L-lysine pro-prodrug containing trans-ferulic acid for 5-amino salicylic acid colon delivery: Synthesis, characterization and in vitro antioxidant activity evaluation

Article abstract of DOI:10.1248/cpb.58.103

In the present work, we report the synthesis of a new 5-amino salicylic acid (5-ASA) pro-prodrug, useful in Crohn disease treatment, and the evaluation of its antioxidant activity. Using as pharmacological carrier L-lysine amino acid and taking advantage of its intrinsic chemical reactivity, due to the presence of two amino groups, placed on the chiral center and in ε-position, we inserted trans-ferulic acid in ε-position, through amidation reaction, esterified with methanol the carboxylic group and, finally, submitted the free amino group to diazotation with 5-ASA, principal drug for inflammatory bowel diseases (IBD) care. All intermediates of synthesis and the final product (derivative A) were characterized with usual spectroscopic techniques, as FT-IR, GC/MS and ¹HMNR. Finally, the derivative A antioxidant activity in inhibiting the lipid peroxidation, in rat-liver microsomal membranes, induced in vitro by two different sources of free radicals, 2,2′-azobis (2-amidinopropane) (AAPH) and tert-butyl hydroperoxide (tert-BOOH), was evaluated. Our pro-prodrug could be successfully applied in pharmaceutical field both as prodrug of 5-ASA than as carrier of trans-ferulic acid.

Full text of DOI:10.1248/cpb.58.103

January 2010
Notes
Chem. Pharm. Bull. 58(1) 103—105 (2010)
103
L-Lysine Pro-Prodrug Containing trans-Ferulic Acid for 5-Amino Salicylic
Acid Colon Delivery: Synthesis, Characterization and in Vitro Antioxidant
Activity Evaluation
Roberta CASSANO, Sonia TROMBINO,* Alessia CILEA, Teresa FERRARELLI, Rita MUZZALUPO, and
Nevio PICCI
Department of Pharmaceutical Sciences, University of Calabria; 87036 Arcavacata di Rende, Cosenza 87100, Italy.
Received July 10, 2009; accepted October 7, 2009; published online October 8, 2009
In the present work, we report the synthesis of a new 5-amino salicylic acid (5-ASA) pro-prodrug, useful in
Crohn disease treatment, and the evaluation of its antioxidant activity. Using as pharmacological carrier L-lysine
amino acid and taking advantage of its intrinsic chemical reactivity, due to the presence of two amino groups,
placed on the chiral center and in e-position, we inserted trans-ferulic acid in e-position, through amidation re-
action, esterified with methanol the carboxylic group and, finally, submitted the free amino group to diazotation
with 5-ASA, principal drug for inflammatory bowel diseases (IBD) care. All intermediates of synthesis and the
1
final product (derivative A) were characterized with usual spectroscopic techniques, as FT-IR, GC/MS and H-
MNR. Finally, the derivative A antioxidant activity in inhibiting the lipid peroxidation, in rat-liver microsomal
membranes, induced in vitro by two different sources of free radicals, 2,2ꢀ-azobis (2-amidinopropane) (AAPH)
and tert-butyl hydroperoxide (tert-BOOH), was evaluated. Our pro-prodrug could be successfully applied in
pharmaceutical field both as prodrug of 5-ASA than as carrier of trans-ferulic acid.
Key words colon specific; 5-amino salicylic acid; pro-prodrug; L-lysine; trans-ferulic acid
Creation of pharmaceutical forms able to selectively re- fasalazine, composed by 5-amino salicylic acid (5-ASA) (or
lease therapeutic agent, is of great interest to reduce collat- mesalazine) and sulfapyridine, is used as standard therapy in
eral effects and increase therapeutic efficacy. In particular, in- IBD. The active moiety is 5-ASA, whereas sulfapyridine acts
flammatory bowel diseases (IBD), characterized of chronic as a carrier that protects active substance, but it is the most
inflammation and unknown etiology, expressed in Crohn responsible of adverse effects. For this reason the idea to
disease and ulcerative colitis, needs pharmaceutical forms change sulfapyridine with L-lysine amino acid, an absolutely
poorly absorbed in gastric mucosa and in superior portions of non toxic carrier, reducing to minimum the collaterals ef-
intestinal part to protect active substance and to arrive in in- fects.
tact form into the colon. For this reason, it has recourse to
5-ASA is rapidly absorbed in the small intestine before its
prodrugs, pH-based polymers, hydrogels, multi-layered sys- entering in the colon, then is necessary its administration in
tems with time-dependent degradation and bacteria-degra- modified release systems or as colon specific pro-prodrug,
dated polymers.1)
and is a highly potent scavenger of reactive oxygen species
Actually, pharmaceutical approach to IBD is with salicy- (ROS) and was shown to reduce lipid peroxidation in Crohn’s
lates and corticosteroids which may induce gastric mucosa disease and ulcerative colitis.3) In fact, oxidative stress has
irritation and a lot of adverse reactions, moreover, some of been recognized as one of the key elements of tissue injury in
them cannot be assumed by oral administration because of its IBD.4) So, in order to improve 5-ASA antioxidant action, we
sensibility to gastric juice’s enzymes.2) In particular, sul- also linked to L-lysine trans-ferulic acid (Fig. 1), which
Fig. 1. Pro-Prodrug Composition and in Vivo Degradation
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: sonia.trombino@unical.it

104
Vol. 58, No. 1
maintains its strong antioxidant activity even if covalently NMR (Table 1). Yield 67%.
bonded, as demonstrated in our previous works.5—7)
0.250 g (0.776 mmol) of derivative 2 were added to
The pro-prodrug idea is due to the presence of two amino 1.61 ml (0.040 mmol) of cool methanol containing 0.028 ml
groups in L-lysine, bonded respectively to chiral center of (3.855·10ꢁ4 mmol) of thyonil chloride. Reaction was given
amino acid and to methyl group in the e-position. Exploiting to 60—70 °C and left to reflux for 7 h under stirring. After
this intrinsic chemical reactivity of L-lysine, we bonded in e- 1 h, 0.008 ml of thyonil chloride (1.102·10ꢁ4 mmol) (30% in
position ferulic acid, esterified with methanol the carboxylic excess) were added to reaction mixture. To remove dimethyl
group and, finally, submitted the free amino group to diazota- sulfite excess, raw product was treated with 20 ml (0.192
tion with 5-ASA, released in the colon by azoreductase en- mmol) of ethylic ether at 0 °C. Derivative 3 (Chart 1), puri-
zymes action. All intermediates of synthesis and the final fied by recystallization from methanol (yield 72%), was char-
1
product (derivative A) were characterized with usual spectro- acterized through FT-IR, GC/MS and H-NMR techniques
scopic techniques, as FT-IR, GC-MS and 1H-MNR.
(Table 1) and then submitted to diazotation.
The pro-prodrug antioxidant activity in inhibiting the lipid
0.173 g (0.514 mmol) of 3 were dissolved in 30 ml of
peroxidation in rat-liver microsomal membranes induced in water, then 0.032 ml (1.035·10ꢁ3 mmol) of hydrochloric acid
vitro by 2,2ꢀ-azobis(2-amidinopropane) (AAPH), which ex- were slowly added at 0—5 °C. A sodium nitrite aqueous so-
ogenously produces peroxyl radicals by thermal decomposi- lution (0.007 g, 0.101 mmol of NaNO2 in 0.050 ml of dis-
tion, and tert-butyl hydroperoxide (tert-BOOH), which en- tilled water) was added to obtain acid conditions that were
dogenously produces alkoxyl radicals by Fenton reactions, kept with subsequent insertions of 0.011 ml (3.559·10ꢁ4
was also evaluated.
mmol) of hydrochloric acid to stabilize salt and to minimize
The synthesis of pro-prodrug (derivative A) was effected secondary reactions. To avoid diazonium salt hydrolysis (in-
in the following way: 2.26 g (16 mmol) of L-lysine were dis- termediate 4, Chart 1), reaction temperature is monitored at
solved in 50 ml of dry chloroform and added, under stirring, 0—5 °C.
to trans-ferulic acid (3 g, 16 mmol) previously treated with
Finally, 0.141 g (1.021 mmol) of salicylic acid were dis-
1.864 ml of thyonil chloride (0.015 mmol) in chloroform. Re- solved in a sodium hydroxide aqueous solution (0.082 g,
action mixture was refluxed at 80 °C for 2 h and then carried 2.05 mmol of NaOH in 15 ml of distilled water) and solution
on for more than 24 h to room temperature. To obtain a com- temperature was maintained to 5 °C. Diazonium salt was
plete hydrolysis, 50 ml of distilled water were added to reac- slowly added in alkaline conditions. Reaction is kept to
tion mixture and 3 extractions with chloroform were con- reflux for 24 h and reaction water was removed through
duced. Organic extracts are left one night on dry sodium sul- lyophilization. The residue was loaded on a silica gel open
fate which was removed though filtration. Raw product was column and eluted with CHCl3/MeOH (75 : 25). The final
purified through chromatographic column and we obtained product 5 (derivative A, yield 79%) was initially character-
1
1
derivative 2 (Chart 1), analyzed by FT-IR, GC/MS and H- ized with FT-IR, GC/MS and H-NMR (Table 1) and then,
Chart 1. Synthetic Route for Derivative A Preparation
Table 1. GC-MS, FT-IR and 1H-NMR Data
Compound
m/z
Wavenumber n (cmꢁ1
)
Chemical shift (d) ppm
2
222 (100%), 177 (71%), 77 (6%)
1711 (–CONH)
7.75 (1H, sb), 7.27 (2H, m), 6.85 (1H, d), 6.60 (1H, sb),
3.90 (3H, s), 3.36 (1H, m), 2.78 (2H, t), 1.12—1.72 (6H, m)
3
5
208 (100%), 177 (61%), 145 (38%), 77 (9%)
285 (100%), 177 (65%), 77 (6%)
1735 (–COOCH3)
1742 (–COOCH3)
7.58 (1H, d), 7.13 (2H, m), 6.80 (1H, d), 6.33 (1H, d), 3.90 (3H, s),
3.80 (3H, s), 3.36 (1H, m), 2.78 (2H, t), 1.12—1.72 (6H, m)
7.86 (1H, m), 7.83 (1H, m), 7.28 (2H, m), 6.78 (2H, m),
6.57 (1H, d), 6.19 (1H, d), 3.81 (3H, s), 3.36 (3H, s), 3.13 (1H, m),
2.74 (2H, m), 1.11—1.55 (6H, m)
GC-MS spectra were performed by using Hewlett Packard GC-MS 5972. NMR spectra were acquired on Bruker VM-300 ACP using CD3OD as solvent. FT-IR spectra were
measured on a Jasco 4200 using KBr disks.

January 2010
105
time-dependent and brought as malondialdehyde (MDA)
nmol/mg proteins (Fig. 2). Derivative A was a stronger an-
tioxidant in protecting the membranes from tert-BOOH- than
from AAPH-induced lipid peroxidation, showing in either
case higher efficiency at 30 min of incubation (Fig. 3) and the
preservation of antioxidant activity up to 2 h confirming re-
sults found previously.
In conclusion, the aim of our work is to target 5-ASA to
the colon in a specific way and reduce to minimum the col-
laterals effects, changing sulfapyridine,9) which is mostly re-
sponsible of adverse effects, with L-lysine amino acid, an ab-
solutely non toxic carrier. With this approach and in order to
obtain a pro-prodrug with increased antioxidant properties,
we successfully bonded trans-ferulic acid to the L-lysine e-
amino group obtaining a hydrophilizing promoiety which
was, subsequently, derivatized with 5-ASA, attaining deriva-
tive A, susceptible to cleavage by enzymes secreted by the
gastrointestinal tract bacterial microflora. Increased hy-
drophilicity of our pro-prodrug should allow 5-ASA colon
specific release. In fact, literature data suggest that following
pro-prodrug oral administration, 5-ASA absorption in the
stomach and small intestines is decreased because of the pro-
moiety polar nature. Therefore, greater levels of the drug, in
the form of the pro-prodrug, can reach the colon. Within the
colon, the bacterially derived enzymes catalyze the conver-
sion of the pro-prodrug to the more lipophilic drug which is
now available for absorption through the colonic mem-
brane.10) From the earlier discussion, it is quite natural to pre-
sume that the main objective of this work was to synthesize
safe pro-prodrug of 5-ASA that could be free from all the ad-
Fig. 2. Effects of Pro-Prodrug on MDA Production Induced by (A) tert-
BOOH and (B) AAPH in Rat-Liver Microsomal Membranes
The microsomal membranes were incubated with 0.25·10ꢁ3
M tert-BOOH or
25·10ꢁ3 M AAPH at 37 °C under air in the dark. The results represent meansꢂS.E.M. verse effects, successfully applied in pharmaceutical field
of six separate experiments.
also as prodrug of trans-ferulic acid, and whose in vivo re-
lease will be tested in the future. In this work we effected the
lipid peroxidation assay for antioxidant activity and the re-
sults suggested that our pro-prodrug possesses an excellent
antioxidant activity.
Acknowledgements This work was financially supported by Academic
Funds.
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