Development of a novel sulfonate ester-based prodrug strategy

Article abstract of DOI:10.1016/j.bmcl.2015.11.074

A self-immolative γ-aminopropylsulfonate linker was investigated for use in the development of prodrugs that are reactive to various chemical or biological stimuli. To demonstrate their utility, a leucine-conjugated prodrug of 5-chloroquinolin-8-ol (5-Cl-8-HQ), which is a potent inhibitor against aminopeptidase from Aeromonas proteolytica (AAP), was synthesized. The sulfonate prodrug was considerably stable under physiological conditions, with only enzyme-mediated hydrolysis of leucine triggering the subsequent intramolecular cyclization to simultaneously release 5-Cl-8-HQ and form γ-sultam. It was also confirmed that this γ-aminopropylsulfonate linker was applicable for prodrugs of not only 8-HQ derivatives but also other drugs bearing a phenolic hydroxy group.

Full text of DOI:10.1016/j.bmcl.2015.11.074

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a novel sulfonate ester-based prodrug strategy
a
b
Kengo Hanaya a, , Shohei Yoshioka , Shinya Ariyasu , Shin Aoki b,c, Mitsuru Shoji a, Takeshi Sugai a
⇑
a Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
b Center for Technologies against Cancer, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan
c Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A self-immolative
c-aminopropylsulfonate linker was investigated for use in the development of pro-
Received 24 September 2015
Revised 14 November 2015
Accepted 20 November 2015
Available online xxxx
drugs that are reactive to various chemical or biological stimuli. To demonstrate their utility, a leu-
cine-conjugated prodrug of 5-chloroquinolin-8-ol (5-Cl-8-HQ), which is a potent inhibitor against
aminopeptidase from Aeromonas proteolytica (AAP), was synthesized. The sulfonate prodrug was consid-
erably stable under physiological conditions, with only enzyme-mediated hydrolysis of leucine triggering
the subsequent intramolecular cyclization to simultaneously release 5-Cl-8-HQ and form
also confirmed that this -aminopropylsulfonate linker was applicable for prodrugs of not only 8-HQ
derivatives but also other drugs bearing a phenolic hydroxy group.
c-sultam. It was
Keywords:
Sulfonate
Prodrug
Suicide substrate
Protease
c
Ó 2015 Elsevier Ltd. All rights reserved.
Sultam
Prodrugs are drug precursors that are designed to improve the
bioavailability and/or aqueous solubility of drugs and to avoid
undesirable metabolism. They can undergo in vivo activation
through enzymatic and/or chemical transformation to generate
active drugs, resulting in desirable inhibitory activity with the
reduction of side effects.1–3 The hydroxy group is one of the most
important functional groups for drugs as it exerts its efficiency
by adjusting the hydrophilicity of the drug and/or by forming
hydrogen bonds with receptor proteins.4 It is also responsible for
coordination to metal ions in cooperation with nitrogen or other
heteroatoms.5–10 For these reasons, hydroxy groups in drugs are
often targeted for derivatization toward the development of
prodrugs.
Various strategies for prodrugs using carboxyl esters, carbon-
ates, phosphates, sulfates, and ethers have been developed. How-
ever, these functional groups can be readily hydrolyzed by
ubiquitous esterases, phosphatases, and sulfatases in the blood,
liver, and other organs and tissues.1–3 Furthermore, carboxylate
esters, the most common prodrugs, can be automatically hydro-
lyzed under physiological conditions.11 The esters of phenolic
hydroxy groups are especially unstable, due to the good leaving
ability of phenol groups in aqueous solution. For these reasons,
not only the accurate prediction of pharmacokinetic disposition
but also the design of prodrugs that can be activated by specific
stimuli only in the target organs and tissues are challenging.1–3,11
Sulfonate ester-based prodrugs have been reported in only a
few papers,12–15 despite the fact that sulfonate esters are com-
monly used as good leaving and protecting groups in organic
chemistry. Sulfonates are generally stable under physiological con-
ditions16 and no enzymes that hydrolyze sulfonates are known. In
this study, a novel sulfonate ester-based prodrug (1) containing a
self-immolative linker17–26 was designed, as shown in Figure 1a.
The prodrug comprises three parts: (1) a chemically (pH, redox,
etc.) or enzymatically reactive trigger on the terminal nitrogen
atom, (2) a self-immolative aminoalkylsulfonate linker, and (3) a
drug bearing a hydroxy group. In Waldmann’s18 and Shabat’s19–21
pioneering works on self-immolative linkers, specific stimuli
caused the formation of lactam and cyclic urea to release ligands
or drugs, respectively. In our self-immolative amino sulfonate lin-
ker, specific stimuli such as peptidases caused the degradation of
triggers to generate aminoalkylsulfonate ester 2, followed by
intramolecular cyclization to produce a drug and sultam 3 instead
of lactam and cyclic urea.
We have reported that quinolin-8-ol derivatives (8-HQs, 4) such
as 5-chloroquinolin-8-ol (5-Cl-8-HQ, 4a) demonstrate potent inhi-
bitory activity against aminopeptidase from Aeromonas proteolytica
(AAP).8,15 The 8-HQs coordinate to two zinc ions in the active site
of AAP, and the Ki value of 4a is determined to be 0.19 lM. In
addition, 8-HQ-based compounds have recently been reported as
⇑
drug candidates for bacterial infections,10 HIV,27–29 cancer,9,30 and
Corresponding author.
E-mail address: hanaya-kn@pha.keio.ac.jp (K. Hanaya).
http://dx.doi.org/10.1016/j.bmcl.2015.11.074
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hanaya, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.11.074

2
K. Hanaya et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
(a)
(b)
Ar
O
Ar
O
O2
S
removal
intramolecular
cyclization
3a: n = 2
3b: n = 3
3c: n = 4
of trigger
HN
trigger
N
H
S
H2N
S
Ar OH
n
n
O2
O2
n-2
1
2
R
R
R
N
N
AAP
N
O
O
OH
H2N
S
H2N
N
H
S
n
n
O2
O2
4a: R = 5-Cl ( = 0.19 µM)
4b: R = 2-Me ( > 100 µM)
Ki
O
Ki
5
Figure 1. (a) Proposed scheme for the activation of prodrugs via intramolecular cyclization. (b) AAP-reactive prodrugs of 8-HQs.
neurodegenerative diseases.31,32 However, 8-HQs may also coordi-
nate to various metal ions in vivo in a bidentate manner, causing
serious side effects, such as subacute myelo-optico-neuropathy
(SMON) by clioquinol (5-chloro-7-iodoquinolin-8-ol). Therefore, a
new methodology for the reduction of side effects due to
8-HQ-based drugs is required. Herein, we prepared AAP-reactive
prodrugs of 8-HQs 5 containing leucine (Leu) as a trigger in order
to demonstrate the prodrug strategy mentioned above. Their
activation with AAP (Fig. 1b) and determination of the activation
reaction rates were performed.
1st step
2nd step
LeuNA
(50 μM)
AAP
(0.02 units/mL)
+
incubation
method A:
prodrug
6a-c
at pH 8.0
and 30 ºC
(5 μM)
LeuNA
(50 μM)
+
Hydrolysis of LeuNA was
followed by measurement
of ΔAbs405 at 25 ºC
AAP
(0.02 units/mL)
incubation
Sulfonate-based prodrugs 6a–c and pseudo-prodrugs 7–10
were prepared (Fig. 2 and Scheme 1). It was hypothesized that
the hydrolysis of Leu in 6a–c and the subsequent intramolecular
prodrug
6a-c
method B:
(control)
at pH 8.0
(5 μM)
and 30 ºC
cyclization would occur to release 5-Cl-8-HQ (Ki = 0.19 lM),
Figure 3. Incubation of each prodrug (6a–c) with (method A) and without (method
decreasing the hydrolytic activity of AAP; thus, it acts as a suicide
substrate of AAP. Pseudo-prodrugs 7–10, wherein 2-Me-8-HQ and
4-substituted phenols showed negligible inhibitory activity against
AAP, were synthesized to confirm the activation mechanism.
Scheme 1a shows the synthesis of compounds 6b–c, 7b–c, and
8–10, wherein phenols were converted to chlorides 11–15 using
chloroalkylsulfonyl chloride and subsequent azidation to afford
16–20. Reduction of the azide group and simultaneous condensa-
tion with N-Boc-leucine yielded 21–25, which were then treated
with TFA to afford 6b–c, 7b–c, and 8–10. For the synthesis of 6a
and 7a in Scheme 1b, condensation of phenols with N-Boc-
aminoethanesulfonyl chlorides33 was performed, followed by the
removal of the Boc group with TFA to obtain 26 and 27. The
condensation of the resulting amine and Leu and the subsequent
B) AAP.
removal of the Boc group in 28 and 29 led to the formation of 6a
and 7a.
Prodrugs 6a–c were examined for the AAP-mediated 4a-releas-
ing reaction as follows (Fig. 3). In the 1st step, each prodrug (6a–c)
was incubated with (method A) or without (method B) AAP at pH
8.0 and 30 °C for a given time period. In 2nd step of method A, an
AAP substrate, L-leucine-p-nitroanilide (LeuNA), was then added to
the above mixture and the hydrolysis of LeuNA by AAP was
monitored at 25 °C through the spectroscopic measurement of
the absorption at 405 nm due to released p-nitroaniline
(e405 = 10300). In 2nd step of method B, LeuNA and AAP were
added to the above reaction mixture, and the hydrolysis of LeuNA
was monitored in a similar way to method A. The hydrolysis of
LeuNA with AAP was a very quick reaction, and the initial reaction
rate and the reaction rate constant (k) in the absence of 4a were
Cl
Ar
calculated to be 0.084 l
M sÀ1 and 13.8 sÀ1, respectively. Residual
Ar =
O
N
H2N
N
H
S
n
O2
AAP activity was evaluated as a percentage of the initial reaction
rate. In all cases, the effect of competition between the hydrolysis
of LeuNA and prodrugs by AAP was minimized using excess
O
6a: n = 2
6b: n = 3
6c: n = 4
amounts of LeuNA (50
lM) against the prodrug (5 lM) for mea-
surement of residual AAP activity.
Cl
CF3
SO2NH2
The results of these experiments are summarized in Figure 4.
First, it was confirmed that these sulfonate-based prodrugs were
considerably stable under the reaction conditions in the absence
of AAP (method B). In the case of 6b and 6c (Fig. 4b and c) in
method A, released 4a inhibited AAP. It had been confirmed inde-
pendently that leucine and sultam demonstrated negligible inhibi-
tion of AAP (data not shown). In particular, inhibition of AAP for 6b
N
7a: n = 2
7b: n = 3
7c: n = 4
8
9
10
Figure 2. Structures of prodrugs 6a–c and pseudo-prodrugs 7–10.
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K. Hanaya et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
(a)
Ar
O
Ar
O
i
ii
HO Ar
Cl
S
N3
S
n
n
O2
O2
11a: Ar = 5-chloroquinolin-8-yl, n = 3
11b: Ar = 5-chloroquinolin-8-yl, n = 4
12a: Ar = 2-methylquinolin-8-yl, n = 3
12b: Ar = 2-methylquinolin-8-yl, n = 4
13: Ar = 4-chlorophenyl, n = 3
16a: Ar = 5-chloroquinolin-8-yl, n = 3
16b: Ar = 5-chloroquinolin-8-yl, n = 4
17a: Ar = 2-methylquinolin-8-yl, n = 3
17b: Ar = 2-methylquinolin-8-yl, n = 4
18: Ar = 4-chlorophenyl, n = 3
14: Ar = 4-trifluoromethylphenyl, n = 3
15: Ar = 4-aminosulfonylphenyl, n = 3
19: Ar = 4-trifluoromethylphenyl, n = 3
20: Ar = 4-aminosulfonylphenyl, n = 3
Ar
Ar
O
iv or vi
iii
O
Boc
N
H
N
H
S
n
H2N
N
H
S
n
O2
O2
O
O
21a: Ar = 5-chloroquinolin-8-yl, n = 3
21b: Ar = 5-chloroquinolin-8-yl, n = 4
22a: Ar = 2-methylquinolin-8-yl, n = 3
22b: Ar = 2-methylquinolin-8-yl, n = 4
23: Ar = 4-chlorophenyl, n = 3
6b: Ar = 5-chloroquinolin-8-yl, n = 3
6c: Ar = 5-chloroquinolin-8-yl, n = 4
7b: Ar = 2-methylquinolin-8-yl, n = 3
7c: Ar = 2-methylquinolin-8-yl, n = 4
8: Ar = 4-chlorophenyl, n = 3
24: Ar = 4-trifluoromethylphenyl, n = 3
25: Ar = 4-aminosulfonylphenyl, n = 3
9: Ar = 4-trifluoromethylphenyl, n = 3
10: Ar = 4-aminosulfonylphenyl, n = 3
(b)
Ar
Ar
O
O
Ar
O
v
iv
H2N
S
2
O2
Boc
H2N
N
H
S
N
H
N
H
S
2
2
O2
O2
O
O
26: Ar = 5-chloroquinolin-8-yl
27: Ar = 2-methylquinolin-8-yl
28: Ar = 5-chloroquinolin-8-yl
6a: Ar = 5-chloroquinolin-8-yl
29: Ar = 2-methylquinolin-8-yl
7a: Ar = 2-methylquinolin-8-yl
Scheme 1. Synthesis of prodrugs 6 and pseudo-prodrugs 7–10. Reagents and conditions: (i) chloropropylsulfonyl chloride (n = 3) or chlorobutylsulfonyl chloride (n = 4), Et3N,
CH2Cl2, 0 °C to rt; (ii) NaN3, TBAI, DMF, 50 °C; (iii) H2, Pd/C, N-Boc-Leu-OSu, THF; (iv) TFA, CH2Cl2 (with the exception of 6b–c); (v) N-Boc-Leu-OH, PyBOP, Et3N, DMF; and (vi)
HCl, dioxane (for 6b–c).
was observed even at the beginning of the reaction (Fig. 4b), sug-
gesting that the hydrolysis and subsequent intramolecular cycliza-
tion progressed much faster than that of 6c. In contrast, the
formation of 4a from 6a bearing a C2 linker was negligible. These
Formation rates of c-sultam via intramolecular cyclization were
evaluated as phenol-formation rates by monitoring the change in
UV absorbance. As mentioned above, the hydrolysis of the Leu moi-
ety was very quick. Assuming that the hydrolysis reaction rate con-
results indicated that the formation of
c-sultam, a 5-membered
stants of the Leu moiety in 7b (50 lM) and 8–10 (200 lM) were
ring, was faster than that of 4- and 6-membered rings and was
nearly identical to that in LeuNA, the reaction rate constants (k1)
under the reaction conditions were estimated to be 13.8 and
34.6 sÀ1, respectively (Scheme 2). When excess amounts of AAP
(3.07 Â 10À7 M, 1.05 units/mL) were used, the hydrolysis reaction
rates (v1) of Leu moiety in 7b and 8–10 were calculated to be 4.2
and 10.4
completed within 30 s. Thus, liberation of phenols 34–37 and
c-sultam 3b from 7b and 8–10 via 30–33 was approximated by a
similar to cyclic urea-type self-immolative linkers.19–21,23
In the above cases, it was difficult to isolate and characterize the
products derived from the self-immolative linker, because a small
amount of 4a released from 6a–c at an early stage inhibited the
further hydrolysis of Leu. In contrast, pseudo-prodrugs 7a–c con-
taining 2-Me-8-HQ (4b), which showed negligible inhibitory activ-
l
M sÀ1, respectively, with the hydrolysis of Leu being
ity (Ki > 100
lM), were expected to be completely hydrolyzed by
AAP to produce 4b and sultam or aminoalkylsulfonic acids.
Pseudo-prodrugs 7a–c were treated with an excess amount of
AAP, and the reactions were monitored by 1H NMR (Figs. 5 and
S1). It was confirmed that the hydrolysis of the leucine moiety in
the pseudo-prodrugs was completed immediately following the
addition of AAP. After incubation for 12 h, only 7b with the C3 lin-
pseudo-first order reaction (k1 ꢀ k2). Typical curves for phenol-
formation rates are shown in Figure 6, with the slopes of each
curve providing the reaction rates (v2) of the intramolecular
cyclization. The calculated rate constants (k2) of the intramolecular
cyclization and pKa values of the phenolic hydroxy groups are
shown in Table 1, demonstrating a positive correlation. The results
showed that the intramolecular cyclization was the rate-limiting
step in the degradation of the prodrugs. Furthermore, it was
proved that the intramolecular cyclization progressed indepen-
dently of substituents and structures of the aryl groups. The
hydrolysis of these compounds was negligible in the absence of
AAP under the reaction conditions (pH 8), with k values below
10À7. This suggested that sulfonate itself was remarkably stable
under physiological pH, unlike carboxylate esters.
ker was completely consumed to release 4b and c-sultam 3b with-
out the release of aminopropylsulfonic acid (11) (Fig. 5b). This
strongly suggested that 4b was released not by automatic hydrol-
ysis of the sulfonate ester in aqueous solution but by nucleophilic
attack of the terminal amino group by the sulfonyl ester in an
intramolecular manner. In contrast, for 7a and 7c, further hydroly-
sis or intramolecular cyclization was negligible, although hydroly-
sis of Leu did occur (Fig. S1).
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K. Hanaya et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 4. Residual activity of AAP toward the hydrolysis of LeuNA (50
incubation of prodrugs having (a) a C2 linker (6a), (b) C3 linker (6b), and (c) C4
linker (6c) (5 M) with (method A, open triangle) and without (method B, open
lM) after the
l
circle) AAP (0.02 units/mL) for a given time period in 10 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) buffer (pH 8.0) containing 2% dimethylsul-
foxide (DMSO) at 25 °C.
Figure 5. 1H NMR spectra showing the change in the region from (a) 8.2 to 7.0 ppm
and (b) 4.0 to 2.0 ppm after incubation of 7b (2 mM) with AAP (1.05 units/mL) for a
given time period at 30 °C in 100 mM NH4HCO3 (pD 8.0) containing 2% d6-DMSO.
Finally, we examined the stability of prodrug 7b in human
serum and CD1 mouse liver S9 (rich in a wide variety of both phase
I and phase II drug-metabolizing enzymes) (Fig. 7). After incuba-
tion for 24 h in human serum, ca. 35% of 7b decomposed to form
an intermediate amine 30 and 4b. In the case of the incubation
in liver S9, 7b was completely hydrolyzed to afford amine 30
within 3 h. Conversely, azide 17a was stable in human serum
and 88 3% of 17a was remained even after incubation for 24 h
in liver S9. It was suggested that the sulfonate ester was consider-
ably stable even in the present of various drug-metabolizing
enzymes as expected. Only the Leu moiety was hydrolyzed as a
result of the proteases present in human serum and mouse liver
S9, which triggered the subsequent intramolecular cyclization to
afford 4b.
These results indicated that the aminopropylsulfonate-type
self-immolative linker can be applied to novel sulfonate prodrugs
of various drugs containing phenolic hydroxy groups. To improve
its usability for prodrugs, further development of novel trigger
moiety other than amide was an important issue.
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K. Hanaya et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Ar
O
Ar
O2
S
AAP
v1, k1
HN
O
H2N
N
H
S
n
H2N
S
Ar OH
O2
n
v2, k2
O2
O
3b
7b: Ar = 2-methylquinolin-8-yl
8: Ar = 4-chlorophenyl
30: Ar = 2-methylquinolin-8-yl
31: Ar = 4-chlorophenyl
34: Ar = 2-methylquinolin-8-yl
35: Ar = 4-chlorophenyl
9: Ar = 4-trifluoromethylphenyl
10: Ar = 4-aminosulfonylphenyl
32: Ar = 4-trifluoromethylphenyl
33: Ar = 4-aminosulfonylphenyl
36: Ar = 4-trifluoromethylphenyl
37: Ar = 4-aminosulfonylphenyl
Scheme 2. Evaluation of intramolecular cyclization reaction rate k2.
Figure 6. Typical curves of the liberation of corresponding phenols after incubation of (a) 7b (50
l
M) and (b) 8 (200
l
M), (c) 9 (200
lM), and (d) 10 (200
lM) for a given
period with 1.05 units/mL AAP in 50 mM HEPES (pH 8.0) containing 2% DMSO.
order to demonstrate its utility, Leu-conjugated prodrugs (6) of
5-Cl-8-HQ, a potent inhibitor against AAP, were designed and
synthesized. Only in the case of compound 6b, which contained a
C3 linker, the hydrolysis of Leu by AAP triggered the subsequent
intramolecular cyclization to release 5-Cl-8-HQ (4a), resulting in
Table 1
Compound
pKa
k2 Â 10À3 (sÀ1
0.025 0.003
0.054 0.003
0.42 0.04
1.9 0.2
)
7b
8
9
10.9
9.6
8.9a
8.3
the inhibition of AAP. Simultaneous formation of
was observed, while -aminopropylsulfonic acid (11) was not
detected. It was found that these prodrugs were stable in the
absence of AAP as trigger under physiological conditions.
c-sultam 3b
10
c
a
Refs. 34,35.
a
Furthermore, the self-immolative aminopropylsulfonate linker
would be applicable not only for prodrugs of 8-HQs but also
for other drugs containing phenolic hydroxy groups. These findings
are significant in the development of other sulfonate-based
proinhibitors against various proteases. Moreover, further exami-
nation of this sulfonate-based strategy with other triggering
groups is currently underway in order to obtain prodrugs sensitive
to a variety of biological stimuli.
In summary, we have successfully developed novel prodrugs
through the rational combination of three fragments: (1) a trigger
moiety, (2) an aminoalkylsulfonate linker as a self-immolative lin-
ker, and (3) a drug. The sulfonate esters were remarkably stable
under physiological conditions and cleaved only by intramolecular
cyclization caused by enzymatic hydrolysis of a trigger moiety,
allowing for the release of drugs at desirable organs or tissues. In
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6
K. Hanaya et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
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Acknowledgements
This study is supported by the Platform Project for Supporting
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Supplementary data
Supplementary data associated with this article can be found, in
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Please cite this article in press as: Hanaya, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.11.074