Design, synthesis, and evaluation of novel cyclic phosphates of 5-aminosalicylic acid as cytochrome P450-activated prodrugs

Article abstract of DOI:10.1021/mp300330v

Four novel cyclic phosphates of the anti-inflammatory agent 5-aminosalicylic acid (5-ASA) were designed and synthesized as cytochrome P450 (CYP)-activated prodrugs. These prodrugs can be used for targeting into gut wall, since these types of cyclic phosph

Full text of DOI:10.1021/mp300330v

Brief Article
pubs.acs.org/molecularpharmaceutics
Design, Synthesis, and Evaluation of Novel Cyclic Phosphates of
5‑Aminosalicylic Acid as Cytochrome P450-Activated Prodrugs
Kristiina M. Huttunen,* Niina Tani, Risto O. Juvonen, Hannu Raunio, and Jarkko Rautio
School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
ABSTRACT: Four novel cyclic phosphates of the anti-
inflammatory agent 5-aminosalicylic acid (5-ASA) were
designed and synthesized as cytochrome P450 (CYP)-activated
prodrugs. These prodrugs can be used for targeting into gut
wall, since these types of cyclic phosphates are known to be
activated mainly by CYP3A forms, which are expressed not
only in the liver but also in the small intestine and to a lesser
extent in the colon. The present study shows that aromatic ring
activating substituents, like chlorine, are definitely needed to
obtain the desired enzymatic cleavage of the cyclic phosphate prodrugs of 5-ASA. However, the position of the activating
substituent has also a strong impact on the chemical stability, and therefore, an appropriate balance between the rates of prodrug
bioactivation and chemical stability needs to be taken into consideration in future studies on cyclic phosphate prodrugs of 5-ASA.
KEYWORDS: 5-aminosalicylic acid, bioactivation/bioconversion, cyclic phosphate, cytochrome P450 enzyme, prodrug
byproduct has been associated with cytotoxicity and genotox-
icity. However, it is rapidly and quantitatively detoxified by
intracellular glutathione, as long as the glutathione levels
remain at 20% of normal liver/gut levels.12 The liberated free
phosphate is subsequently hydrolyzed by phosphatases
releasing the parent drug with a free hydroxyl group. In the
present study four cyclic phosphates of 5-ASA were designed
and synthesized to investigate whether these compounds could
serve as CYP-selective prodrugs of 5-ASA. Taking several
structure-related requirements into consideration, these types
of cyclic phosphate prodrugs can undergo a CYP-catalyzed
prodrug cleavage and could be ideal candidates for further
studies to target 5-ASA especially to the small intestinal wall.
INTRODUCTION
■
5-Aminosalicylic acid (5-ASA, mesalamine) is an anti-
inflammatory agent primarily used to treat inflammatory
bowel diseases, such as ulcerative colitis and Crohn’s disease.1
Local concentrations of 5-ASA in the intestinal mucosa are
known to determine the clinical efficacy of 5-ASA. However,
when administrated orally, 5-ASA is rapidly and extensively
absorbed from the upper gastrointestinal tract, causing
unwanted systemic side effects.2 Several approaches, such as
prodrugs and prolonged-released formulations, have been
developed to deliver 5-ASA locally to the site of inflammation,
i.e., to the distal ileum and/or colon.3 Classic examples are azo-
prodrugs of 5-ASA, such as sulfasalazine, that are activated by
colonic bacterial azoreductases. However, all these oral 5-ASA
derivatives cause multiple adverse effects. Other prodrug
strategies, such as amino acid esters,4 acrylic-type polymeric
prodrugs,5 conjugates with dendrimer,6 dextran,7 and cyclo-
dextrin,8 as well as liposomal formulations9 of 5-ASA have also
been studied as colon-specific prodrugs with variable results.
Being a valuable and widely used agent, more selective methods
are needed to improve the targeting of 5-ASA to its site of
action.1
EXPERIMENTAL SECTION
■
General Synthetic Methods. All the reactions were
performed with reagents of commercial high purity quality
without further purification. Reactions were monitored by thin-
layer chromatography using aluminum sheets coated with silica
gel 60 F245 (0.24 mm) with suitable visualization. Purifications
by flash chromatography were performed on silica gel 60
1
(0.063−0.200 mm mesh). H, 13C, and 31P nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker Avance
We have recently reported that cyclic phosphates could serve
as potential cytochrome P450 (CYP) 3A4-selective prodrugs of
drug molecules having a free hydroxyl group.10 To date, this
prodrug approach has been successfully applied to 18β-
glycyrrhetinic acid to gain antiulcer and anti-inflammatory
sustained-release effects.11 Bioconversion of cyclic phosphate
prodrugs have been designed to undergo an initial CYP-
catalyzed oxidation at the C4 methine primarily by CYP3A
forms (Figure 1). The hydroxylated intermediate of the cyclic
phosphate is then converted to a free phosphate by β-
elimination of aryl vinyl ketone. This highly electrophilic
500 spectrometer (Bruker Biospin, Fallanden, Switzerland)
̈
operating at 500.13, 125.75, and 202.46 MHz, respectively.
1
Tetramethylsilane was used as an internal standard for H and
13C spectra and 85% H3PO4 as an external standard for 31P
Special Issue: Prodrug Design and Target Site Activation
Received: June 17, 2012
Revised: June 17, 2012
Accepted: August 23, 2012
© XXXX American Chemical Society
A
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Molecular Pharmaceutics
Brief Article
Figure 1. Proposed bioconversion of cyclic phosphate prodrugs to 5-ASA.
3JHH = 7.8 Hz). 13C NMR: δ 29.63, 58.50, 75.62, 119.62,
119.92, 124.97, 125.02, 125.37, 126.99, 128.54, 129.96, 134.41,
142.96, 143.16, 158.06, 171.45. 31P NMR: δ −5.88 and −5.36
(50:50). Anal. Calcd for (C16H15NO6PCl·1.2DCM): C, 42.54;
H, 3.36; N, 2.88. Found: C, 42.87; H, 3.39; N, 3.28.
5-Amino-2-((4-(4-chlorophenyl)-2-oxido-1,3,2-dioxa-
phosphinan-2-yl)oxy)benzoic Acid (4). 1H NMR (CDCl3):
δ 2.07−2.49 (2H, m), 4.52−4.73 (2H, m), 5.55−5.80 (1H, m),
7.28−7.46 (6H, m), 8.25−8.26 (1H, m). 13C NMR: δ 31.44,
50.76, 81.88, 120.14, 120.19, 120.82, 125.71, 125.91, 126.86,
127.00, 129.14, 135.03, 136.39, 162.59, 163.00, 171.59. 31P
NMR: δ −34.73 and −32.34 (65:35). Anal. Calcd for
(C16H15NO6PCl·0.4H2O·0.2DMF): C, 49.16; H, 4.28; N,
4.14. Found: C, 48.93; H, 3.89; N, 4.12.
High-Performance Liquid Chromatography (HPLC)
Analyses. The analyses were performed on the high-
performance liquid chromatography (HPLC) system, which
consisted of an Agilent 1100 binary pump (Agilent
Technologies Inc., Wilmington, DE, USA), a 1100 micro
vacuum degasser, an 1100 autosampler, a 1200 variable
wavelength detector SL (λ = 200 nm), an 1100 fluorescence
detector (λexc = 310 nm, λemiss = 500 nm), and an Agilent
Zorbax ODS analytical column (4.6 mm × 250 mm, 5 μm).
The chromatographic separations for samples from biological
matrixes were achieved by using an isocratic elution of 50 mM
phosphate buffer pH 2.0 and acetonitrile (7:3, v/v). The
chromatography was carried out at flow rate of 1.0 mL/min and
at room temperature.
Chemical Stability. The chemical stability of the cyclic
phosphate prodrugs 1−4 was studied in 50 mM (ion strength
0.15) buffered aqueous solutions (HCl buffer at pH 1.0, acetate
buffer at pH 5.0, phosphate buffer at pH 7.4, and borate buffer
at pH 9.0) 37 °C for three weeks. The incubation mixture was
prepared by dissolving the prodrug stock solution (10 mM) in
ethanol to preheated buffer solutions. The final prodrug
concentration in the incubation mixtures was 200 μM and
the ethanol concentration 2%. The samples were withdrawn at
appropriate intervals, and acetonitrile was added to the samples
(1:1, v/v) to minimize further hydrolysis prior to HPLC
analyses. The hydrolysis followed first-order kinetics, and the
pseudo-first-order half-lives (t1/2) for the hydrolysis of the
prodrugs were calculated from the slope of the linear portion of
the plotted logarithm of remaining prodrug versus time.
In Vitro Bioconversion. The bioactivation of the cyclic
phosphate prodrugs 1−4 was determined in isolated rat liver
microsomes prepared by standard differential ultracentrifuga-
tion and stored at −80 °C until use.19 The incubation mixture
spectra. Furthermore, the products were characterized by
elemental analysis (C, H, N) with a ThermoQuest CE
Instruments EA 1110-CHNS-O elemental analyzer (CE
Instruments, Milan, Italy).
General Procedure for the Synthesis of Cyclic
Phosphates (1−4). The amine group of 5-ASA was protected
by stirring 5-ASA (1 equiv) with di-tert-butyl-dicarbonate (1
equiv) and NaHCO3 (1 equiv) in anhydrous DMF at +50 °C
overnight, and the residue was purified by flash chromatog-
raphy eluting with 1−5% MeOH in DCM solution.13
A
solution of the compound 5 (1 equiv) in anhydrous DMF (5
mL) was treated with 1 M tert-BuMgCl in THF (1.3 equiv) and
stirred at room temperature under argon for 30 min. A selected
4-aryl-2-(4-nitrophenoxy)-2-oxo-1,3,2- dioxaphosphorinane 6−
9 (1.3 equiv), prepared as previously described,14−18 in
anhydrous THF (2−5 mL) was added to the reaction mixture
and stirred overnight at room temperature. The mixture was
quenched with saturated NH4Cl (10 mL) and extracted with
EtOAc (20 mL). The organic phase was washed with 1 M
NaOH (2 × 20 mL), water (2 × 20 mL), and brine (2 × 20
mL), dried over Na2SO4, and evaporated under reduced
pressure. The t-Boc protecting group was removed by stirring
the obtained compounds in 3 M HCl solution at room
temperature for 1−2 h. The cyclic phosphates 1−4 were
purified by flash chromatography eluting with 1−5% MeOH in
DCM solution.
5-Amino-2-((2-oxido-4-phenyl-1,3,2-dioxaphos-
1
phinan-2-yl)oxy)benzoic Acid (1). H NMR (CDCl3): δ
2.17−2.35 (2H, m), 4.09−4.27 (2H, m), 4.87−4.91 (1H, m),
7.12−7.32 (6H, m), 8.04−8.08 (2H, m). 13C NMR: δ 39.99,
59.04, 65.58, 120.50, 120.54, 125.63 (2C), 126.81, 128.64,
128.82 (2C), 140.48, 144.57, 155.49, 161.92, 171.04. 31P NMR:
δ
−6.33 and −6.01 (84:16). Anal. Calcd for
(C16H16NO6P·1.2H2O·0.2DCM): C, 50.16; H, 4.83; N, 3.61.
Found: C, 49.92; H, 4.68; N, 3.59.
5-Amino-2-((4-(2-chlorophenyl)-2-oxido-1,3,2-dioxa-
phosphinan-2-yl)oxy)benzoic Acid (2). 1H NMR (CDCl3):
δ 2.21−2.35 (2H, m), 4.46−4.74 (2H, m), 5.87−6.04 (1H, m),
7.28−7.62 (5H, m), 8.12−8.23 (2H, m). 13C NMR: δ 32.35,
69.02, 79.25, 120.31, 125.74, 125.74, 125.85, 126.93, 127.78,
129.59, 130.11, 131.06, 135.61, 144.78, 155.07, 171.33. 31P
NMR: δ −13.69 and −11.26 (81:19). Anal. Calcd for
(C16H15NO6PCl 0.3 H2O): C, 49.39; H, 4.04; N, 3.60.
Found: C, 49.06; H, 4.01; N, 3.42.
5-Amino-2-((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxa-
phosphinan-2-yl)oxy)benzoic Acid (3). 1H NMR (CDCl3):
δ 1.94−2.26 (2H, m), 3.35−4.09 (2H, m), 4.86−5.31 (1H, m),
3
7.02−7.29 (5H, m), 7.93 (1H, d, JHH = 8.1 Hz), 8.07 (1H, d,
B
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Molecular Pharmaceutics
Brief Article
a
Scheme 1. Synthesis of Cyclic Phosphates of 5-ASA
a
Reagents and conditions: (a) di-tert-butyl dicarbonate, NaHCO3, DMF, + 50 °C, 20 h, 80%; (b) LiAlH4, Et2O, reflux, 6−8 h, 33−98%; (c) 4-
nitrophenyl phosphorodichloridate, Et3N, THF, 0 °C to rt, 3−4 h, 83−99%; (d) (1) 1 M tert-BuMgCl, DMF, rt, 20 h, (2) 3 M HCl (aq), rt, 1−2 h,
33−50%.
was prepared by mixing 1 M Tris-HCl buffer (pH 7.4) and 100
μg of microsomal protein to water and preincubating the
mixture for 2 min at 37 °C. Prodrug stock solutions (5 mM) in
50% ethanol (the final concentration of prodrug 1 was 100 μM
and the ethanol concentration 1%) and a NADPH regenerating
system (1.15 mM NADP, 12.5 mM isocitric acid, 56.25 mM
KCl, 187.5 mM Tris-HCl, 12.5 mM MgCl2, 0.0125 mM MnCl2,
and 0.77 μL/mL isocitric acid dehydrogenase, pH 7.4) were
added, and the mixture was incubated approximately 6 to 24 h
at 37 °C. In blank solutions, liver microsomes or the NADPH
regenerating system was replaced with the same volume of
water. The samples were withdrawn at appropriate intervals,
and the enzymatic reactions were terminated by the addition of
ice-cold acetonitrile (1:1, v/v). The samples were centrifuged
for 15 min at 12000g and kept on ice until the supernatants
were analyzed by HPLC. The first-order half-lives (t1/2) for the
reaction of the prodrugs were calculated from the slope of the
linear portion of the plotted logarithm of remaining prodrug
versus time.
Hydrolysis in alkaline phosphatase solution was determined
by incubating a mixture of the cyclic phosphate prodrugs 1−4
in ethanol (1 mM), 1 M Tris-HCl buffer (pH 7.4), and alkaline
phosphatase EC 3.1.3.1 (type VII-S: from bovine intestinal
mucosa, 2.590 units/mg protein) purchased from Sigma (St.
Louis, MO, USA) in water at 37 °C for 24 h. The final
concentration of the prodrugs was 100 μM and the ethanol
concentration 1%, and the incubation mixture contained ca.
100 mU of alkaline phosphatase. In blank solutions, alkaline
phosphatase was replaced with the same volume of water. The
samples were withdrawn at appropriate intervals, and the
enzymatic reactions were terminated by the addition of ice-cold
acetonitrile (1:1, v/v). The samples were centrifuged for 15
min at 12000g and kept on ice until the supernatants were
analyzed by HPLC.
RESULTS AND DISCUSSION
■
As previously described, the desired CYP-catalyzed prodrug
cleavage of cyclic phosphates requires a C4-aryl ring attached to
the six-membered heterocyclic ring.10,14 The benzylic carbon
renders the cyclic phosphate more susceptible to CYP3A-
mediated oxidation. This makes the cyclic phosphates highly
selective prodrugs, since they are bioactivated only inside the
cells that are rich in CYP3A forms. Therefore, cyclic phosphate
derivatives are ideal for targeting drugs to tissues expressing
CYP3As. Although CYP3A forms reside predominantly in the
liver, they are also abundantly expressed in the small intestine
and with lower levels in the colon.20−22 Lately, CYP enzymes
have become widely exploited in prodrug research. CYPs
account for up to 75% of all enzymatic metabolism of drugs,
with a predominant role played by the CYP3A4 form.
Numerous clinically important drug−drug interactions occur
through inhibition or induction of CYPs, especially CYP3A4. In
addition, genetic polymorphisms in CYP genes have a
significant effect on disposition of a number of commonly
used drugs.23,24 These aspects need to be taken into account
when developing prodrugs activated by any specific CYP form,
especially CYP3A4.
The cyclic phosphates of 5-ASA, which are taken up into the
enterocytes and selectively bioconverted to their corresponding
free phosphates, are retained as charged intermediates in the
mucosal tissues. This augments the targeting effect and is
especially beneficial in Crohn’s disease, which is manifested in
both small and large intestine.25 In mucosal tissues the
phosphate intermediates are subsequently bioconverted to 5-
ASA by intestinal alkaline phosphatase (IAP). Although IAP is
mainly expressed in the apical membrane of enterocytes, it is
known to be released to the luminal side by lysophosphati-
dylcholine.26 Therefore, increased mucosal 5-ASA concen-
trations could be achieved by the cyclic phosphate prodrug
approach.
C
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Molecular Pharmaceutics
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Erion et al. have reported that several electron withdrawing
substituents, like chlorine in the C4-aryl ring or pyridine as an
aromatic ring, augment the prodrug conversion rate, while
electron donating substituents lower the rate of bioactia-
tion.14−16 Although electron donating substituents may
increase oxidation rate, the slow β-elimination makes the
total bioconversion rate much slower than with electron
withdrawing substituents, which in contrast may decrease
oxidation rate, but have much faster β-elimination and total
bioconversion rate. On the basis of this information four cyclic
phosphate prodrugs of 5-ASA were designed and synthesized in
the present study (Scheme 1): one with a plain C4-phenyl ring
(1) and three with a chlorine substituent in the ortho-, meta-
and para-positions of the C4-phenyl ring (2−4). The cyclic
phosphates of 5-ASA were prepared by protecting the amino
group of 5-ASA with tert-butyloxycarbonyl group (t-Boc) in
basic conditions and then reacting the obtained compound 513
with cyclic phosphate nitrobenzenes 6−9 in the presence of
tert-butylmagnesium chloride (Scheme 1). The t-Boc-protect-
ing group was finally cleaved by treatment with aqueous HCl to
obtain the cyclic phosphates 1−4 with moderate to good yields.
As previously described, the cyclic phosphate nitrobenzenes 6−
9 were prepared by reducing commercial β-keto esters with
lithium aluminum hydride (LiAlH4) to provide racemic
mixtures of diols 10−13, which were then reacted as
unseparated mixtures with 4-nitrophenyl phosphorodichlor-
idate in the presence of triethylamine to yield the compounds
6−9.14−18 Unseparated diastereomers of cyclic phosphates 1−4
were used to evaluate the nonenzymatic and enzymatic stability
as well as primary enzymatic bioconversion by CYP enzymes.
Due to the amphoteric properties of 5-ASA and its
derivatives, it was challenging to develop a proper HPLC
method to evaluate these compounds.27 Especially 5-ASA
phosphate (the intermediate of the prodrug bioconversion
process) had poor retention in reverse-phased columns, and
when using ion-pair reagents or a normal-phase column the
peak symmetries were unsatisfactory. As free phosphates are
well-known to be hydrolyzed by ubiquitous phosphatases28 and
since we have previously shown that these cyclic phosphates are
bioconverted in two steps, first by CYP enzymes and then by
phosphatases,10 we did not quantify the free phosphate, only 5-
ASA, the end product of the total prodrug bioconversion
process. Agilent Zorbax ODS analytical column (250 mm × 4.6
mm, 5 μm) was found to produce the most satisfactory
chromatographic separation of the prodrugs 1−4 and 5-ASA by
isocratic elution of 50 mM phosphate buffer pH 2.0 and
acetonitrile (7:3; 1.0 mL/min). The absorption maximum for
all cyclic phosphates was near to nonspecific wavelength, 200
nm, which in its part complicated the detection of these
compounds by UV. Moreover, none of the cyclic phosphates
emitted fluorescent light, unlike 5-ASA, which was detected by
the fluorescence detector (λexc = 310 nm, λemiss = 500 nm) in
our studies. Therefore, mass spectrometry should be used, if
possible, to detect these compounds in further studies.
Figure 2. Proposed hydrolysis reaction of cyclic phosphates of 5-ASA
in buffer solutions.
electronegative chlorine most probably weakened the bond
between phosphorus and oxygen atoms after the nucleophilic
attack of the water molecule to the phosphorus atom, which led
to the formation of a ring-opened product. These products
1
were identified on the basis of H, 13C, and 31P NMR spectra
(data not shown). Since the hydrolysis produced stable ring-
opened forms, which did not release any 5-ASA, it can be
concluded that the bioactivation reaction can only occur in the
presence of prodrug activating enzymes. Although the chlorine
substituent in the C4-aryl ring lowered the chemical stability of
cyclic phosphates of 5-ASA and at least the cyclic phosphate 4
was considered to be too unstable for targeting purposes, all the
prodrugs were selected for the bioactivation studies to evaluate
how the position of this electron withdrawing group affects the
bioconversion process.
Enzymatic stability of the prodrugs 1−4 toward hydrolysis by
alkaline phosphatase (from bovine intestinal mucosa) was
studied to confirm that these cyclic phosphates were not
susceptible for possible premature bioconversion by IAP. As
shown in Table 2, all four prodrugs were highly stable in
intestinal alkaline phosphatase solution at 37 °C and obviously
poor substrates for phosphatases. Since these experiments were
carried out in Tris-HCl buffer at pH 7.4, it clearly shows that
phosphate ions increase the rate of hydrolysis in aqueous media
(Table 1).
Bioactivation of the prodrugs 1−4 was studied as previously
described with isolated liver microsomes known to be rich in
CYP enzymes.10 Comparison of the bioconversion rates in
untreated rat liver microsomes (control) with liver microsomes
obtained from rats treated with the CYP3A-inducing agent,
dexamethasone, gave us initial information whether these
prodrugs behaved similarly as the cyclic phosphates published
previously, i.e., these prodrugs were activated mainly by CYP3A
forms. Unexpectedly, the cyclic phosphate 1 was not oxidized in
the rat liver microsomes at all (Table 2), although it has been
reported that a simple phenyl ring at the C4-position of the
cyclic phosphate should fulfill the requirements for the CYP-
catalyzed oxidation.10,14 In contrast, the cyclic phosphates 2, 3,
and 4 with a chlorine substituent at the ortho-, meta- and para-
positions, respectively, were oxidized in rat liver microsomes
(Table 2). Hence, in the case of 5-ASA an aromatic activating
substituent is definitely needed to obtain the desired
bioconversion of the cyclic phosphates. The bioconversion
reaction of the prodrugs 2−4 followed first-order kinetics and
released small amounts of 5-ASA. The reason why only low
levels of 5-ASA (10−60%) were detected in our experiments
was most probably due to other cellular reactions, such as N-
acetylation, which produced other derivatives of 5-ASA that
were not monitored in our experiments. Since the reaction did
not occur in the blank samples (without microsomes or
NAPDH, an essential component for the CYP-catalyzed
reactions), we concluded that the reaction was CYP-mediated.
Chemical stability of the prodrugs 1−4 was studied in 50
mM aqueous buffer solutions at pH values ranging from 1.0 to
9.0 at 37 °C. When hydrolyzed, the degradation of prodrugs
followed pseudo-first-order kinetics and released stable ring-
opened forms (Figure 2). The rate of hydrolysis was
remarkably faster with the para-substituted prodrug 4 than
with meta- and ortho-substituted prodrugs 3 and 2,
respectively, whereas the prodrug 1 did not hydrolyze at all
during the three weeks’ experimental time (Table 1). The
D
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Brief Article
Table 1. Half-Lives of the Cyclic Phosphate Prodrugs 1−4 in 50 mM Buffer Solutions at 37 °C (Mean SD, n = 3)
t1/2 (h)
compd
pH 1.0
pH 5.0
pH 7.4
pH 9.0
1
2
3
4
a
a
a
a
b
88.73 5.23
4.69 0.04
0.48 0.20
91.96 11.82
4.14 0.08
75.13 13.37
5.78 0.50
0.98 0.32
82.60 8.05
5.57 0.20
0.56 0.34
0.58 0.14
a
b
No degradation was observed during the three weeks’ incubation. n = 2.
CONCLUSION
Table 2. Half-Lives of the Cyclic Phosphate Prodrugs 1−4 in
Rat Liver Microsomes (RLM), Dexamethasone-Pretreated
Rat Liver Microsomes (DEX), and Bovine Intestinal Alkaline
Phosphatase Solution (IAP) at 37 °C (Mean SD, n = 3)
■
In conclusion, the results of the present study are consistent
with the earlier ones, i.e., drugs having a free hydroxyl group
can be converted into cyclic phosphates that can serve as
CYP3A-activated prodrugs. In the case of cyclic phosphates of
5-ASA, an unsubstituted phenyl prodrug was found to be inert
to CYP-mediated bioactivation and, therefore, a ring activating
substituent has to be incorporated to the phenyl ring of the
cyclic phosphate to achieve the desired CYP-catalyzed
oxidation. However, different ring activating substituents may
have a strong impact not only on the bioactivation rate but also
on the chemical stability of these prodrugs, and therefore an
appropriate balance between the rates of bioactivation and
chemical stability needs to be taken into consideration when
adding an electron withdrawing substituent to the aromatic ring
of the cyclic phosphates. Based on this study, the cyclic
phosphates of 5-ASA with a chlorine substituent in the ortho-
or meta-position of the C4-aryl ring may be suitable for further
evaluation to deliver higher local concentrations of 5-ASA to
the mucosal cells, especially to the small intestine.
t1/2 (min)
compd
RLM
DEX
IAP
1
2
3
4
a
a
a
a
a
a
702.28 265.44
292.50 41.70
23.03 1.06
455.44 135.57
156.90 31.50
26.65 0.71
a
No degradation was observed during the 24 h incubation.
The enzymatic reaction rates were much faster with the cyclic
phosphate 4 than with the cyclic phosphates 3 and 2, which
strongly implies that the chlorine substituent at the para-
positions speeds up the oxidation rate remarkably. As expected,
the cyclic phosphates 2 and 3 were oxidized more rapidly in
dexamethasone-induced rat liver microsomes, which indicates
that CYP3A forms were involved in the bioactivation process
(Figure 3). However, in the case of the para-substituted
AUTHOR INFORMATION
■
Corresponding Author
*School of Pharmacy, Faculty of Health Sciences, University of
Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.
Tel: +358 403 553 684. Fax: +358 17 162456. E-mail: kristiina.
huttunen@uef.fi.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mrs. Tiina Koivunen and Eeva Kemppainen, M.Sc.,
for skillful technical assistance with synthesis and Mrs. Helly
Rissanen and Tarja Matikainen, M.Sc., for valuable technical
assistance with the in vitro studies. The work was financially
supported by the National Graduate School of Organic
Chemistry and Chemical Biology and Jenny and Antti Wihuri
Foundation.
Figure 3. First order reaction rate constants of the enzymatic reaction
of the prodrugs 2 and 3 in rat liver microsomes (RLM) and
dexamethasone-induced rat liver microsomes (DEX).
prodrug, no difference was seen between the reaction rates in
control rat liver microsomes and dexamethasone-induced ones
(Table 2). Most likely the oxidation of the cyclic phosphate 4
was mediated by other CYP forms or other enzymes. Together
with low chemical stability, the cyclic phosphate 4 was
considered as an unideal prodrug for targeting of 5-ASA. In
contrast, the cyclic phosphate 3 with chlorine at the meta-
position in the C4-aryl ring seems to have both reasonable
bioactivation rate and sufficient chemical stability to target 5-
ASA to the mucosal tissues of intestine, while the ortho-
substituted cyclic phosphate 2 may have too slow a
bioconversion rate. However, both of these cyclic phosphate
prodrugs of 5-ASA should be carried on to future studies.
ABBREVIATIONS USED
■
5-ASA, 5-aminosalicylic acid (mesalamine); CYP, cytochrome
P450 enzymes; IAP, intestinal alkaline phosphatase
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Molecular Pharmaceutics
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dx.doi.org/10.1021/mp300330v | Mol. Pharmaceutics XXXX, XXX, XXX−XXX