Investigation of solvolysis kinetics of new synthesized fluocinolone acetonide C-21 esters-an in vitro model for prodrug activation

Article abstract of DOI:10.3390/molecules16032658

In this study the solvolysis of newly synthesized fluocinolone acetonide C-21 esters was analysed in comparison with fluocinonide during a 24-hour period of time. The solvolysis was performed in an ethanol-water (90:10 v/v) mixture using the excess of NaHCO₃. The solvolytic mixtures of each investigated ester have been assayed by a RPHPLC method using isocratic elution with methanol-water (75:25 v/v); flow rate 1 mL/min; detection at 238 nm; temperature 25 °C. Solvolytic rate constants were calculated from the obtained data. Geometry optimizations and charges calculations were carried out by Gaussian W03 software. A good correlation (R = 0.9924) was obtained between solvolytic rate constants and the polarity of the C-O2 bond of those esters. The established relation between solvolytic rate constant (K) and lipophilicity (cLogP) with experimental anti-inflammatory activity could be indicative for topical corticosteroid prodrug activation.

Full text of DOI:10.3390/molecules16032658

Molecules 2011, 16, 2658-2671; doi:10.3390/molecules16032658
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Investigation of Solvolysis Kinetics of New Synthesized
Fluocinolone Acetonide C-21 Esters—An In Vitro Model for
Prodrug Activation
Bojan D. Markovic 1,*, Vladimir D. Dobricic 1, Sote M. Vladimirov 1, Olivera A. Cudina 1,
Vladimir M. Savic 2 and Katarina D. Karljikovic-Rajic 3
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode
Stepe 450, 11221 Belgrade, Serbia
2
Department of Organic Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe
450, 11221 Belgrade, Serbia
3
Department of Analytical Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe
450, 11221 Belgrade, Serbia
* Author to whom correspondence should be addressed; E-Mail: bojan@pharmacy.bg.ac.rs;
Tel.: +381-11-3951-335; Fax: +381-11-3972-840.
Received: 18 January 2011; in revised form: 16 March 2011 / Accepted: 21 March 2011 /
Published: 23 March 2011
Abstract: In this study the solvolysis of newly synthesized fluocinolone acetonide C-21
esters was analysed in comparison with fluocinonide during a 24-hour period of time. The
solvolysis was performed in an ethanol-water (90:10 v/v) mixture using the excess of
NaHCO3. The solvolytic mixtures of each investigated ester have been assayed by a RP-
HPLC method using isocratic elution with methanol-water (75:25 v/v); flow rate
1 mL/min; detection at 238 nm; temperature 25 °C. Solvolytic rate constants were
calculated from the obtained data. Geometry optimizations and charges calculations were
carried out by Gaussian W03 software. A good correlation (R = 0.9924) was obtained
between solvolytic rate constants and the polarity of the C-O2 bond of those esters. The
established relation between solvolytic rate constant (K) and lipophilicity (cLogP) with
experimental anti-inflammatory activity could be indicative for topical corticosteroid
prodrug activation.
Keywords: prodrug; fluocinolone acetonide esters; solvolysis; descriptor; in vitro model

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1. Introduction
Corticosteroids are used topically for their anti-inflammatory and immunosuppressive actions.
Applied in this fashion, particularly to large areas, when the skin is injured, or under occlusive
dressings, corticosteroids may be absorbed in sufficient amounts to cause systemic effects [1].
Therefore, the therapy with corticosteroids is limited both by systemic and local side effects. In order
to increase benefit/risk ratio [2] the series of novel α-alkoxyalkanoyl and α-aryloxyalkanoyl esters
with higher lipophilicity have been synthesized in our laboratory as analogues of fluocinolone
acetonide 21-acetate (fluocinonide) [3]. Fluocinolone acetonide 21-acetate (FA-21-Ac) is about five
times more potent than its active form fluocinolone acetonide (FA) due to enhanced lipophilicity and
percutaneous absorption [4,5]. Thus, the new synthesized corticosteroid esters: fluocinolone acetonide
21-(2’-ethoxypropionate) (FA-21-EP), fluocinolone acetonide 21-(2’-ethoxybutyrate) (FA-21-EB),
fluocinolone acetonide 21-(2’-methoxypropionate) (FA-21-MP), fluocinolone acetonide 21-(2’-
methoxybutyrate) (FA-21-MB), fluocinolone acetonide 21-(2’-phenoxypropionate) (FA-21-PhP) could
be potential excellent prodrug candidates to improve the drug’s physical and pharmaceutical
properties. Prodrugs become pharmacologically active after the ester hydrolysis, since the
glucocorticoid actions are caused only by corresponding glucocorticoid alcohols [6]. Thus, the
hydrolysis of C17 or/and C21 esterified corticosteroids in biological media has a significant impact on
their local potency and the systemic availability.
The hydrolysis of carboxylic acid esters is one of the most thoroughly studied chemical reactions in
chemistry and biochemistry. Zhang et al. [7] have analyzed the hydrolysis of 41 different alkyl esters
of aliphatic and aromatic carboxylic acids. They have calculated hydrolytic rate constants for all of the
esters and established a model that correlates hydrolytic rate constant with several molecular
descriptors (carbonyl carbon charge, carbonyl oxygen charge and LUMO energy). Methanol-mediated
hydrolysis (e.g. methanolysis followed by hydrolysis) of β-lactams mimics the enzymatic pathway and
could be studied in the gas phase and solution as a model reaction for hydrolysis of β-lactams by β-
lactamases [8].
Our previous work was related to the application of derivative spectrophotometry for the study of
FA-21-PhP solvolysis [9]. The aim of this study is to establish kinetic rate orders and the
corresponding solvolytic rate constants of the novel esters and comparison with fluocinonide.
Correlations between experimentally determined solvolytic rate constants and descriptors of steric and
electronic properties of those esters have been established. The solvolytic model could be efficient as
an in vitro model for corticosteroid C-21 ester prodrug activation. The significance of this model was
established by obtaining a good correlation between the anti-inflammatory activity (determined in the
test of inhibition of croton oil induced mice ear edema) [3] with solvolytic rate constants and
lipophilicity. These correlations could be used in a rational design of novel esters.
2. Results and Discussion
2.1. Solvolytic rate constants determination
The solvolytic processes were followed after 0, 2, 4, 6, 8 and 24 hours using a HPLC method.
Concentrations of fluocinolone acetonide (FA) in each solvolytic mixture were determined via FA

Molecules 2011, 16
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peak areas, using the method of regression analysis. The concentration of corresponding ester in each
solvolytic mixture was calculated using equation (1):
c(t) = c(0)−c
(1)
where: c(t) – concentration of corresponding ester after 2, 4, 6, 8 or 24 hours.
c(0) – concentration of corresponding ester in freshly prepared mixtures (t = 0)
c – concentration of FA (equal to the concentration of solvolyzed ester fraction)
Figure 1. Solvolysis of six fluocinolone acetonide esters.
Solvolytic rate constants are obtained from the slope of the plot of ln[ester concentration] versus
time, as shown in Figure 1.

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2.2. Mechanism of solvolysis of fluocinolone acetonide esters
Corticosteroid-21-esters are prodrugs and undergo in vivo hydrolysis by esterases. Products of THE
hydrolysis are the pharmacologically active alcohol and the corresponding acid. Due to the poor water
solubility of the analyzed FA esters, selected conditions of solvolysis included an ethanol-water (90:10
v/v) solvent mixture with an excess of sodium hydrogen carbonate. Under these conditions, the
degradation products are the FA and the corresponding ethyl esters (transeesterification) [10-12]. The
general pathway of fluocinolone acetonide C-21 ester solvolysis is shown in Scheme 1.
Scheme 1. The general pathway of fluocinolone acetonide C-21 esters solvolysis.
2.3. Kinetics of esters solvolysis
The graphs were plotted using ln[ester concentration] versus time (Figure 1) with denoted data of
regression and correlation analysis. Correlation coefficients indicate good linearity. These results
confirmed pseudo-first-order kinetics, which means that solvolytic rate depends on ester concentration
when the concentration of NaHCO3 is significantly higher.
2.4. Descriptor selection
All the tested esters differ from each other in the acid used for esterification. Thus, their
susceptibility to solvolysis can be affected by the steric, electronic or physico-chemical properties of

Molecules 2011, 16
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corresponding acids. Considering this, following descriptors have been selected: cLogP, carbonyl
carbon steric hindrance (steric hindrance), C=O1 bond length and C-O2 bond length, charge of
carbonyl carbon (C), charge of ester oxygen (O2), charge of carbonyl oxygen (O1), charge differences
C-O1, charge differences C-O2, polarity [13] of C-O2 bond (PC-O2), polarity of C=O1 bond (PC=O1).
The values of calculated descriptors and their coefficients of correlation (R) with solvolytic rate
constants (K) are presented in Table 1.
Table 1. Solvolytic rate constants (K) and descriptor values.
FA-21-Ac FA-21-EB
FA-21-EP
1.370
FA-21-MB
1.369
FA-21-MP FA-21-PhP
R
C-O2 bond length
1.381
1.229
1.369
1.232
1.370
1.232
1.374 0.9627
1.228 -0.9168
0.2777 -0.5215
-0.2851 -0.8342
-0.3227 -0.0956
0.5628 0.9023
0.6004 -0.5807
0.7732 0.9247
0.7373 -0.6644
0.3025 0.7510
-0.2767 -0.7309
-0.2575 0.1229
0.5792 0.8315
0.5599 0.4620
0.7958 0.8771
0.6876 0.3561
0.8161 -0.4782
-0.7509 -0.5802
-0.5598 0.2239
1.5670 0.1414
1.3759 -0.4076
2.1531 0.2631
1.6896 -0.4605
0.2232 0.4277
-0.2010 -0.7492
-0.2207 0.4194
0.4242 0.8752
0.4439 -0.0451
0.5828 0.9118
0.5452 -0.3450
0.5518 -0.0727
-0.5214 -0.5609
-0.4059 0.4213
1.0731 0.3315
0.9577 -0.4469
1.4745 0.4106
1.1760 -0.4686
C=O1 bond length
C
1.232
1.232
0.2991
-0.2580
-0.2830
0.5571
0.5821
0.7694
0.7154
0.3080
-0.2595
-0.2407
0.5676
0.5488
0.7838
0.6745
0.7654
-0.6927
-0.5207
1.4582
1.2862
2.0137
1.5807
0.2314
-0.1879
-0.2095
0.4193
0.4409
0.5790
0.5418
0.5089
-0.4682
-0.3784
0.9771
0.8874
1.3494
1.0906
0.2990
-0.2237
-0.2950
0.5226
0.5940
0.7155
0.7318
0.2971
-0.2475
-0.2489
0.5446
0.5460
0.7455
0.6727
0.7898
-0.6792
-0.5390
1.4689
1.3288
2.0110
1.6371
0.2233
-0.1783
-0.2172
0.4016
0.4405
0.5498
0.5427
0.5138
-0.4582
-0.3959
0.9720
0.9097
1.3306
1.1207
0.3062
-0.2284
-0.2949
0.5346
0.6011
0.7324
0.7405
0.3027
-0.2515
-0.2478
0.5542
0.5505
0.7592
0.6782
0.8195
-0.6837
-0.5423
1.5033
1.3618
2.0595
1.6778
0.2277
-0.1807
-0.2167
0.4084
0.4443
0.5595
0.5474
0.5387
-0.4610
-0.3997
0.9997
0.9383
1.3696
1.1560
0.2991
-0.2234
-0.2943
0.5225
0.5934
0.7153
0.7311
0.2975
-0.2471
-0.2481
0.5446
0.5457
0.7456
0.6723
0.7917
-0.6784
-0.5383
1.4701
1.3300
2.0126
1.6386
0.2237
-0.1778
-0.2165
0.4015
0.4402
0.5496
0.5423
0.5167
-0.4574
-0.5167
0.9742
1.0334
1.3336
1.2732
0.3069
-0.2282
-0.2940
0.5351
0.6009
0.7331
0.7403
0.3033
-0.2511
-0.2468
0.5544
0.5502
0.7595
0.6778
0.8212
-0.6833
-0.5413
1.5044
1.3625
2.0611
1.6786
0.2283
-0.1802
-0.2157
0.4085
0.4440
0.5596
0.5470
0.5410
-0.4605
-0.3988
1.0016
0.9398
1.3722
1.1578
O2
O1
C-O2
C-O1
PC-O2
PC=O1
C
O2
O1
C-O2
C-O1
PC-O2
PC=O1
C
O2
O1
C-O2
C-O1
PC-O2
PC=O1
C
O2
O1
C-O2
C-O1
PC-O2
PC=O1
C
O2
O1
C-O2
C-O1
PC-O2
PC=O1

Molecules 2011, 16
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Table 1. Cont.
C
O2
0.5089
-0.4682
-0.3784
0.9771
0.8874
1.3494
1.0906
0.3188
-0.2633
-0.2719
0.5821
0.5907
0.8038
0.7260
0.2370
-0.1910
-0.2395
0.4280
0.4766
0.5911
0.5857
1.24
0.5138
-0.4582
-0.3959
0.9720
0.9097
1.3306
1.1207
0.3057
-0.2597
-0.2765
0.5655
0.5822
0.7741
0.7173
0.2277
-0.1877
-0.2424
0.4154
0.4701
0.5687
0.5791
1.36
0.5387
-0.4610
-0.3997
0.9997
0.9383
1.3696
1.1560
0.3086
-0.2616
-0.2765
0.5702
0.5850
0.7811
0.7208
0.2287
-0.1883
-0.2426
0.4171
0.4713
0.5714
0.5807
1.34
0.5167
-0.4574
-0.5167
0.9742
1.0334
1.3336
1.2732
0.3062
-0.2591
-0.2752
0.5654
0.5815
0.7740
0.7164
0.2280
-0.1872
-0.2943
0.4153
0.5223
0.5685
0.6435
1.37
0.5410
-0.4605
-0.3988
1.0016
0.9398
1.3722
1.1578
0.3089
-0.2612
-0.2748
0.5701
0.5837
0.7810
0.7192
0.2293
-0.1881
-0.2940
0.4174
0.5233
0.5719
0.6447
1.35
0.5518 -0.0727
-0.5214 -0.5609
-0.4059 0.4213
1.0731 0.3315
0.9577 -0.4469
1.4745 0.4106
1.1760 -0.4686
0.3107 0.9101
-0.2736 -0.6169
-0.2745 0.8871
0.5843 0.9243
0.5852 0.8641
0.8028 0.9578
0.7186 0.7311
0.2281 0.7636
-0.1979 -0.6912
-0.3227 -0.0345
0.4260 0.9853
0.5508 0.1160
0.5853 0.9924
0.6764 0.0966
1.40 -0.5356
O1
C-O2
C-O1
PC-O2
PC=O1
C
O2
O1
C-O2
C-O1
PC-O2
PC=O1
C
O2
O1
C-O2
C-O1
PC-O2
PC=O1
Steric hindrance
cLogP
K
2.79
3.79
3.26
3.40
2.87
4.59 0.0533
0.0615
0.0049
0.0039
0.0040
0.0089
0.0434 1.0000
2.6. Relationships between solvolytic rate constant and selected descriptors
There are two mechanisms for base-catalyzed hydrolysis: BAC2 (base-catalyzed, acyl oxygen
cleavage, bimolecular) and BAL2 (base-catalyzed alkyl oxygen cleavage, bimolecular). In most cases,
BAC2 prevails over the BAL2 reaction path [14,15]. In accordance with the fact that solvolysis is a
similar process to hydrolysis and with respect to the BAC2 mechanism, the carbonyl carbon is the site
of the ethoxide ion attack and the cleavage site for alcoholic group formation, any changes at the
carbonyl group will directly affect the rate of solvolysis. In the following study the C(=O1)-O2 moiety
and its properties were used to correlate reaction rate data. Since many substituents affect charge
distribution of carbonyl group, they can have impact on solvolysis rate. Therefore, different acids used
for corticosteroid esterification, can affect ester stability and solvolysis rate.
Starting from AM1 optimized structures the best correlations were obtained between solvolytic rate
constant and C-O2 distance (R = 0.9627). Slight changes in bond length could affect bond dissociation.
A similar correlation (R = 0.9247) has been found between solvolytic rate constants and polarity of C-
O2 bonds. In order to find the most accurate model ab initio Hartree-Fock (HF) and Density
Functional Theory (DFT) with B3LYP model calculations have been performed. In these calculations
different basis sets (STO-3G and 6-31G) were used. To assess the influence of solvatation effects these
calculations have been carried out in the gas phase and in the solvent - ethanol (the conductor-like
screening model COSMO and the conductor-like polarizable continuum model CPCM).

Molecules 2011, 16
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Analyzing the data from Table 1 a certain correlation between solvolytic rate constant and carbonyl
carbon charge could be noted. If the carbonyl carbon charge is higher, nucleophilic ethoxide ion will
attack it easily. Since this phase of base-catalyzed ester solvolysis is rate limiting, higher carbonyl
charge accelerates this process (R = 0.9101 in HF/6-31G computation model using CPCM solvatation
method with ethanol as solvent). Among the tested FA esters, FA-21-Ac and FA-21-PhP have
significantly higher solvolytic rate constants and corresponding carbonyl carbon charges than the other
esters. Acetic acid has three hydrogen atoms in the α-position which negligibly affect its carbonyl
carbon charge, whereas α-phenoxypropionic acid has a group with positive (methyl) and strong
negative inductive effect (phenoxy) and the overall effect is electron withdrawing, which increases
carbonyl carbon charge. In the molecules of acids which have been selected for synthesis (α-
methoxybutyric, α-methoxypropionic, α-ethoxypropionic and α-ethoxybutyric acid) there is one group
in the α-position with a negative inductive effect (methoxy or ethoxy group) and one group with
positive inductive effect (methyl or ethyl group). These groups have opposite effects on carbonyl
carbon charge and make it less positive comparing to carbonyl carbon of acetic and
α−phenoxypropionic acids.
Gas phase Ab initio calculations with different levels of theory and different basis sets did not give
significantly better correlations than semi-empirical AM1. However, good correlations were achieved
if these calculations are carried out in the presence of solvent. Correlation analysis showed that the
most accurate model was obtained by using polarity of C-O2 bond calculated by B3LYP/STO-3G
model with ethanol as a solvent in COSMO solvatation model (Figure 2). The polarity presents a
descriptor calculated by multiplication of charge differences with the distance between two atoms and
a good correlation polarity versus rate of hydrolysis of N-acetylazoles has been obtained [13]. In our
study, polarity of C-O2 band (PC-O2) was calculated by multiplication of charge differences between
carbonyl carbon charge (C) and ester oxygen charge (O2) with C-O2 bond length (bond length
measured on AM1 optimized structures, Mulliken charges calculated by B3LYP/STO-3G model using
ethanol as solvent in COSMO solvatation model).
The equation (2) was obtained using single linear regression analysis:
K = (2.5808±0.1604) × PC-O2 – (1.4658 ± 0.0924)
(2)
where R = 0.9924, SE = 0.0035, F = 258.855; K – pseudo-first order solvolytic rate constant; PC-O2
polarity of C-O2 bond
–
Figure 2. Correlation between polarity of C-O2 bond (B3LYP/STO-3G SCRF(COSMO,
ethanol)) and solvolytic rate constant.
0,08
0,06
0,04
0,02
0,00
0,565
0,57
0,575
0,58
0,585
0,59
0,595
Polarity of C-O2 bond, PC-O2

Molecules 2011, 16
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2.7. Relationship between solvolitic rate constant and anti-inflammatory activity
Solvolysis kinetics of the new synthesized fluocinolone acetonide C-21 esters have been
investigated as a simple, rapid and low cost in vitro model for topical corticosteroid prodrug activation.
Anti-inflammatory activity (represented as a percentage of reduction of mass edema) has been
determined in a test of croton oil induced mice ear edema. To assess whether this model can be used to
estimate the prodrugs activation, a regression analysis between local anti-inflammatory activity and
solvolytic rate constants was performed. There is no significant correlation between these two
parameters, but it could be noted that there are two groups (Figure 3A) – the first with high solvolysis
rate constants (FA-21-Ac and FA-21-PhP) and the second with low solvolysis rate constants (FA-21-
MP, FA-21-EP, FA-21-MB and FA-21-EB). Topical prodrug activity depends on several factors: rate
of hydrolysis, interactions of the active form with target structures, permeability, etc. James et al. [16]
studied the effect of various aliphatic esters of testosterone on rat prostate and seminal vesicles and
correlated with lipophilicity and rate of ester hydrolysis by liver esterase. In accordance with this
manuscript anti-inflammatory activity was linked (Figure 3B) with solvolysis rate constant (K) and
lipophilicity (cLogP – values are of significance ranged from 2.79 to 4.59).
Figure 3. A. Experimental anti-inflammatory activity versus solvolytic rate constant. B.
Predicted versus experimental anti-inflammatory activity of fluocinolone acetonide C-21
esters.
Good correlation (R = 0.7214) obtained by multiple regression analysis is indicative that this in
vitro solvolytic model could be used as a model for topical corticosteroid prodrugs activation.
3. Experimental
3.1. General
Fluocinolone acetonide 21-acetate (FA-21-Ac) was purchased from Galenika a.d. Serbia.
Fluocinolone acetonide 21-(2’-ethoxypropionate) (FA-21-EP), fluocinolone acetonide 21-(2’-ethoxy-
butyrate) (FA-21-EB), fluocinolone acetonide 21-(2’-methoxypropionate) (FA-21-MP), fluocinolone
acetonide 21-(2’-methoxybutyrate) (FA-21-MB) and fluocinolone acetonide 21-(2’-phenoxy-
propionate) (FA-21-PhP) were synthesized in our laboratory. Synthesis of the fluocinolone acetonide
esters was carried out using racemic α-alkoxyalkanoyl or α-aryloxyalkanoyl acids thus affording the
corresponding mixtures of two diastereomeric products. Acids used for esterifications, as well as the

Molecules 2011, 16
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corresponding esters, are in the form of racemic mixtures. The diastereomers did not show separated
1
signals in H-NMR spectra, most likely due to their overlap. Lack of mutual interactions between the
acid and the steroid moieties caused by the distance between them is presumably the reason for the
observed 1H-NMR results. On the other hand, 13C-NMR spectra clearly suggested the presence of two
diastereomeric products, although it is difficult to differentiate them unambiguously.
Reagents: fluocinolone acetonide, absolute ethanol Chromasolv HPLC purity, methanol
Chromasolv HPLC purity (Sigma-Aldrich Chemie GmbH), sodium hydrogen carbonate,
thionylchloride (Merck), ethyl 2-bromopropionate, 2-bromobutiric acid, 2-phenoxypropionic acid
(Fluka), dichlormethane, ethyl acetate (Carlo Erba), N,N-dimethylformamide (Alfa Aesar) and
deionized water (TKA water purification system, Niederelbert, Germany) were used throughout
this study.
Melting points were determined on a Boetius PHMK 05 apparatus. 1H-NMR and 13C-NMR spectra
were recorded on a Bruker AVANCE instrument at 500 and 125 MHz using CDCl3 as a solvent and
TMS as an internal standard, chemical shifts (δ) are given in ppm, coupling constants (J) in Hz. IR
spectra were recorded on a Thermo Scientific NICOLET iS10 by using diamond ATR. Direct ESI-MS
spectra were recorded on Agilent Technologies 6210-1210 TOF-LC-ESI-MS instrument in positive
mode.
3.2. General procedure for synthesis of fluocinolone acetonide α-alkoxyalkanoyl and α-aryloxy-
alkanoyl C-21 monoesters
In this study five structurally related acids were used for the esterification of FA. 2-
Methoxypropionic, 2-ethoxypropionic, 2-methoxybutyric and 2-ethoxybutyric acid were prepared
from the corresponding 2-bromopropionic and 2-bromobutyric acids with sodium methoxide or
sodium ethoxide, according to the patent literature [17]. The mentioned acids were transformed into
the corresponding chlorides by reaction with an excess of thionyl chloride. Due to high volatility of
prepared acylchlorides, the losses resulting compounds were significant during removal of the last
traces of thionyl chloride, so the yields of pure compounds were about 15%. Typically, to a solution of
FA (220.0 mg, 0.486 mmol) in dichloromethane (15 mL) triethylamine (0.1 mL, 0.721 mmol) was
added. The mixture was cooled at 0 °C and corresponding acyl chloride (134.6 mg, 0.729 mmol) was
added dropwise. The resulting mixture was stirred 4 h at room temperature. Dichloromethane (50 mL)
was added and the solution washed with hydrochloric acid (1 M, 50 mL), aqueous solution of sodium
hydrogen carbonate (5%, 50 mL) and water (twice with 50 mL) and dried with anhydrous Na2SO4. The
crude product was purified by silica gel chromatography using chloroform as mobile phase to afford
the pure product.
(6α,11β,16α)-6,9-Difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene) bis(oxy)]-pregna-1,4-diene-
3,20-dione-21-(2′-ethoxypropionate) (FA-21-EP). White solid, melting point 231–232.5 °C. 1H-NMR:
0.94 (3H, d, J = 7 Hz, H-18), 1.23 (3H, s, H-23), 1.44 (3H, s, H-24), 1.47 (3H, dd, J = 17 Hz, J = 7 Hz,
H-2’’), 1.53 (3H, s, H-19), 1.59–2.01 (7H, m, H-7α, H-12β, H-15, H-3’), 2.15–2.56 (4H, m, H-7β, H-
8, H-12α, H-14), 3.49–3.72 (2H, m, H-1’’), 4.09–4.15 (1H, m, H-2’), 4.42 (1H, d, J = 8 Hz, H-11),
4.90–5.03 (3H, m, H-16, H-21), 5.32-5.46 (1H, dq, J = 48.5 Hz, J = 6 Hz, H-6), 6.37 (1H, dd,

Molecules 2011, 16
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13
J = 10.5 Hz, J = 1.5 Hz, H-2), 6.44 (1H, s, H-4), 7.12 (1H, d, J = 10 Hz, H-1). C-NMR: 15.2, 16.3,
18.9, 23.0, 23.1, 25.8, 26.5, 29.7, 31.8, 31.9, 32.0, 33.6, 33.8, 34.0, 37.1, 42.7, 45.6, 47.9, 48.0, 65.8,
67.8, 67.9, 71.6, 71.9, 74.5, 81.8, 85.6, 87.1, 97.4, 97.9, 99.3, 111.8, 121.2, 121.3, 130.3, 150.3, 161.1,
173.5, 185.4, 203.2. IR (ATR) 857.3, 896.7, 934.3, 996.2, 1,068.5, 1,128.3, 1,160.7, 1,209.9, 1,265.6,
1,374.1, 1,455.3, 1,609.1, 1,627.5, 1,666.8, 1,727.9, 2,925.1, 3,333.8 cm-1. MS [M+H]+ calculated for
C29H39F2O8 = 553.26075; observed = 553.26104.
(6α,11β,16α)-6,9-Difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene) bis(oxy)]-pregna-1,4-diene-
1
3,20-dione-21-(2′-ethoxybutyrate) (FA-21-EB). White solid, melting point 255–257 °C. H-NMR:
0.94 (3H, t, H-18), 1.00–1.06 (2H, m, H-4’) 1.23–1.27 (3H, s, H-23), 1.44 (3H, s, H-24), 1.53 (3H, s,
H-19), 1.59–2.01 (6H, m, H-7α, H-12β, H-15, H-3’), 2.13–2.56 (4H, m, H-7β, H-8, H-12α, H-14),
3.49–4.20 (2H, m, H-1’’), 3.90–4.21 (1H, m, H-2’), 4.42 (1H, t, J = 3 Hz, H-11), 4.93–5.01 (3H, m, H-
16, H-21), 5.32–5.46 (1H, dq, J = 48.5 Hz, J = 6 Hz, H-6), 6.37 (1H, dd, J = 10 Hz, J = 2 Hz, H-2),
13
6.43 (1H, s, H-4), 7.12 (1H, d, J = 10 Hz, H-1). C-NMR: 9.6, 15.1, 16.3, 18.14, 23.0, 25.7, 26.3,
26.4, 26.5, 26.9, 31.7, 31.8, 31.9, 32.0, 33.4, 33.6, 33.8, 34.0, 37.1, 37.4, 42.7, 45.3, 45.6, 48.0, 59.7,
63.8, 66.1, 67.4, 67.8, 71.5, 71.8, 79.8, 81.6, 81.8, 85.6, 87.1, 97.3, 98.0, 99.4, 111.7, 121.2, 121.5,
130.2, 150.4, 161.2, 172.9, 185.5, 203.2. IR (ATR) 856.6, 897.6, 934.9, 996.4, 1,068.0, 1,179.9,
1,264.0, 1,374.5, 1,626.7, 1,664.9, 1,725.9, 2,937.8, 3,336.1 cm-1. MS [M+H]+ calculated for
C33H39F2O8 = 567.27640; observed = 567.27683.
(6α,11β,16α)-6,9-Difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene)bis(oxy)]-pregna-1,4-diene-
1
3,20-dione-21-(2′-methoxypropionate) (FA-21-MP). White solid, melting point 236–238 °C. H-
NMR: 0.94 (3H, d, J=10, H-18), 1.22 (3H, s, H-23), 1.44 (3H, d, J = 2.5 Hz, H-24), 1.53 (3H, s, H-19),
1.60–2.22 (7H, m, H-7α, H-12β, H-15, H-3’), 2.28–2.55 (4H, m, H-7β, H-8, H-12α, H-14), 3.45 (3H,
d, J = 3.5 Hz, H-1’’), 4.03 (1H, m, H-2’), 4.42 (1H, m, H-11), 4.85–5.01 (3H, m, H-16, H-21), 5.32–
5.46 (1H, dq, J = 49 Hz, J = 6.5 Hz, H-6), 6.37 (1H, 1H, dd, J = 10 Hz, J = 1.5 Hz, H-2), 6.44 (1H, s,
13
H-4), 7.12 (1H, dd, J = 10.5 Hz, J = 1.5 Hz, H-1). C-NMR: 16.3, 18.5, 20.5, 23.0, 23.1, 25.8, 26.5,
29.7, 31.7. 31.8, 31.9, 32.0, 33.6, 33.8, 34.0, 37.1, 42.7, 45.5, 45.6, 47.9, 48.1, 57.8, 67.6, 71.6, 71.9,
76.2, 81.8, 85.6, 87.1, 97.4, 98.0, 99.4, 111.7, 111.8, 121.2, 121.3, 130.2, 150.4, 161.1, 161.2, 170.7,
185.5, 203.6. IR (ATR) 859.5, 897.6, 994.0, 1,038.7, 1,068.2, 1,130.2, 1,161.1, 1,181.5, 1,209.3,
1,265.4, 1,233.7, 1,374.6, 1,408.1, 1,455.5, 1,610.3, 1,627.4, 1,667.4, 1,727.2, 1,746.3, 2,940.0, 3350.8
cm-1. MS [M+H]+ calculated for C33H39F2O8 = 539.24450; observed = 539.24450.
(6α,11β,16α)-6,9-Difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene) bis(oxy)]-pregna-1,4-diene-
1
3,20-dione-21-(2′-methoxybutyrate) (FA-21-MB). White solid, melting point 224–225 °C. H-NMR:
0.95 (3H, d, J = 7 Hz, H-18), 1.02 (3H, dt, J = 15 Hz, J = 5 Hz, H-4’), 1.23 (3H, s, H-23), 1.44 (3H, s,
H-24), 1.54 (3H, s, H-19), 1.6–1.97 (6H, m, H-7α, H-12β, H-15, H-3’), 2.17–2.56 (4H, m, H-7β, H-8,
H-12α, H-14), 3.45 (3H, d, J = 3 Hz, H-1’’), 3.83-3.88 (1H, m, H-2’), 4.43 (1H, t, J = 3 Hz, H-11),
4.91–5.07 (3H, m, H-16, H-21), 5.32–5.46 (1H, dq, J = 50 Hz, J = 4 Hz, H-6), 6.37 (1H, dd, J = 10,
J = 1.5 Hz, H-2), 6.44 (1H, s, H-4), 7.14 (1H, d, J = 10 Hz, H-1). 13C-NMR: 9.3, 16.3, 23.0, 25.8, 26.1,
26.5, 30.9, 31.8, 32.0, 33.6, 34.0, 37.0, 42.7, 45.6, 47.9, 48.1, 58.1, 67.8, 71.5, 71.8, 81.4, 81.8, 85.6,
87.1, 97.4, 98.0, 99.4, 111.7, 121.1, 121.2, 130.2, 150.6, 161.2, 161.3, 172.6, 185.5, 203.2. IR (ATR)

Molecules 2011, 16
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857.3, 896.5, 996.3, 1,067.2, 1,131.2, 1,262.8, 1,243.6, 1,374.4, 1,456.1, 1,627.1, 1,665.0, 1,727.3,
2,938.4, 3,336.4 cm-1. MS [M+H]+ calculated for C33H39F2O8 = 553.26075; observed = 553.26135.
(6α,11β,16α)-6,9-Difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene) bis(oxy)]-pregna-1,4-diene-
3,20-dione-21-(2′-phenoxypropionate) (FA-21-PhP). White solid, melting point 222–224 °C.
1H-NMR: 0.89 (3H, d, J = 24.5 Hz, H-18), 1.22 (3H, s, H-23), 1.43 (3H, s, 24H), 1.50 (3H, d,
J = 4 Hz, H-19), 1.59–1.80 (7H, m, H-7α, H-12β, H15, H-3’), 1.99 (1H, bs, OH), 2.13–2.50 (4H, m,
H-7β, H-8, H-12α, H-14), 4.37 (1H, bs, H-11), 4.88 (1H, q, J = 3 Hz, H-2’), 4.93–5.02 (3H, m, H-16,
H-21), 5.37 (1H, qd, J = 48.7 Hz, J = 3 Hz, H-6), 6.35 (1H, d, J = 10 Hz, H-2), 6.42 (1H, s, H-4), 6.92
(2H, m, H-2’’, H-6’’), 6.99 (1H, m, H-4’’), 7.09 (1H, dd, J = 10 Hz, J = 1.5 Hz, H-1), 7.29 (2H, t,
J = 8 Hz, H-3’’, H-5’’). 13C-NMR: 16.3, 18.7, 23.0, 25.8, 26.5, 29.7, 31.8, 33.6, 33.8, 34.0, 37.03,
42.7, 45.6, 47.9, 68.0, 71.6, 72.1, 81.8, 85.6, 87.1, 97.3, 97.9, 99.3, 111.8, 115.0, 115.1, 121.2, 121.7,
129.6, 130.2, 150.4, 161.2, 172.2, 185.5, 203.0, IR (ATR) 693.3, 858.9, 900.6, 936.3, 969.1, 999.9,
1,067.5, 1,132.8, 1,186.8, 1,232.8, 1,297.5, 1,373.5, 1,385.8, 1,447.3, 1,496.0, 1,603.4, 1,665.3,
1,729.0, 1,765.9, 2,946.0, 3,302.0 cm-1. MS [M+H]+ calculated for C33H39F2O8 = 601.26075;
observed = 601.25983.
3.3. Solvolytic process evaluation method
HPLC analysis were performed on a HP 1100 (Agilent Technologies, Palo Alto, CA, USA) system
equipped with binary pump, Rheodyne injector (sample loop 20 μL), 1100 UV detector, controlled by
IBM PC Pentium Vectra XA computer. Analyses were made on Zorbax Extend-C18 column
(150 mm × 4.6 mm, i.d., 3.5 μm) Agilent, USA, using isocratic elution with methanol-water (75:25
v/v) at a flow rate of 1 mL/min. The experiments were conducted at 25 °C. The data were collected
and processed using the Chem Station software package. Solvolysis sample mixtures were stirred with
a Tehtnica Rotamix SHP-10 (Zelezniki, Slovenia) magnetic stirrer.
Stock aqueous solution of sodium hydrogen carbonate was prepared by dissolving NaHCO3
(280 mg) in a 50 mL volumetric flask (0.067 M).
Preparation of FA stock solution (for calibration curve) – accurately weighted FA (8 mg) was
quantitatively transferred into a 5 mL volumetric flask with ethanol (approximate volume 3 mL) for
complete dissolution and the flask was filled up to mark with ethanol (solution A). Aliquots of
0.1, 0.4, 0.7, 1 and 1.3 mL of solution A were transferred separately into five 10 mL volumetric flasks
followed by ethanol (6 mL). NaHCO3 stock aqueous solution (1 mL) was added and each flask was
filled up to mark with ethanol.
Preparation of solvolysis sample mixtures – accurately weighted FA-21-Ac (16 mg), FA-21-MB
(18.5 mg), FA-21-MP (17.7 mg), FA-21-EB (18.35 mg), FA-21-EP (18 mg) and FA-21-PhP (20 mg)
were quantitatively transferred into separate 100 mL volumetric flasks with ethanol (approximately
volume 60 mL) for complete dissolution, NaHCO3 stock aqueous solution (10 mL) was added and the
flasks were filled up to mark with ethanol. The prepared mixtures were immediately vigorously stirred
(200 rpm).
For HPLC analysis aliquots of each solvolysis sample mixture (20 μL) were withdrawn and injected
at time intervals 0, 2, 4, 6, 8 and 24 h. Chromatograms were recorded immediately using the UV

Molecules 2011, 16
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detector set at 238 nm. The solvolysis percentages were calculated via the obtained peak areas
corresponding to FA contents in the solvolytic mixtures after 0, 2, 4, 6, 8 and 24 h.
3.3. Computational detail
Initially, molecular mechanic optimizations of corticosteroid C-21 esters’ structures using MM2
algorithm have been carried out using Chem3D Ultra 7.0.0 software (Cambridge, MA, USA). The
MM2 optimized structures were used for further optimization by semi-empirical AM1 method [18]
implemented in Gaussian W03 [19]. AM1 optimized structures was used for calculation lengths of
C=O1 and C-O2 bonds. Mulliken charges on carbonyl carbon (C) and both oxygen atoms of ester
moiety (O1 and O2) were calculated using AM1 method. In order to find the most accurate model the
ab initio Hartree-Fock (HF) [20] and Density Functional Theory (DFT) calculations with the hybrid
exchange functional of Becke [21,22] and the Lee, Yang, and Parr [23] correlation functional (B3LYP)
have been performed. In these calculation different basis sets (STO-3G and 6-31G) were used. To
assess the influence of solvatation effects these calculations have been carried out in the gas phase and
in the solvent - ethanol (the conductor-like screening model COSMO and the conductor-like
polarizable continuum model CPCM). Steric hindrance is calculated in MarvinSketch 5.1.3
(ChemAxon Ltd., Budapest, Hungary). Lipophilicity (cLogP) has been calculated using ChemDraw
Ultra 7.0.0 software (Cambridge, MA, USA).
3.4. Determination of anti-inflammatory activity in the test of inhibition of croton oil induced mice ear
edema
Topical anti-inflamatory activities of fluocinolone acetonide C-21 esters have been determined by
measuring inhibition of croton oil induced mice ear edema. Animals have been divided in groups of
five mice. Croton oil solution in acetone (35 μg/mL) has been applied topically to the inner aspect of
the right ear in a dose of 20 μL using a Hamilton syringe. Acetone (20 μL) has been applied in the left
ear. Solutions of test compounds in acetone (28 μM) have been applied only to the right ear in doses of
20 μL, 30 minutes after croton oil application. The control group only received the solution of croton
oil in the right ear, but no solution of corticosteroid ester. Four hours later, animals were sacrificed
with diethyl ether and 6 mm diameters disks were cut from the middle part of ears. Anti-inflammatory
activities were expressed as percentages of mass reduction of edema. The results have been recently
published [3].
4. Conclusions
Corticosteroid esters, such as fluocinolone acetonide 21-esters, are excellent prodrug candidates to
improve the parent drug’s physical and pharmaceutical properties. The solvolysis of new synthesized
FA-21-esters was analyzed using rapid, simple and sensitive HPLC method. Solvolytic rate constants
are found to be in good correlation with polarity of C-O2 bond (PC-O2) indicating that choice of acid
used for prodrug synthesis affects ester stability and pharmacological action. This study shows that this
in vitro solvolytic model obtained by multiple regression analysis between solvolytic rate constant and

Molecules 2011, 16
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lipophilicity with anti-inflammatory activity, could be used as a model for prediction of prodrug
activation.
Acknowledgements
This work was partially supported by the Ministry of Science and Technological Development,
Belgrade, Serbia, as part of Project No.172041.
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Sample Availability: Samples of the compounds are available from the authors.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).