Synthesis and stability study of a modified phenylpropionic acid linker-based esterase-sensitive prodrug

Article abstract of DOI:10.1016/S0960-894X(02)00750-3

An esterase-sensitive amide prodrug 1 with a modified phenylpropionic acid linker was synthesized. The prodrug can be converted to the drug using isolated porcine esterase and human plasma. Paraoxon, an esterase inhibitor, can inhibit prodrug-to-drug conv

Full text of DOI:10.1016/S0960-894X(02)00750-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3439–3442
Synthesis and Stability Study of a Modified Phenylpropionic Acid
Linker-Based Esterase-Sensitive Prodrug
Xiaoping Song and Teruna J. Siahaan*
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047, USA
Received 3 July 2002; accepted 15 August 2002
Abstract—An esterase-sensitive amide prodrug 1 with a modified phenylpropionic acid linker was synthesized. The prodrug can be
converted to the drug using isolated porcine esterase and human plasma. Paraoxon, an esterase inhibitor, can inhibit prodrug-to-
drug conversion. The conversion of prodrug 1 was via phenol intermediate 9 followed by a lactonization reaction to give lactone 2
and the drug.
# 2002 Elsevier Science Ltd. All rights reserved.
Prodrug strategies have been developed and utilized to
transiently alter the physicochemical properties of
drugs. Previously, we have used cyclic prodrugs to
enhance cell membrane permeation and metabolic sta-
bility of peptides and peptidomimetics.1ꢀ7 These cyclic
prodrugs were formed by linking the C-and Nt-ermini
of the peptide using three different linkers, such as
acyloxy(alkoxy),1ꢀ3 coumarinic acid4ꢀ5 and phenylpro-
pionic acid4ꢀ7 linkers. Conversion of these cyclic pro-
drugs to the parent drugs was initiated by esterase
hydrolysis of the ester bond between the peptide and the
linker followed by a fast chemical reaction (Scheme 1).
Unfortunately, the coumarinic acid-and phenylpro-
pionic acid-based cyclic prodrugs have low water solu-
bility, largely due to their hydrophobicity. Therefore,
there is a need to modify these linkers to balance
the solubility and membrane permeation of the cyclic
prodrugs.
In this work, the phenylpropionic acid (1a) linker was
modified to increase its hydrophilicity by substituting
the dimethyl groups with dimethoxy groups at posi-
tions-3 and -5 of the aromatic ring to give a linker used
in prodrug 1 (Scheme 1). In the future, the methoxy
groups at position-3 or -5 can be used as a handle for
other functional groups to balance the solubility and
membrane permeation of the prodrugs. However, this
modification may change prodrug-to-drug conversion
properties of this linker. Thus, we synthesized a model
amide prodrug 1 using the modified linker (Scheme 1).
Scheme 1.
*Corresponding author. Tel.: +1-785-864-7327; fax: +1-785-864-5736; e-mail: siahaan@ku.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00750-3

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X. Song, T. J. Siahaan / Bioorg. Med. Chem. Lett. 12 (2002) 3439–3442
Finally, the rate of prodrug-to-drug conversion was
evaluated in isolated esterase solution and human
plasma in the absence and presence of paraoxon, an
esterase inhibitor. The chemical stability study of the
cyclic prodrug 1 was conducted in phosphate buffer,
pH 7.4.
KMnO4 gave an acid 8 in 65% yield. Finally, a coupling
reaction between carboxylic acid 8 and 4-methoxy ani-
line using DCC produced the desired prodrug 1 in 87%
yield.9
The applicability of this linker was appraised by study-
ing the prodrug-to-drug conversion in isolated esterase
or human plasma solution. For chemical stability, pro-
drug 1 was incubated in 50 mM phosphate buffer with
ionic strength of 0.15 at pH 7.4 and 37 ꢁC. The
disappearance of prodrug 1 and the appearance of
4-methoxy aniline and lactone 2 were monitored by
HPLC. In buffered solution, prodrug 1 was degraded in
a pseudo-first-order fashion with stoichiometric pro-
duction of lactone 2 and 4-methoxy aniline (Fig. 1). The
half-life of prodrug 1 is 3529 min, which is comparable
to the half-life (4039 min) of parent prodrug 1a (Scheme
1).8,10 In this degradation, the intermediate 9 (Scheme 1)
was not observed because it was rapidly converted to
lactone 2 and 4-methoxyaniline after hydrolysis of the
ester. This result is consistent with the conversion of
phenylpropionic acid-derived prodrugs previously
reported by Borchardt et al.8,10
The synthesis of prodrug 1 was initiated by alkylation of
the 3,5-dimethoxy phenol at the 2-position with 3,3-
dimethyl acrylate in CH3SO3H at 70 ꢁC to produce lac-
tone 2 in 40% yield (Scheme 2). The use of CH3SO3H
gave a better yield of lactone 2 than did H2SO4, which
was used previously.8 Reduction of lactone 2 with
LiAlH4 produced diol 3 in 70% yield. The primary
alcohol in 3 was selectively protected with t-butyl dime-
thylsilyl group (TBDMS) to give compound 4 in 78%
yield. The phenolic group in compound 4 was acetylated
using acetic anhydride to yield 81% of compound 5.
Treatment of compound 5 with a mixture of acetic acid,
water and THF removed the TBDMS-protecting group,
resulting in compound 6 in 63% yield. The primary
alcohol in compound 6 was oxidized to aldehyde 7 with
PCC in CH2Cl2; further oxidation of the aldehyde with
Figure 2. The stability of prodrug 1 in porcine liver esterase. It shows
the entire time course for the disappearance of the prodrug 1 (&), the
appearance of the intermediate 9 (~), and the appearance of product 2
(*).
Figure 1. The stability of prodrug 1 in phosphate buffer at pH 7.4. It
shows the entire time course for the disappearance of prodrug 1 (&)
and the appearance of product 2 (*).
Scheme 2. (a) CH3SO3H, 40%; (b) LiAlH4, THF, 70%; (c) TBDMS–Cl, Et3N, CH2Cl2, 78%; (d) (CH3CO)2O, DMAP, CH2Cl2, 81%; (e) HOAc,
TFA/H2O, 63%; (f) PCC, CH2Cl2, 70%; (g) KMnO4, acetone/H2O, 65%; (h) H2NR, DCC, DMAP, CH2Cl2, 87%.

X. Song, T. J. Siahaan / Bioorg. Med. Chem. Lett. 12 (2002) 3439–3442
3441
Finally, the conversion of prodrug 1 was evaluated in
90% human plasma for its applicability in vivo. In
human plasma, the half-life of prodrug 1 is 52 min, 66
times faster than the chemical degradation (Fig. 3). This
half-life was very similar to the half-life (53.7 min) of
parent prodrug 1a (Scheme 1) in plasma.10 Similar to
conversion in isolated esterase, a small amount of
intermediate 9 was also observed, suggesting that ester
hydrolysis is the first step of the conversion. To confirm
the ester hydrolysis of prodrug 1 in human plasma,
paraoxon (a potent esterase inhibitor) was used to inhi-
bit the esterase activity in the human plasma (Fig. 4).
The inclusion of paraoxon did increase the stability of
prodrug 1, and the degradation was found to be
approximately 2.3 times slower. This result suggests that
prodrug 1 most probably degrades in 90% human
plasma by hydrolysis of the ester bond catalyzed by
esterase.
Figure 3. The stability of prodrug 1 in 90% human plasma. It shows
the entire time course for the disappearance of the prodrug 1 (&), the
appearance of the intermediate 9 (~), and the appearance of product 2
(*).
In conclusion, we have synthesized prodrug 1 with a
modified phenylpropionic acid promoiety. This esterase-
sensitive prodrug 1 can be converted to drug by esterase
hydrolysis with a similar rate to that of parent prodrug
1a. Modification of the 3-and 5p-ositions of the
aromatic ring in the promoiety maintained its ability to
form a rapid lactonization reaction. This makes it an
attractive linker for making cyclic prodrugs of peptides.
In the future, the methoxy groups at the 3-and 5p- osi-
tions can be utilized to balance the solubility and
transmembrane permeation of the prodrug.
Acknowledgements
This work was supported by NIH grant HL-59931 and
Self Faculty Scholar Award to T.J.S. from The Uni-
versity of Kansas. We thank Dr. Todd Williams, Mr.
Robert Drake and Ms. Homigol Biesada at the Mass
Spectroscopy Laboratory for their technical support.
Figure 4. The stability of prodrug 1 in 90% human plasma in the
presence of paraoxon. It shows the entire time course for the dis-
appearance of the prodrug 1 (&) and the appearance of product 2 (*).
References and Notes
1. Gangwar, S.; Pauletti, G. M.; Siahaan, T. J.; Stella, V. J.;
Borchardt, R. T. J. Org. Chem. 1997, 62, 1356.
Next, the prodrug-to-drug conversion was examined
using isolated porcine esterase. Incubation of prodrug 1
in porcine esterase solution converted prodrug 1 to lac-
tone 2 and 4-methoxy aniline with a half-life of 10 min
(Fig. 2). For a comparison, the half-life of the parent
prodrug 1a in porcine esterase solution was 11.9 min.10
The enzymatic degradation of prodrug 1 exhibited
pseudo-first order kinetics to completion. A small
amount of intermediate 9 was observed in this enzy-
matic reaction, suggesting that prodrug-to-drug con-
version is via intermediate 9. Furthermore, the release
of 4-methoxy aniline was due to the lactonization reac-
tion, which gives lactone 2 as well. The presence of the
intermediate 9 also suggests that the rate of ester
hydrolysis is faster than that of the lactonization reac-
tion. In addition, since no intermediate 8 (Scheme 2)
was observed during the prodrug-to-drug conversion,
the release of 4-methoxy aniline was not due to direct
amide hydrolysis.
2. Bak, A.; Siahaan, T. J.; Gudmundsson, O. S.; Gangwar, S.;
Friis, G. J.; Borchardt, R. T. J. Pept. Res. 1999, 53, 393.
3. Pauletti, G. M.; Gangwar, S.; Okumu, F. W.; Siahaan, T. J.;
Stella, V. J.; Borchardt, R. T. Pharm. Res. 1996, 13, 1613.
4. Wang, B.; Nimkar, K.; Wang, W.; Zhang, H.; Shan, D.;
Gudmundsson, O. S.; Gangwar, S.; Siahaan, T. J.; Borchardt,
R. T. J. Pept. Res. 1999, 53, 370.
5. Gudmundsson, O. S.; Jois, S. D. S.; Vander Velde, D. G.;
Siahaan, T. J.; Wang, B.; Borchardt, R. T. J. Pept. Res. 1999,
53, 383.
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Borchardt, R. T. J. Org. Chem. 1997, 62, 1363.
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5867.
9. 1: MS/FAB 402 (M+H)+, 441 (M+K)+; 1H NMR
(400 MHz, CDCl3) d 1.58 [6H, s, 3,3-(CH3)2], d 2.28 (3H, s,
acetyl–CH3), d 2.87 (2H, s, 2-CH2), d 3.77 (3H, s, 60-OCH3), d
3.79 (3H, s, 40-OCH3), d 3.83 (3H, s,–OCH3), d 6.12 (1H, d,

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X. Song, T. J. Siahaan / Bioorg. Med. Chem. Lett. 12 (2002) 3439–3442
J=3.0 Hz, 60-ArH), d 6.43 (1H, d, J=2.6 Hz, 40-ArH), d 6.78
(2H, d, J=9 Hz, Ar0H), d 6.81 (1H, s, NH), d 7.15 (2H, d,
J=9.0 Hz, Ar0H); 2: MS/FAB 237 (M+H)+; 1H NMR
(400 MHz, CDCl3) d 1.42 [6H, s, 4, 4-(CH3)2], d 2.59 (2H, s,
CH2), d 3.79 (3H, s, 5-OCH3), d 3.82 (3H, s, 7-OCH3), d 6.24
(1H, d, J=3.0 Hz, 6-ArH), d 6.27 (1H, d, J=2.5 Hz, 8-ArH);
3: MS/FAB 240 M+, 241 (M+H)+; 1H NMR (400 MHz,
CDCl3) d 1.54 [6H, s, 3, 3-(CH3)2], d 2.13 (2H, t, J=7.0 Hz, 1-
CH2), d 3.63 (2H, t, J=7.0 Hz, 2-CH2), d 3.75 (3H, s, 60-
OCH3), d 3.76 (3H, 40-OCH3), d 5.93 (1H, d, J=3.0 Hz, 50-
ArH), d 6.09 (1H, d, J=2.5 Hz, 70-ArH); 4: MS/FAB 355
(M+H)+; 1H NMR (400 MHz, CDCl3) d 0.01 [6H, s, Si–
(CH3)2], d 0.86 (9H, s, t-butyl), d 1.53 [6H, s, 3, 3-(CH3)2], d
2.05 (2H, t, J=6.8 Hz, 1-CH2), d 3.60 (2H, t, J=6.8 Hz, 2-
CH2), d 3.72 (3H, s, 60-OCH3), d 3.74 (3H, 40-OCH3), 5.98
(1H, d, J=2.6 Hz, 50-ArH), d 6.07 (1H, d, J=2.6 Hz, 70-ArH),
CH2), d 2.27 (3H, s, acetyl–CH3), d 3.43 (2H, t, J=7.5 Hz, 2-
CH2), d 3.77 (3H, s, 60-OCH3), d 3.80 (3H, 40-OCH3), 6.02
(1H, d, J=2.6 Hz, 50-ArH), d 6.36 (1H, d, J=2.6 Hz, 70-ArH);
6: MS/FAB 283 (M+H)+; 1H NMR (400 MHz, CDCl3) d
1.44 [6H, s, 3, 3-(CH3)2], d 2.10 (2H, t, J=6.9 Hz, 1-CH2), d
2.29 (3H, s, acetyl–CH3), d 3.51 (2H, t, J=6.9 Hz, 2-CH2), d
3.77 (3H, s, 60-OCH3), d 3.82 (3H, s, 40-OCH3), 6.02 (1H, d,
J=2.6 Hz, 50-ArH), d 6.39 (1H, d, J=2.6 Hz, 70-ArH); 7: MS/
1
FAB 281 (M+H)+; H NMR (400 MHz, CDCl3) d 1.52 [6H,
s, 3, 3-(CH3)2], d 2.25 (3H, s, acetyl–CH3), d 2.85 (2H, d,
J=2.6 Hz, CH2), d 3.78 (3H, s, 60-OCH3), d 3.82 (3H, s, 40-
OCH3), 6.08 (1H, d, J=2.6 Hz, 50-ArH), d 6.40 (1H, d, J=2.6
Hz, 70-ArH), d 9.54 (1H, t, J=2.6 Hz, ald.); 8: MS/FAB
296 M+; 1H NMR (400 MHz, CDCl3) d 1.54 [6H, s, 3, 3-
(CH3)2], d 2.29 (3H, s, acetyl-CH3), d 2.92 (2H, s, CH2), d 3.77
(3H, s, 60-OCH3), d 3.81 (3H, s, 40-OCH3), 6.06 (1H, d, J=2.6
Hz, 50-ArH), d 6.38 (1H, d, J=2.6 Hz, 70-ArH).
d 6.23 (1H, br, 20-OH); 5: MS/FAB 397 (M+H)+; H NMR
1
(400 MHz, CDCl3) d ꢀ0.01 [6H, s, Si-(CH3)2], d 0.86 (9H, s, t-
10. Amsberry, K. L.; Gerstenberger, A. E.; Borchardt, R. T.
Pharm. Res. 1991, 8, 455.
butyl), d 1.42 [6H, s, 3, 3-(CH3)2], d 2.08 (2H, t, J=6.8 Hz, 1-