5. Although prodrug 14 released its parent drug
quinine in a time-dependent manner with no byproduct, the rate
was very slow (t1/2 value: 1.7 h, Figure 6) in pH 7.4 PBS at 37°C.
Most of the well-known ester-type prodrugs require the presence
enzymes, such as alkaline phosphatase and esterase.32 No
spontaneously cleavable ester-type prodrugs have been used in
clinical practice owing to the slow drug release rate. Although
prodrug 14 (t1/2 = 1.7 h) forms the same five-membered ring as 8
(t1/2 < 1 min), the t1/2 value of 14 was more than hundred-fold of
that of 8. This result appears to demonstrate the advantage of our
prodrug strategy.
The water-solubility and stability of synthetic prodrugs 3, 7,
and 9 was evaluated using HPLC analysis (see Supporting
Information). In all cases, the tested prodrugs exhibited water-
solubility better than that of the corresponding parent drugs
(Table 1). Prodrugs 3, 7, and 9 were stable as TFA salts for 1
month. Although storage of prodrugs 3, 7, and 9 in physiological
saline solution for 1 day resulted in 4%, 0%, and 6% drug
release, respectively, such release does not seem to present an
obstacle to clinical use of the prodrugs as injectable formulations.
In conclusion, we designed and synthesized a series of novel
water-soluble prodrugs based on a decomposition reaction of
arginine methyl ester. These prodrugs could be converted to the
respective parent drugs between the t1/2 values of 1 min and 40
min. Phenytoin prodrug 3 (t1/2 value; 13 min) and sulfathiazole
prodrug 7 (t1/2 value; 40 min) are suitable for an injectable
formulation and an orally-administered drug, respectively.
Although the t1/2 of quinine prodrug 8 was too short to allow
clinical use of the compound (<1 min), quinine prodrug 9 had a
longer t1/2 of 1.1 min, demonstrating that alteration of the
Although the cleavage of an amide bond is difficult in the
absence of an enzyme, many ester-type prodrugs are known. A
conventional ester-type prodrug of quinine, 14, was synthesized
for comparison with our ester-type prodrug of quinine 8. Prodrug
guanidino-acyl
moiety
altered
the
pharmacokinetic
characteristics of these prodrugs. These results suggest that a
quinine prodrug with a longer and clinically suitable t1/2 can be
potentially developed by utilizing our prodrug strategy. Malaria
is a disease caused by parasitic protozoa of the genus
Plasmodium, which are transmitted to humans by the Anopheles
mosquito. Malaria is a potent threat to human beings; indeed, the
range of the Anopheles mosquito is expanding as a result of
global warming. Recently, chloroquine-resistant strains of
Plasmodium protozoa have been identified, while the use of
quinine has increased. Patients with severe malaria require
intravenous administration of quinine for a period of 4 h. If the
current quinine treatment regimen can be modified to consist of a
single injection through the use of our prodrug approach, the
burden of malaria on many patients might be reduced.
Acknowledgments
This study was supported in part by the Grants-in-Aid for
Scientific Research (C) from MEXT (Ministry of Education,
Culture, Sports, Science and Technology), Japan (KAKENHI
No.23590137 and No.26460163). We thank Dr. O. Miyata and
Dr. A. Takeuchi for their help in preparing the manuscript and
measuring ESI-Mass spectra, respectively.
Figure 6. Formation rate of quinine from conventional ester-type
prodrug of quinine, 14, in pH 7.4 PBS at 37°C.

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Table 1. Drug release rate and solubility of prodrugs
t1/2 value*1
solubility (mg/mL)
parent drug*3 prodrug
ratio
of solubility*2
prodrug
parent drug
R1
R2
n
(min)
2
3
4
5
7
8
9
2-aminobenzimidazole
phenytoin
-H
-H
-H
-H
0
0
0
0
0
0
1
38
13
—
6.3 × 10-3
—
—
10.2
—
—
1620
—
phenytoin
-CH3
-H
-H
1.1
0.78
40
phenytoin
-CH3
-H
—
—
—
sulfathiazole
quinine
-CH3
-H
1.4 × 10-3
—
7.1
5071
—
-H
<1
—
quinine
-H
-H
1.1
2.1 × 10-1
>10
>48
*1t1/2 is the time required for 50% release of parent drugs in pH 7.4 PBS at 37°C.
*2prodrug/parent drug
*3The parent drugs, sulfathiazole and quinine, were used as a TFA salt and an HCl salt, respectively, for measuring the solubilities.

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