Synthesis of Nitro Esters of Prednisolone, New Compounds Combining Pharmacological Properties of Both Glucocorticoids and Nitric Oxide

Article abstract of DOI:10.1021/jm030964b

Glucocorticoids (GC) are widely used in therapy for their many pharmacological properties including antiinflammatory and immunosuppressive actions. However, their use over long periods is hampered by a number of severe side effects. Given the biological properties of nitric oxide (NO) and previous experience with nonsteroidal antiinflammatory agents, we synthesized new chemical entities combining both NO and GC properties. Here we report the synthesis of nitro esters of prednisolone obtained through the esterification, with different linkers, on the hydroxy group at C-21 position of the corticosteroid structure. The alkyl chain, as of the nitrooxy derivative (2), or aromatic linkers, as of o-, m-, and p-nitrooxymethylbenzoate derivatives (3-5), respectively, furnish stable compounds that release NO and inhibit the GC receptors in biological assays. To improve solubility we introduced a more water-soluble linker such as the nitrooxyalkylpiperidine or -piperazine group (6-9). Also these compounds retained properties of both NO and prednisolone. Compound 5 (NCX 1015) was selected for its better profile: enhanced antiinflammatory properties and reduced side effects compared with prednisolone. NCX 1015 is currently under preclinical development.

Full text of DOI:10.1021/jm030964b

J . Med. Chem. 2004, 47, 711-719
711
Syn th esis of Nitr o Ester s of P r ed n isolon e, New Com p ou n d s Com bin in g
P h a r m a cologica l P r op er ties of Both Glu cocor ticoid s a n d Nitr ic Oxid e
Pier Giovanni Baraldi,*,† Romeo Romagnoli,† Maria del Carmen Nun˜ez,† Mauro Perretti,§ Mark J . Paul-Clark,§
Massimiliano Ferrario,‡ Mirco Govoni,‡ Francesca Benedini,‡ and Ennio Ongini‡
Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara, 44100 Ferrara, Italy, Nicox Research Institute,
Via Ariosto 21, 20091 Bresso, Milan, Italy, and The William Harvey Research Institute,
Pharmacology Division of St. Bartholomew’s & The Royal London School of Medicine and Dentistry,
Charterhouse Square, London EC1M 6BQ, United Kingdom
Received J uly 21, 2003
Glucocorticoids (GC) are widely used in therapy for their many pharmacological properties
including antiinflammatory and immunosuppressive actions. However, their use over long
periods is hampered by a number of severe side effects. Given the biological properties of nitric
oxide (NO) and previous experience with nonsteroidal antiinflammatory agents, we synthesized
new chemical entities combining both NO and GC properties. Here we report the synthesis of
nitro esters of prednisolone obtained through the esterification, with different linkers, on the
hydroxy group at C-21 position of the corticosteroid structure. The alkyl chain, as of the nitrooxy
derivative (2), or aromatic linkers, as of o-, m-, and p-nitrooxymethylbenzoate derivatives (3-
5), respectively, furnish stable compounds that release NO and inhibit the GC receptors in
biological assays. To improve solubility we introduced a more water-soluble linker such as the
nitrooxyalkylpiperidine or -piperazine group (6-9). Also these compounds retained properties
of both NO and prednisolone. Compound 5 (NCX 1015) was selected for its better profile:
enhanced antiinflammatory properties and reduced side effects compared with prednisolone.
NCX 1015 is currently under preclinical development.
In tr od u ction
of dissociation between transactivation and trans-
repression, for example, strong transrepression/low
transactivation. Recent reviews illustrate the ongoing
effort toward the realization of more selective gluco-
corticoid-related therapeutic agents.2,4
Glucocorticoids (GC) have been used successfully for
more than 50 years for their potent antiinflammatory
and immunosuppressive action. Use of GC in therapy
is limited by their side effects, which depend on the dose
used and duration of treatment. The continued use of
GC leads to an array of unwanted effects ranging from
alteration of the endocrine homeostasis to a number of
complications such as hypertension, hyperglycemia,
fluid and electrolyte abnormalities, osteoporosis, gastro-
intestinal damage, increased susceptibility to infection,
etc.1,2
Based on the knowledge of the mechanism of action
of the GC, a medicinal chemistry effort was done in
order to identify new compounds possessing potent
antiinflammatory properties and reduced complications.
Specifically, GC bind to a specific member of the nuclear
receptor superfamily, the glucocorticoid receptor (GR),
which has been cloned and more recently the crystal
structure has been determined.3 Upon ligand binding,
the GR translocates to the nucleus where it interacts
with specific DNA sequences. The net result is a
regulation of gene transcription, through either activa-
tion or repression mechanisms. Currently, it is believed
a better drug profile can be achieved through a degree
Prompted by the successful results with nitric oxide-
(NO-) releasing nonsteroidal antiinflammatory agents
(NSAIDs),5-7 we identified a strong rationale to apply
the same synthetic approach to glucocorticoids. More
than a decade of intense research has demonstrated that
NO exerts antiinflammatory effects, has cytoprotective
actions on gastric mucosa, and is relevant to bone
metabolism, to name a few of its biological actions.8,9
Thus, we introduced a NO-releasing moiety into the GC
structure so as to have new chemical entities that could
release NO slowly over time, the first step being the
ester bond cleavage. Structural changes were introduced
in position 21 of the GC so as to avoid interference with
molecular recognition of the GC.
Here, we describe a group of NO-releasing derivatives
of prednisolone (1) that appear of interest as a new class
of improved GC-related agents.
On the basis of previous experience with NO-releasing
NSAIDs, we started with the introduction of the nitro-
oxybutyryl (2) and the isomers of nitrooxymethylbenzoyl
groups (3-5) into the 21 position of prednisolone. The
compounds displayed inhibitory activity on GR, there-
fore showing that they retain GC-related effects. As a
further step, with the aim to improve water solubility,
we synthesized the compounds 6-9 by linking the
nitrooxy (-ONO2) moiety to C-21 of prednisolone through
heterocycles such as piperidine (6 and 7) and piperazine
(8 and 9). To determine the effect of the nitrooxymethyl
* To whom correspondence should be addressed at the Dipartimento
di Scienze Farmaceutiche, Universita` di Ferrara, Via Fossato di
Mortara 17-19, 44100 Ferrara, Italy. Tel +39-0532-291293; fax +39-
0532-291296; e-mail pgb@ifeuniv.unife.it.
† Universita` di Ferrara.
‡ Nicox Research Institute.
§ The William Harvey Research Institute, Pharmacology Division
of St. Bartholomew’s & The Royal London School of Medicine and
Dentistry.
10.1021/jm030964b CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/20/2003

712 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Baraldi et al.
Sch em e 2a
a
Reagents: (a) corresponding chloromethylbenzoyl chloride,
Et3N, THF, rt, 18 h; (b) AgNO3, CH3CN/THF, reflux, 4 h; (c)
4-hydroxymethylbenzoyl chloride, Et3N, THF, rt, 1 h.
base, furnished the corresponding prednisolone 21-
chloromethylbenzoate derivatives 12, 13, and 14, re-
spectively. These three chloro derivatives were con-
verted into the target nitro ester compounds 3, 4, and
5 by treatment with silver nitrate in a refluxing mixture
of acetonitrile/tetrahydrofuran as solvent.
Synthesis of compounds 6-9, characterized by the
presence of a heterocyclic ring, was achieved from a
common chloro intermediate (15). The prednisolone (1)
was selectively acylated on its 21-position by chloro-
acetyl chloride in tetrahydrofuran, with triethylamine
as base, to give compound 15. Two different strategies
were employed for the synthesis of piperidyl derivatives
6 and 7. The first approach for the preparation of 6 and
7 is reported in Scheme 3.
F igu r e 1. Structure of the different NO-releasing derivatives
of prednisolone. Compound 5 (NCX 1015) was selected for
further development.
Sch em e 1a
The known 2-[4-(N-Boc-piperidyl)]-1-ethanol 1611 and
3-[4-(N-Boc-piperidyl)]-1-propanol 1712 were trans-
formed into the corresponding chloro derivatives 1811
and 19, respectively, by direct chlorination under stand-
ard conditions (carbon tetrachloride, triphenylphos-
phine). The subsequent removal of the t-Boc protecting
group by anhydrous HCl in ethyl acetate furnished 4-
(2-chloroethyl)piperidine 2011 and 4-(3-chloropropyl)-
piperidine 21 as hydrochloride salts.
a
Reagents: (a) 4-bromobutyryl chloride, Et3N, THF, rt, 4 h; (b)
AgNO3, CH3CN/THF, reflux, 8 h.
These compounds were condensed with 15 to give the
piperidyl derivatives 22 and 23, respectively, which
were submitted to the subsequent reaction with silver
nitrate. While the derivative 7 was obtained in accept-
able yield, although after long time reaction (48 h) and
using an excess of silver nitrate, the same reaction
performed on compound 22 did not yield the desired
product 6 but only the degradation of the starting
material.
Due to the low versatility of this method, which
depends on the reactivity of the chloro alkyl chain joined
to the piperidine ring, an alternative convergent ap-
proach, reported in Scheme 4, was employed for the
synthesis of derivatives 6 and 7. In this way, the
alcohols 16 and 17 were transformed into the corre-
sponding bromo derivatives 2413 and 25,14 respectively,
in good yields. The subsequent SN2 reaction with silver
nitrate in tetrahydrofuran proceeded in a short time
(less than 1 h) to afford the compounds 26 and 27,
respectively. The t-Boc protecting group was removed
moiety in derivative 5, the corresponding hydroxymethyl
derivative 10 has been also prepared (Figure 1).
Ch em istr y
The synthesis of compound 2 is represented in Scheme
1.
The alkyl chain was introduced in the position 21 of
prednisolone (1) by reaction with the commercially
available 4-bromobutyryl chloride. The 21-bromobutyryl
derivative (11) was converted into the final nitro ester
(2) by treatment with silver nitrate (AgNO3) in a
refluxing mixture of acetonitrile/tetrahydrofuran as
solvent.
The derivatives 3, 4, and 5, characterized by the
presence of an aromatic spacer, were prepared following
a two-step procedure reported in Scheme 2.
The condensation of the ortho, meta, and para isomers
of chloromethylbenzoyl chloride10 with prednisolone (1)
in tetrahydrofuran, in the presence of triethylamine as

Synthesis of Nitro Esters of Prednisolone
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 713
Sch em e 3a
a
Reagents: (a) ClCOCH2Cl, Et3N, THF, rt, 18 h; (b) CCl4, PPh3, DCM, rt, 18 h; (c) HCl/AcOEt, 0 °C, 3 h; (d) Et3N, DMF, rt, 24 h; (e)
AgNO3, CH3CN/THF, reflux, 48 h.
Sch em e 4a
a
Reagents: (a) CBr4, PPh3, THF, rt, 18 h; (b) AgNO3, THF, rt, 1 h; (c) HCl/AcOEt, 0 °C, 3 h; (d) Et3N, DMF, rt, 24 h.
Sch em e 5a
with HCl in ethyl acetate to give the corresponding
piperidines 28 and 29 (as hydrochloride salts). Finally,
the condensation of the chloroacetyl prednisolone de-
rivative 15 with 28 and 29 furnished in a good yield
the compounds 6 and 7, respectively, which were
isolated as trifluoroacetic salts after purification by
reversed-phase HPLC and immediate lyophilization.
Also for the preparation of piperazinyl derivatives 8
and 9, two different strategies were employed. In the
first route (Scheme 5), 1-(2-chloroethyl)piperazine 3015
and 1-(3-chloropropyl)piperazine 3116 (as bishydrochlo-
ride salts) were joined to 15 by use of triethylamine in
N,N′-dimethylformamide to furnish the intermediates
32 and 33, respectively. Only for the latter compound
(33) did the reaction with silver nitrate in the standard
conditions furnish the nitro ester (9) in a good yield,
a
Reagents: (a) Et3N, DMF, rt, 24 h; (b) AgNO3, CH3CN/THF,
while for derivative 32 the reaction with silver nitrate
reflux, 48 h.
did not furnish the desired product (8), due to a probable
intramolecular SN2 reaction of the chloroethylamino
moiety, with the generation of a chemically reactive
species (an aziridinium ion) corresponding to compound
34 (Scheme 5).
All the attempts to synthesize 8 under different
reaction conditions failed; thus a new synthetic route,
depicted in Scheme 6, was developed for the synthesis
of 8, along with the corresponding homologue 9. The
bromo derivative 37 was prepared by direct bromination
from the corresponding alcohol 3517 under standard
conditions (carbon tetrabromide/triphenylphosphine with
tetrahydrofuran as solvent), while 38 was synthesized
by the reaction of N-Boc-piperazine 3618 with an equi-
molar amount of 1,3-dibromopropane. These bromoalkyl-
N-Boc-piperazines 37 and 38 were subsequently trans-

714 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Baraldi et al.
Sch em e 6a
Ta ble 1. Effect of Prednisolone, Compounds 1-10 on
Glucocorticoid Receptor Binding with [3H]Dexamethasone as
Ligand in Human Blood Mononuclear Cells
% displacement of
compound
(at 1 µM)
[3H]dexamethasone
bounda (fmol/mL)
[3H]dexamethasone
specific bindinga
control
1
2
3
4
5
6
7
12.4 ( 1.5
5.5 ( 2.3
1.6 ( 0.8
8.7 ( 0.6
19.8 ( 4.2
6.6 ( 1.4
1.7 ( 0.4
2.3 ( 1.2
4.2 ( 0.8
2.1 ( 1.0
5.3 ( 1.7
56
87
30
47
86
81
66
83
57
8
9
10
a
Binding data are expressed as mean ( SEM of n ) 3.
a
Reagents: (a) CBr4, PPh3, THF, rt, 18 h; (b) 1,3-dibromopro-
pane, DCM, Et3N, rt, 18 h; (c) AgNO3, THF, rt, 2 h; (d) HCl/AcOEt,
0 °C, 4 h; (e) Et3N, DMF, rt, 24 h.
formed into the corresponding nitro esters 39 and 40,
respectively, after treatment with an excess of silver
nitrate in tetrahydrofuran at room temperature. In
these latter compounds, the t-Boc protecting group was
removed with anhydrous HCl in ethyl acetate to give
41 and 42. These nitrooxy derivatives were condensed
with 15 to yield 8 and 9, respectively, isolated as
trifluoroacetic salts after purification by reversed-phase
HPLC.
To determine the effect of nitrooxy moiety, we synthe-
sized the hydroxy derivative 10 (Scheme 2). Predniso-
lone (1) was condensed with the commercially available
4-hydroxymethylbenzoyl chloride in the presence of
triethylamine to give the target compound 10.
F igu r e 2. Kinetics of nitrosylhemoglobin formation in whole
rat blood following treatment with S-nitroso-N-acetylpenicill-
amine (SNAP) (O) and (5, NCX 1015) (9). SNAP induced NO
release rapidly with HbFe(II)NO formation, which was stable
over 4 h, 9.1 ( 0.6 µM at 15 min and 8.7 ( 4.3 µM at 4 h.
HbFe(II)NO formation from 5 was slow and ranged from 0.4
( 0.3 µM at 15 min to 28.2 ( 6.6 µM at 4 h. Values are mean
( SEM of three independent experiments.
Resu lts
selected for further studies, clearly shows that NO is
released slowly (Figure 2). The example of Figure 2
shows that a reference NO donor such as SNAP led to
the formation of the bioactive NO form, detected as the
complex NO-hemoglobin [HbFe(II)NO], which occurred
rapidly (15 min) and was stable over 4 h (9.1 ( 0.6 µM
at 15 min and 8.7 ( 4.3 µM at 4 h). Compound 5
released bioactive NO with a time-dependent formation
of the paramagnetic adduct HbFe(II)NO, and the time
course of HbFe(II)NO formation ranged from 0.4 ( 0.3
µM after 15 min of incubation to 28.2 ( 6.6 µM at 4 h.
Introduction of a lipophilic ester moiety into position
21 led, however, to the decrease of solubility. Thus, a
further step was made by using spacers having different
solubility properties. To achieve this we replaced the
phenyl ring with piperidine or piperazine rings (com-
pounds 6-9). Compounds 6-9 showed good water
solubility as salts with trifluoroacetic acid. The substi-
tution of the phenyl (5, NCX-1015) with a piperidine (6
and 7) increased the solubility by 2 orders of magnitude,
while the presence of a piperazine furnished more
soluble derivatives (8 and 9), which are 104-fold more
soluble than 5. The buffer solubilities of NCX-1015 (5)
and piperine/piperazine derivatives (6-9) were deter-
mined by the protocol reported by Casini et al.21 (Table
2).
The compounds synthesized (2-10) were stable either
in buffer solution or in the standard vehicle used in the
assays, whereas in the biological environment such as
blood or cell cultures they were subject to cleavage of
the ester bond. Binding affinity to the human GR was
determined by the displacement of 50 nM [3H]dexa-
methasone, a reference GR agonist, by a 1 µM concen-
tration of test compounds 2-10 and compared with that
of prednisolone (1). The results are shown in Table 1.
Thus, in the peripheral blood mononuclear cells
(PBMC) assay, compounds 2, 3, and 5-10 displaced GR
binding. The percent displaced of [3H]dexamethasone
binding ranged from 30% to 87%. The data show that
all compounds retained the capacity to interact with the
recognition site of GR. Differences in potency may well
depend on the time to dissociate the prednisolone part
from the nitro ester moiety. GR binding inhibition
appeared to be independent of the presence of the
nitrate group as shown by comparing compound 5 with
10, the latter being the denitrated form of 5 (Figure 1,
Table 1). Previous data on NSAIDs19,20 showed that one
key feature of nitro esters linked to the drug through
the butyryl or benzoyl moiety is the release of NO
slowly, whereas classic nitrovasodilators release NO
more rapidly. Analysis of compound 5, which was

Synthesis of Nitro Esters of Prednisolone
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 715
Ta ble 2. Solubility Data of Prednisolone and Selected
NO-Releasing Derivatives
than prednisolone while it displayed lesser liability to
induce side effects such as hypertension, gastrointesti-
nal damage, and bone metabolism complications. This
achievement provided a stimulus to extend the approach
to other compounds, for example, dexamethasone or
budesonide, whose activity profile is being character-
ized. Overall, NO-releasing GC derivatives offer a new
opportunity to improve significantly the therapeutic
benefits of the GC class of drugs.
compound
solubilitya (mM)
1
0.27
0.007
0.71
0.15
21.3
53.3
5 (NCX 1015)
6
7
8
9
a
In pH 7.4 buffer, at 25 °C.
Ta ble 3. Antiinflammatory Properties of Prednisolone and
Exp er im en ta l Section
Selected NO-Releasing Derivativesa
Gen er a l Meth od s. All reactions were carried out under
argon atmosphere, unless otherwise described. Standard
syringe techniques were applied for transferring anhydrous
solvents. Reaction courses and product mixtures were routinely
monitored by thin-layer chromatography on silica gel (pre-
coated F254 Merck plates); the spots were examined with UV
light and visualized with aqueous KMnO4. 1H NMR spectra
were recorded in the given solvent with a Bruker AC 200
spectrometer. Chemical shifts are reported as δ values in parts
per million. The splitting pattern abbreviations are as fol-
lows: s (singlet), d (doublet), dd (double doublet), t (triplet),
br (broad), and m (multiplet). Melting points (mp) were
determined on a Buchi-Tottoli apparatus and are uncorrected.
All products reported showed 1H NMR spectra in agreement
with the assigned structures. Column chromatography were
carried out on Merck silica gel (230-240 mesh). All compounds
obtained commercially were used without further purification.
Organic solutions were dried over anhydrous MgSO4. Methanol
was distilled from magnesium turnings, dioxane was distilled
from calcium hydride, and anhydrous DMF was distilled from
calcium chloride and stored over molecular sieves (3 Å). HPLC
separations were conducted with a Waters Delta Prep 3000 A
reversed-phase column (30 × 3 cm; 15 mm). The compounds
were eluted with a gradient of 0-60% B in 25 min at a flow
rate of 30 mL/min; the mobile phases were solvent A [10% (v/
v) acetonitrile in 0.1% TFA] and solvent B [60% (v/v) aceto-
nitrile in 0.1% TFA]. Analytical HPLC analyses were per-
formed on a Bruker liquid chromatography (LC) 21-C instru-
ment with a Vydac 218 TP 5415 C18 column (250 × 4 mm, 5
mm particle size) and equipped with a Bruker LC 313 UV
variable-wavelength detector. Recording and quantification
were accomplished with a chromatographic data processor
coupled to an Epson computer system (QX-10).
P r ed n isolon e 21-[4′-(Br om o)bu tyr a te] (11). To a solution
of prednisolone (1) (2.5 g, 7 mmol) in tetrahydrofuran (150 mL)
were added triethylamine (3.9 mL, 28 mmol) and 4-bromo-
butyryl chloride (3.25 mL, 28 mmol). The reaction was stirred
for 4 h at room temperature and the solvent was evaporated
under vacuum. The residue was dissolved in ethyl acetate and
water. The two phases were separated and the organic phase
was washed with brine (5 mL) and dried (Na2SO4) and the
solvent was removed under reduced pressure. The crude
product was purified by crystallization with n-hexane. Yield
82%; white solid, mp ) 178-180 °C; 1H NMR (DMSO-d6) δ
0.85 (s, 3H), 1.45 (s, 3H), 0.90-1.15 (m, 2H), 1.30-1.60 (m,
2H), 1.60-1.80 (m, 3H), 1.80-2.20 (m, 5H), 2.35 (m, 1H), 2.50-
2.63 (m, 4H), 3.35 (t, 2H), 4.35 (s, 1H), 4.76 (d, J ) 3.7 Hz,
1H), 4.83 (d, J ) 17.5 Hz, 1H), 5.15 (d, J ) 17.5 Hz, 1H), 5.46
(s, 1H), 5.97 (s, 1H), 6.22 (dd, J ) 10.1 and 1.6 Hz), 7.38 (d, J
) 10.1 Hz, 1H).
treatmentb
PMN per mouse (106)
% inhibition
vehicle control
prednisolone
16.4 ( 1.2
9.1 ( 0.6c
9.1 ( 1.0c
4.1 ( 0.5c,d
9.0 ( 0.7c
7.8 ( 0.9c
45
45
75
46
52
2
5 (NCX-1015)
8
9
a
As measured on the inhibition of neutrophil (PMN) extrava-
sation in the zymosan-induced peritonitis mouse model. Equi-
b
molar doses of drugs (13.8 µmol/kg) or vehicle (0.25 mL) were given
i.p. 1 h prior to zymosan (1 mg/peritoneal cavity). Neutrophil
(PMN) influx into the cavities was measured at the 4 h time point.
Data are mean ( SEM of n ) 5 mice per group. c P < 0.05 vs
vehicle control (one-way analysis of variance plus Bonferroni post-
d
hoc analysis). P < 0.05 vs prednisolone (one-way analysis of
variance plus Bonferroni post-hoc analysis).
The compounds were also effective in vivo and dis-
played potent antiinflammatory properties. For in-
stance, when tested on the intense recruitment of
neutrophils provoked by intraperitoneal zymosan injec-
tion, all derivatives were able to produce significant
inhibitory effects (Table 3). While prednisolone and
compounds 2, 8, and 9 produced approximately 40-50%
inhibition, compound 5 was significantly more effective,
producing >70% inhibition of white blood cell recruit-
ment (Table 3).
On the basis of different criteria ranging from in vitro
to in vivo properties, compound 5, NCX 1015, was
selected for further investigation. In addition, this
prednisolone derivative displayed higher potency than
prednisolone in animal models of acute and chronic
inflammation.22,23 Interestingly, initial data showed that
NCX 1015 can spare the bone compartment, possibly
through the release of NO, thereby suggesting that
treatment with this new class of compounds can be
associated with a reduced risk of secondary osteoporo-
sis.24 Unlike prednisolone, prolonged NCX 1015 treat-
ment did not appear to cause hypertension in normo-
tensive animals (Dr. M. D’Amico, unpublished data) or
gastrointestinal damage.25 Other studies are ongoing.
Interestingly, like prednisolone, NCX 1015 was effective
when given orally.22 Overall, the data with the lead
compound (5) support the concept that NO-releasing
glucocorticoids possess properties that make them of
interest for their wider antiinflammatory properties and
reduced side effects.
P r ed n isolon e 21-[4′-(Nitr ooxy)bu tyr a te] (2). To a solu-
tion of compound 11 (2.8 g, 5.5 mmol) dissolved in acetonitrile/
tetrahydrofuran (150 mL, 4/1 v/v) was added silver nitrate
(1.87 g, 11 mmol). The mixture was stirred at reflux in the
dark for 8 h. The precipitate was filtered off, the solvent was
evaporated under vacuum, and the residue was dissolved in
ethyl ether and water. The organic phase was washed with
water and brine, dried (Na2SO4), and evaporated under
reduced pressure. The crude product was purified by silica gel
chromatography with ethyl acetate/n-hexane (6/5 v/v) as
eluent. Yield 41%; white solid, mp ) 80-85 °C; 1H NMR
Con clu sion
The attempts made to improve the overall profile of
GC have led to the synthesis of a series of prednisolone
derivatives that combine the biological properties of both
GC and NO. Interestingly, one lead compound (5, NCX
1015) has shown greater antiinflammatory properties

716 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Baraldi et al.
(DMSO-d6) δ 0.82 (s, 3H), 1.42 (s, 3H), 0.90-1.15 (m, 2H),
1.30-1.55 (m, 2H), 1.60-1.80 (m, 3H), 1.90-2.15 (m, 5H), 2.35
(m, 1H), 2.48-2.65 (m, 4H), 4.29 (s, 1H), 4.58 (t, J ) 6.4 Hz,
2H), 4.74 (d, J ) 3.8 Hz, 1H), 4.77 (d, J ) 17.5 Hz, 1H), 5.11
(d, J ) 17.5 Hz, 1H), 5.42 (s, 1H), 5.91 (s, 1H), 6.16 (dd, J )
10.1 and 1.6 Hz, 1H), 7.32(d, J ) 10.1 Hz, 1H). Anal. Calcd
for (C25H33NO9): C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
12-14. A solution of 1 (721 mg, 2 mmol) in tetrahydrofuran
(5 mL) was added to a mixture of chloromethylbenzoyl chloride
(756 mg, 4 mmol) and triethylamine (0.56 mL, 4 mmol) cooled
with an ice bath. The reaction was stirred for 18 h at room
temperature and the solvent was evaporated under vacuum.
The residue was dissolved in ethyl acetate (20 mL) and water
(5 mL). The two phases were separated and the organic phase
was washed with brine (5 mL) and dried (Na2SO4), and the
solvent was removed under reduced pressure. The crude
product was purified to furnish the derivatives 12-14, used
for the next step.
P r ed n isolon e 21-[2′-(Ch lor om eth yl)ben zoa te] (12). This
compound was purified by flash chromatography with ethyl
acetate/petroleum ether (6/4 v/v) as eluent. Yield 78%; yellow
solid, mp ) 88-90 °C; 1H NMR (DMSO-d6) δ 0.80 (s, 3H), 0.89
(m, 1H), 1.02 (m, 1H), 1.35 (m, 2H), 1.35 (s, 3H), 1.66 (m, 3H),
2.02 (m, 3H), 2.29 (m, 1H), 2.44 (m, 2H), 4.28 (s, 1H), 4.55 (s,
2H), 4.70 (d, J ) 3.6 Hz, 1H), 4.86 (d, J ) 17.2 Hz, 1H), 5.14
(d, J ) 17.2 Hz, 1H), 5.47 (s, 1H), 5.92 (s, 1H), 6.18 (dd, J )
10.0 and 1.2 Hz, 1H), 7.30 (d, J ) 10.0 Hz, 1H), 7.43 (m, 4H).
P r ed n isolon e 21-[3′-(Ch lor om eth yl)ben zoa te] (13). This
compound was purified by crystallization with n-hexane. Yield
82%; white solid, mp ) 240-245 °C; 1H NMR (DMSO-d6) δ
0.83 (s, 3H), 1.41 (s, 3H), 0.85-1.15 (m, 2H), 1.25-1.60 (m,
2H), 1.60-1.80 (m, 3H), 1.80-2.10 (m, 3H), 2.29 (m, 1H), 2.48-
2.56 (m, 2H), 4.33 (m, 1H), 4.77 (d, J ) 3.8 Hz, 1H), 4.89 (s,
2H), 5.05 (d, J ) 17.5 Hz, 1H), 5.36 (d, J ) 17.5 Hz, 1H), 5.51
(s, 1H), 5.93 (s, 1H), 6.18 (dd, J ) 10.1 and 1.6 Hz, 1H), 7.35
(d, J ) 10.1 Hz, 1H), 7.58 (t, J ) 7.7 Hz, 1H), 7.77 (d, J ) 7.7
Hz, 1H), 7.97 (d, J ) 7.7 Hz, 1H), 8.08 (s, 1H).
2.56 (m, 2H), 4.31 (m, 1H), 4.76 (d, J ) 3.8 Hz, 1H), 5.08 (d, J
) 17.5 Hz, 1H), 5.31 (d, J ) 17.5 Hz, 1H), 5.50 (s, 1H), 5.67 (s,
2H), 5.92 (s, 1H), 6.18 (dd, J ) 10.1 and 1.6 Hz, 1H), 7.33 (d,
J ) 10.1 Hz, 1H), 7.66 (t, J ) 7.6 Hz, 1H), 7.77 (d, J ) 7.9 Hz,
1H), 8.04 (d, J ) 7.9 Hz, 1H), 8.11 (s, 1H). Anal. Calcd for
(C29H33NO9): C, H, N.
P r edn isolon e 21-[4′-(Nitr ooxym eth yl)ben zoate] (5). This
compound was purified by crystallization with tetrahydro-
furan/n-hexane. Yield 54%; white solid, mp ) 233-234 °C; 1H
NMR (DMSO-d6) δ 0.84 (s, 3H), 0.85-1.15 (m, 2H), 1.41 (s,
3H), 1.20-1.60 (m, 2H), 1.60-1.80 (m, 3H), 1.80-2.10 (m, 3H),
2.29 (m, 1H), 2.50-2.55 (m, 2H), 4.33 (s, 1H), 4.76 (s, 1H), 5.04
(d, J ) 17.7 Hz, 1H), 5.35 (d, J ) 17.7 Hz, 1H), 5.48 (s, 1H),
5.69 (s, 2H), 5.92 (s, 1H), 6.17 (d, J ) 10.1 Hz, 1H), 7.34 (d, J
) 10.1 Hz, 1H), 7.64 (d, J ) 8.3 Hz, 2H), 8.04 (d, J ) 8.3 Hz,
2H). Anal. Calcd for (C29H33NO9): C, H, N.
P r ed n isolon e 21-Ch lor oa ceta te (15). To a solution of
prednisolone (1) (1 mmol, 360 mg) in tetrahydrofuran (10 mL)
was added triethylamine (1.1 mmol, 153 µL). The mixture was
cooled at 0 °C, and chloroacetyl chloride (1.1 mmol, 87 µL) was
added dropwise. The mixture was stirred for 18 h at room
temperature (TLC ethyl acetate/petroleum ether 7/3), diluted
with ethyl acetate (10 mL), and then washed with water (5
mL) and brine (5 mL). The separated organic phase was dried
(Na2SO4) and concentrated under reduced pressure, and the
residue was precipitated with petroleum ether (10 mL). After
filtration, the final product (15) (420 mg, 95% yield) was
obtained as a pale yellow solid. This product was used without
1
any purification for the next reaction: mp ) 245-247 °C, H
NMR (DMSO-d6) δ 0.79 (s, 3H), 0.89 (m, 1H), 0.99 (m, 1H),
1.33 (m, 2H), 1.38 (s, 3H), 1.66 (m, 3H), 2.01 (m, 3H), 2.29 (m,
1H), 2.49 (m, 4H), 4.28 (s, 1H), 4.74 (d, J ) 3.8 Hz, 1H), 4.83
(d, J ) 17.4 Hz, 1H), 5.18 (d, J ) 17.4 Hz, 1H), 5.47 (s, 1H),
5.92 (s, 1H), 6.15 (dd, J ) 10.0 and 1.2 Hz, 1H), 7.32 (d, J )
10.0 Hz, 1H).
N-Boc-4-(3-ch lor op r op yl)p ip er id in e (19). A solution of
(17) (729 mg, 3 mmol) and triphenylphosphine (2.36 g, 9 mmol)
in dry dichloromethane (20 mL) at room temperature under
argon was treated with freshly distilled carbon tetrachloride
(0.45 mL, 4.5 mmol). After 18 h, the mixture was filtered, and
the filtrate was washed with water (5 mL) and brine (5 mL),
dried (Na2SO4), and concentrated at reduced pressure. The
residue was purified by flash chromatography (ethyl acetate/
petroleum ether 2/8 v/v) to give compound 19 as a yellow oil
(668 mg, yield 85%). 1H NMR (CDCl3) δ 1.13 (m, 2H), 1.38 (m,
2H), 1.45 (s, 9H), 1.55 (m, 3H), 1.76 (m, 2H), 2.68 (t, J ) 11.8
Hz, 2H), 3.53 (t, J ) 8.4 Hz, 2H), 4.06 (br s, 2H).
4-(3-Ch lor op r op yl)p ip er id in e Dih yd r och lor id e (21).
Compound 19 (524 mg, 2 mmol) was dissolved in ethyl acetate
(10 mL) and the solution was saturated with dry hydrogen
chloride at 0 °C. The mixture was stirred for 3 h to complete
the deprotection, and the solvent was evaporated to give a solid
residue that was suspended in ethyl ether (30 mL) and stirred
for 1 h at room temperature. After filtration, compound 21 was
obtained as a crystalline white salt (mp ) 160-161 °C) used
without any purification for the next reaction.
P r ed n isolon e 21-[4-(2-Ch lor oeth yl)p ip er id in -1-yl]a ce-
ta te (22). To a stirred solution of 15 (436 mg, 1 mmol) and 20
(201 mg, 1.1 mmol) in N,N′-dimethylformamide (5 mL) cooled
at 0 °C was added triethylamine (0.3 mL, 2.2 mmol) dropwise.
The mixture was stirred overnight at room temperature, the
solvent was removed under vacuum, and the resulting residue
was dissolved in a mixture of ethyl acetate (10 mL) and water
(5 mL). The organic phase was washed with brine (2 mL), dried
(Na2SO4), and then evaporated. Purification by flash chroma-
tography (dichloromethane/methanol 9/1 v/v) furnished 22 as
a white solid (350 mg, yield 64%); mp ) 195-197 °C; 1H NMR
(DMSO-d6) δ 0.78 (s, 3H), 0.87 (m, 2H), 0.92 (m, 1H), 1.23 (m,
1H), 1.39 (s, 3H), 1.66 (m, 4H), 2.00 (m, 8H), 2.32 (m, 1H),
2.50 (m, 5H), 2.62 (t, J ) 6.8 Hz, 2H), 3.33 (t, J ) 4.6 Hz, 2H),
3.66 (t, J ) 6.8 Hz, 2H), 4.28 (s, 1H), 4.73 (s, 1H), 4.78 (d, J )
13.2 Hz, 1H), 5.08 (d, J ) 13.2 Hz, 1H), 5.43 (s, 1H), 5.92 (s,
1H), 6.18 (dd, J ) 10.4 and 1.4 Hz, 1H), 7.32 (dd, J ) 10.4
and 1.4 Hz, 1H).
P r ed n isolon e 21-[4′-(Ch lor om eth yl)ben zoa te] (14). This
compound was purified by silica gel chromatography with
dichloromethane/acetone (8/2 v/v) as eluent. Yield 97%; white
1
solid, mp ) 242-247 °C; H NMR (DMSO-d6) δ 0.83 (s, 3H),
1.41 (s, 3H), 0.85-1.15 (m, 2H), 1.25-1.60 (m, 2H), 1.60-1.80
(m, 3H), 1.80-2.10 (m, 3H), 2.29 (m, 1H), 2.48-2.55 (m, 2H),
4.33 (s, 1H), 4.76 (d, J ) 3.6 Hz, 1H), 4.78 (s, 2H), 5.04 (d, J
) 17.5 Hz, 1H), 5.35 (d, J ) 17.5 Hz, 1H), 5.50 (s, 1H), 5.93 (s,
1H), 6.19 (dd, J ) 10.1 and 1.7 Hz, 1H), 7.35 (d, J ) 10.1 Hz,
1H), 7.62 (d, J ) 8.2 Hz, 2H), 8.01 (d, J ) 8.2 Hz, 2H).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
3-5. A solution of 12, 13, or 14 (1 mmol) in acetonitrile/
tetrahydrofuran (6 mL, 4/2 v/v) was treated with silver nitrate
(2 mmol, 340 mg) at reflux in the dark for 4 h. The precipitate
was filtered off, the solvent was evaporated under vacuum,
and the residue was dissolved in ethyl acetate (10 mL) and
water (2 mL). The organic phase was washed with water (3 ×
2 mL) and brine (2 mL), dried (Na2SO4), and evaporated under
reduced pressure. The crude product purified furnished com-
pound 3, 4, or 5).
P r edn isolon e 21-[2′-(Nitr ooxym eth yl)ben zoate] (3). This
compound was purified by flash chromatography with ethyl
acetate/petroleum ether (1/1 v/v) as eluent. Yield 67%; amor-
1
phous white solid, mp ) 158-160 °C; H NMR (DMSO-d6) δ
0.80 (s, 3H), 0.89 (m, 1H), 1.02 (m, 1H), 1.35-1.60 (m, 2H),
1.41 (s, 3H), 1.66-1.76 (m, 3H), 1.99-2.08 (m, 3H), 2.29 (m,
1H), 2.50-2.54 (m, 2H), 4.33 (s, 1H), 4.78 (d, 1H), 4.79 (d, J )
17.4 Hz, 1H), 5.35 (d, J ) 17.4 Hz, 1H), 5.50 (s, 1H), 5.92 (m,
3H), 6.19 (d, J ) 10.1 Hz, 1H), 7.35 (d, J ) 10.1 Hz, 1H), 7.62-
7.73 (m, 2H), 7.90 (d, J ) 7.75 Hz, 1H), 8.03 (d, J ) 7.75 Hz,
1H). Anal. Calcd for (C29H33NO9): C, H, N.
P r edn isolon e 21-[3′-(Nitr ooxym eth yl)ben zoate] (4). This
compound was purified by crystallization with n-hexane. Yield
83%; white solid, mp ) 208-216 °C; 1H NMR (DMSO-d6) δ
0.83 (s, 3H), 1.40 (s, 3H), 0.83-1.15 (m, 2H), 1.25-1.60 (m,
2H), 1.60-1.80 (m, 3H), 1.80-2.10 (m, 3H), 2.29 (m, 1H), 2.48-

Synthesis of Nitro Esters of Prednisolone
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 717
P r edn isolon e 21-[4-(3-Ch lor opr opyl)piper idin -1-yl]ace-
ta te (23). By following the same procedure reported for the
synthesis of 22 but with 21 in place of 20, compound 23 was
(s, 1H), 5.92 (s, 1H), 6.17 (d, J ) 10.0 Hz, 1H), 7.34 (d, J )
10.0 Hz, 1H). Anal. Calcd for (C32H43F3N2O11): C, H, N.
P r ed n isolon e 21-[4-(3-Nitr ooxyp r op yl)p ip er id in -1-yl]-
a ceta te Tr iflu or oa cetic Sa lt (7). By following the same
procedure reported for the synthesis of 6 but with 29 in place
of 28, compound 7 was isolated as a white crystalline solid
(yield 45%).
1
obtained as a brown solid (yield 76%), mp ) 170-171 °C. H
NMR (DMSO-d6) δ 0.78 (s, 3H), 0.86 (m, 2H), 1.08 (t, J ) 7.4
Hz, 2H), 1.17 (m, 1H), 1.24 (m, 1H), 1.38 (s, 3H), 1.66 (m, 4H),
1,86 (m, 2H), 1,98 (m, 6H), 2.28 (m, 1H), 2.50 (m, 5H), 2.62 (t,
J ) 6.8 Hz, 2H), 3.39 (t, J ) 4.6 Hz, 2H), 3.67 (t, J ) 6.8 Hz,
2H), 4.28 (s, 1H), 4.73 (s, 1H), 4.77 (d, J ) 13.2 Hz, 1H), 5.08
(d, J ) 13.2 Hz, 1H), 5.46 (s, 1H), 5.92 (s, 1H), 6.15 (dd, J )
10.4 and 1.4 Hz, 1H), 7.33 (d, J ) 10.4 Hz, 1H).
P r ed n isolon e 21-[4-(2-Ch lor oeth yl)p ip er a zin -1-yl]a ce-
ta te (32). To a solution of 15 (436 mg, 1 mmol) and 30 (265
mg, 1.2 mmol) in N,N′-dimethylformamide (5 mL) cooled in
an ice bath was added triethylamine (0.46 mL, 3.3 mmol); the
mixture was stirred overnight and then concentrated. The
residue was taken up in ethyl acetate (15 mL), washed with
water (5 mL) and brine (5 mL), dried (Na2SO4), and concen-
trated. Purification by flash chromatography (dichloromethane/
methanol 9/1 v/v) furnished 32 as a brown solid (423 mg, yield
P r ed n isolon e21-[4-(3-(Nitr ooxy)p r op yl)p ip er id in -1-yl]-
a ceta te Tr iflu or oa cetic Sa lt (7). To a solution of 23 (270
mg, 0.5 mmol) in a mixture of acetonitrile (12 mL) and
tetrahydrofuran (8 mL) was added silver nitrate (255 mg, 3
equiv, 1.5 mmol), and after 48 h at reflux the reaction mixture
was cooled to room temperature, the salts were filtered off,
and the solvent was evaporated under reduced pressure. The
crude product was purified by preparative HPLC and submit-
ted to immediate lyophilization to give 7 as a white crystalline
solid (312 mg, yield 45.4%), mp ) 120-121 °C. 1H NMR
(DMSO-d6) δ 0.80 (s, 3H), 0.91 (m, 1H), 1.23 (m, 3H), 1.39 (s,
3H), 1.66 (m, 8H), 1.97 (m, 7H), 2.34 (m, 1H), 2.49 (m, 4H),
3.49 (m, 3H), 3.51 (d, J ) 7 Hz, 2H), 4.28 (m, 1H), 4.51 (t, J )
6.4 Hz, 2H), 4.75 (s, 1H), 4.81 (d, J ) 17.4 Hz, 1H), 5.19 (d, J
) 17.4 Hz, 1H), 5.46 (s, 1H), 5.92 (s, 1H), 6.16 (dd, J ) 10.0
and 1.6 Hz, 1H), 7.34 (d, J ) 10.0 Hz, 1H). Anal. Calcd for
(C33H45F3N2O11): C, H, N.
1
77%); mp ) 218-220 °C. H NMR (DMSO-d6) δ 0.78 (s, 3H),
0.87 (m, 2H), 0.92 (m, 1H), 1.23 (m, 1H), 1.39 (s, 3H), 1.66 (m,
5H), 2.00 (m, 6H), 2.32 (m, 1H), 2.50 (m, 5H), 2.62 (t, J ) 6.8
Hz, 2H), 3.33 (t, J ) 4.6 Hz, 2H), 3.66 (t, J ) 6.8 Hz, 2H), 4.28
(s, 1H), 4.73 (s, 1H), 4.78 (d, J ) 13.2 Hz, 1H), 5.08 (d, J )
13.2 Hz, 1H), 5.43 (s, 1H), 5.92 (s, 1H), 6.18 (dd, J ) 10.4 and
1.4 Hz, 1H), 7.32 (dd, J ) 10.4 and 1.4 Hz, 1H).
P r edn isolon e 21-[4-(3-Ch lor opr opyl)piper azin -1-yl]ace-
ta te (33). By following the same procedure reported for the
synthesis of 32 but with 31 in place of 30, compound 33 was
isolated as a white crystalline solid (yield 68%); mp ) 228-
1
230 °C. H NMR (DMSO-d6) δ 0.78 (s, 3H), 0.83 (m, 2H), 0.91
(m, 1H), 1.26 (m, 1H), 1.43 (s, 3H), 1.68 (m, 6H), 1.81 (t, J )
7 Hz, 2H), 2.02 (m, 1H), 2.37 (t, J ) 6.6 Hz, 4H), 2.49 (m, 8H),
3.33 (t, J ) 4.8 Hz, 2H), 3.65 (t, J ) 6.4 Hz, 2H), 4.28 (s, 1H),
4.73 (s, 1H), 4.78 (d, J ) 17.6 Hz, 1H), 5.09 (d, J ) 17.6 Hz,
1H), 5.42 (s, 1H), 5.92 (s, 1H), 6.18 (dd, J ) 10.2 and 1.2 Hz,
1H), 7.31 (dd, J ) 10.2 Hz, 1H).
N-Boc-4-(2-n itr ooxyeth yl)p ip er id in e (26). A solution of
24 (873 mg, 3 mmol) and silver nitrate (765 mg, 4.5 mmol) in
dry tetrahydrofuran (15 mL) was stirred at room temperature
for 1 h. The salts were filtered off and the solvent was removed
under reduced pressure. The crude product was dissolved in
dichloromethane (15 mL), the organic phase was washed with
water (5 mL) and dried (Na2SO4), and the solvent was
evaporated under reduced pressure. The crude product was
purified by flash chromatography (eluent ethyl acetate/
petroleum ether 2/8 v/v) to afford 26 (641 mg, yield 78%) as a
P r ed n isolon e 21-[4-(3-Nitr ooxyp r op yl)p ip er a zin -1-yl]-
a ceta te Ditr iflu or oa cetic Sa lt (9). A solution of 33 (563 mg,
1 mmol) and silver nitrate (510 mg, 3 mmol) in a mixture of
acetonitrile (12 mL) and tetrahydrofuran (8 mL) was refluxed
in the dark for 48 h. The precipitate was filtered off, and the
solvent was evaporated under vacuum. The crude product was
purified by reversed-phase HPLC to give the desired product
(425 mg, yield 52%) as a white crystalline powder after
1
yellow oil. H NMR (CDCl3) δ 1.16 (m, 2H), 1.46 (s, 9H), 1.66
(m, 5H), 2.69 (t, J ) 11.6 Hz, 2H), 4.17 (br s, 2H), 4.51 (t, J )
6.4 Hz, 2H).
N-Boc-4-(3-n itr ooxyp r op yl)p ip er id in e (27). By following
the same procedure reported for the synthesis of 26 but with
25 in place of 24, compound 27 was obtained as a yellow oil
1
lyophilization; mp ) 200-202 °C. H NMR (DMSO-d6) δ 0.78
(s, 3H), 0.86 (d, J ) 10.4 Hz, 1H), 1.06 (m, 1H), 1.27 (m, 1H),
1.39 (s, 3H), 1.65 (m, 4H), 2.07 (m, 6H), 2.33 (m, 4H), 2.49 (m,
4H), 2.83 (m, 2H), 3.16 (m, 2H), 3.36 (m, 1H), 3.52 (t, J ) 6. 2
Hz, 2H), 4.16 (m, 2H), 4.59 (t, J ) 6 Hz, 2H), 4.74 (s, 1H), 4.81
(d, J ) 17.6 Hz, 1H), 5.15 (d, J ) 17.6 Hz, 1H), 5.45 (s, 1H),
5.92 (s, 1H), 6.16 (d, J ) 10 Hz, 1H), 7.33 (dd, J ) 10 Hz, 1H).
Anal. Calcd for (C34H45F6N3O13): C, H, N.
1
(708 mg, yield 82%). H NMR (CDCl3) δ 1.37 (m, 2H), 1.44 (s,
9H), 1.58 (m, 2H), 1.77 (m, 5H), 2.72 (m, 2H), 4.21 (br s, 2H),
4.44 (t, J ) 6.6 Hz, 2H).
4-(2-Nitr ooxyeth yl)piper idin e Hydr och lor ide (28). Com-
pound 26 (548 mg, 2 mmol) was dissolved in a saturated
solution of hydrogen chloride in ethyl acetate (10 mL) and
stirred for 3 h at 0 °C. The solvent was removed by rotary
evaporation at reduced pressure and the resulting residue was
triturated with dry ethyl ether to give 28 as a white solid (630
mg, 100%); mp ) 143-145 °C.
4-(3-Nitr ooxyp r op yl)p ip er id in e Hyd r och lor id e (29). By
following the same procedure reported for the synthesis of 28
but with 27 in place of 26, compound 29 was isolated as a white
solid (630 mg, 100%); mp ) 150-152 °C.
P r edn isolon e 21-[4-(2-Nitr ooxyeth yl)piper idin -1-yl]ace-
ta te Tr iflu or oa cetic Sa lt (6). To a solution of prednisolone
21-chloroacetate (15) (436 mg, 1 mmol) and 28 (231 mg, 1.1
mmol) in N,N′-dimethylformamide (5 mL) cooled to 0 °C was
added triethylamine (0.31 mL, 2.2 mmol). The reaction mixture
was stirred overnight at room temperature and evaporated to
dryness. The residue was stirred with ethyl acetate (10 mL),
insoluble material was filtered off, and the filtrate was
evaporated to dryness. A final purification by preparative
HPLC gave 7 as a white crystalline solid (358 mg, yield 52%),
mp ) 126-127°C. 1H NMR (DMSO-d6) δ 0.80 (s, 3H), 0.92 (m,
1H), 1.39 (s, 3H), 1.48 (m, 3H), 1.66 (m, 6H), 1.89 (m, 7H),
2.34 (m, 1H), 2.51 (m, 4H), 3.12 (m, 2H), 3.49 (m, 1H), 3.51 (d,
J ) 7 Hz, 2H), 4.31 (m, 1H), 4.51 (t, J ) 6.2 Hz, 2H), 4.75 (s,
1H), 4.92 (d, J ) 17.4 Hz, 1H), 5.25 (d, J ) 17.4 Hz, 1H), 5.47
N-Boc-4-(2-br om oeth yl)p ip er a zin e (37). N-Boc-4-(2-hy-
droxyethyl)piperazine (35) (1.15 g., 5 mmol) was dissolved in
dry tetrahydrofuran (20 mL), and carbon tetrabromide (1.82
g., 5.5 mmol) was added. Then a solution of triphenylphosphine
(1.44 g, 5.5 mmol) in dry tetrahydrofuran (5 mL) was added
dropwise over 2 h. The mixture was stirred at room temper-
ature for 18 h,and then n-hexane (10 mL) was added to the
mixture, which was washed with saturated aqueous NaHCO3
(2 × 5 mL), water (5 mL), and brine (5 mL) and dried (Na2SO4).
The organic phase was evaporated to give an oil that was
purified by flash chromatography (ethyl acetate/petroleum
ether 2/8) to afford 37 as an oil that slowly crystallized on
1
storage (979 mg, yield 67%). H NMR (CDCl3) δ 1.45 (s, 9H),
2.45 (t, J ) 5.2 Hz, 4H), 2.79 (t, J ) 7.2 Hz, 2H), 3.43 (m, 6H).
N-Boc-4-(3-br om op r op yl)p ip er a zin e (38). To a solution
of N-Boc-piperazine (1 g, 5 mmol) and triethylamine (0.77 mL,
5.5 mmol) in dichloromethane (15 mL) cooled in an ice bath
was added 1,3-dibromopropane (0.56 mL, 5.5 mmol), and the
solution was stirred for 18 h at room temperature. The mixture
was diluted with dichloromethane (5 mL), washed with a
saturated aqueous solution of NaHCO3 (5 mL), water (5 mL),
and brine (5 mL), dried (Na2SO4), and then concentrated. The
residue, purified by flash chromatography (ethyl acetate/
petroleum ether 3/7) furnished 38 as an oil (1.18 g, yield 77%).

718 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Baraldi et al.
1H NMR (CDCl3) δ 1.46 (s, 9H), 1.94 (m, 2H), 2.38 (t, J ) 5.0
Hz, 4H), 2.49 (t, J ) 7.0 Hz, 2H), 3.43 (t, J ) 5.0 Hz, 4H), 3.60
(t, J ) 6.6 Hz, 2H).
(m, 2H); 1.41 (s, 3H); 1.20-1.60 (m, 2H); 1.60-1.90 (m, 3H);
1.90-2.15 (m, 4H); 2.30 (m, 1H); 2.50-2.60 (m, 2H); 4.32 (s,
1H); 4.76 (d, 1H), 4.86 (d, J ) 3.8 Hz, 2H); 5.03 (d, J ) 17.5
Hz, 1H); 5.34 (d, J ) 17.5 Hz, 1H); 5.50 (s, 1H); 5.93 (s, 1H);
6.17 (dd, J ) 10.1 and 1.6 Hz, 1H); 7.34 (d, J ) 10.1 Hz, 1H);
7.62 (d, J ) 8.2 Hz, 2H); 8.02 (d, J ) 8.2 Hz, 2H). Anal. Calcd
for (C29H34O7): C, H.
N-Boc-4-(2-Nitr ooxyeth yl)p ip er a zin e (39). To a solution
of 37 (880 mg, 3 mmol) in dry tetrahydrofuran (10 mL) was
added silver nitrate (765 mg, 4.5 mmol) in one portion. The
mixture was stirred for 2 h at room temperature, the salts
were filtered off, and then the filtrate was concentrated under
reduced pressure. The crude product was dissolved in di-
chloromethane (20 mL), the organic phase was washed with
water (5 mL) and dried (Na2SO4), and the solvent was
evaporated under reduced pressure. The crude product was
purified by flash chromatography (eluent ethyl acetate/
petroleum ether 1/1) to give 39 as an oil (704 mg, yield 92%).
1H NMR (CDCl3) δ 1.46 (s, 9H), 2.46 (t, J ) 5 Hz, 4H), 2.73 (t,
J ) 5.6 Hz, 2H), 3.43 (t, J ) 5.2 Hz, 4H), 4.58 (t, J ) 5.6 Hz,
2H).
Solu bility Assa ys. The assay was carried out according to
the method described by Casini et al.21 A standard solution
was prepared by dissolving a precisely weighed amount
(generally 1 mg) of compound 5-9 in 10 mL of methanol. The
UV absorption maximum of each compound was determined
(with a PerkinElmer-Lambda 25 spectrophotometer), eventu-
ally diluting the solution (with MeOH) as necessary. A
saturated solution of each compound was then prepared by
stirring magnetically a small volume of phosphate buffer (pH
7.4) in the presence of excess compound for 3 h. The saturated
solution was filtered to remove solid compound (through a
Gelman GHP Acrodisc 0.45-µm filter) and scanned by UV at
the wavelength of the absorption maximum previously deter-
mined. Total solubility was determined by the relationship C′
) A′C/A, where C ) concentration of standard solution
(milligrams per milliliter), A ) absorbance of standard solu-
tion, A′ ) absorbance of saturated solution, and C′ ) concen-
tration of saturated solution (milligrams per milliliter).
Biologica l Assa ys: (A) Glu cocor ticoid Recep tor Bin d -
in g. Human peripheral blood mononuclear cells (PBMC) were
isolated from whole blood by the histopaque density gradient
method. Cells were counted and 1 × 106 cells were transferred
to assay tubes in RPMI containing 1% glutamine. [3H]Dexa-
methasone (50 nM) was added to each tube, followed im-
mediately by test compounds at the concentrations stated, and
tubes were vortexed and incubated for 1 h at 37 °C. Cells were
then washed four times with 0.01 M ice-cold phosphate-
buffered saline (PBS), and the amount of [3H]dexamethasone
was determined by liquid scintillation. Concentration of
radioactive dexamethasone bound was calculated by subtract-
ing nonspecific binding values from test values and multiplying
by the molar/radioactivity ratio. Data (mean ( SEM) are
expressed as femtomoles of [3H]dexamethasone bound per
milliliter of n ) 3 determinations for each group. The com-
pounds were dissolved in DMSO to prepare a 10 mM stock
solution and diluted in buffer prior to experimentation. Control
cells received vehicle alone (0.1% DMSO).
(B) In fla m m a tion Mod els. The effect of selected com-
pounds on the process of neutrophil extravasation into an
inflammatory site was determined as described by Paul-Clark
et al.22 Briefly, compounds were administered at the anti-
inflammatory dose of 13.8 µmol/kg (corresponding to 5 mg/kg
prednisolone) given intraperitoneally 1 h prior to zymosan; the
vehicle control group was injected with 100 µL of peanut oil.
Zymosan (1 mg in 0.5 mL of saline) was given i.p. and the
extent of cell recruitment determined 4 h later as reported.22
Data (mean ( SEM of 5 mice/group) are reported as number
of polymorphonuclear cells migrated per cavity as well as
percentage of inhibition vs control migration (vehicle group).
Mea su r em en ts of NO Relea se. We used an electron
paramagnetic resonance (EPR) spectroscopic approach to
measure bioactive NO as nitrosylhemoglobin [HbFe(II)NO] in
rat whole blood. The method used is a modification of that of
Carini et al.20 Venous rat blood was taken by withdrawal with
heparinized syringe from an abdominal vein and deoxygenated
with a gas mixture of nitrogen (95%) and carbon dioxide (5%)
for 30 min in a sealing glass purge system equipped with an
inlet and outlet valve and a water warming jacket to maintain
controlled temperature (37 °C). S-Nitroso-N-acetylpenicill-
amine (SNAP) was used as reference NO donor. Drugs were
dissolved in DMSO. The drug was added to blood at a final
concentration of 100 µM. The blank was prepared by adding
the vehicle (final content 1% v/v).
N-Boc-4-(3-n itr ooxyp r op yl)p ip er a zin e (40). By following
the same procedure reported for the synthesis of 39 but with
38 in place of 37, compound 40 was isolated as an oil (yield
1
95%). H NMR (CDCl3) δ 1.45 (s, 9H), 1.90 (m, 2H), 2.36 (t, J
) 5.0 Hz, 4H), 2.42 (t, J ) 7.2 Hz, 2H), 3.41 (t, J ) 5.0 Hz,
4H), 4.53 (t, J ) 6.4 Hz, 2H).
1-(2-Nitr ooxyeth yl)piper azin e Hydr och lor ide (41). Com-
pound 39 (550 mg, 2 mmol) was dissolved in a 2 M solution of
hydrogen chloride in ethyl acetate (20 mL) cooled in an ice
bath and stirred for 4 h at room temperature. The solvent was
removed at reduced pressure and the residue was triturated
with dry ethyl ether to give 41 as a white solid (496 mg, yield
100%); mp ) 180-181 °C. 1H NMR (DMSO-d6) δ 3.45 (m, 9H),
3.54 (t, J ) 6.6 Hz, 2H), 4.04 (t, J ) 6.6 Hz, 2H), 10.01 (br s,
2H).
1-(3-Nitr ooxyp r op yl)p ip er a zin e Hyd r och lor id e (42).
By following the same procedure reported for the synthesis of
41 but with 40 in place of 39, compound 42 was isolated as a
1
white solid (yield 95%); mp ) 180-181 °C, H NMR (DMSO-
d6) δ 2.19 (t, J ) 7 Hz, 4H), 3.22 (t, J ) 7.4 Hz, 2H), 3.62 (m,
7H), 3.75 (t, J ) 6.4 Hz, 2H), 9.78 (br s, 2H).
P r ed n isolon e 21-[4-(2-Nitr ooxyeth yl)p ip er a zin -1-yl]-
a ceta te Bistr iflu or oa cetic Sa lt (8). To a stirred solution of
15 (436 mg, 1 mmol) and 41 (372 mg, 1.5 mmol) in N,N′-
dimethylformamide (5 mL) cooled at 0 °C was added triethyl-
amine (0.84 mL, 6 mmol) dropwise. The mixture was stirred
overnight at room temperature, the solvent was removed under
vacuum, and the resulting residue was dissolved in a mixture
of ethyl acetate (15 mL) and water (5 mL). The organic phase
was then washed with brine (2 mL), dried (Na2SO4), and then
evaporated. Purification on reversed-phase HPLC furnished
8 as a white solid after lyophilization (370 mg, yield 46%); mp
1
) 108-110 °C. H NMR (DMSO-d6) δ 0.79 (s, 3H), 0.88 (d, J
) 11.6 Hz, 1H), 0.94 (m, 1H), 1.28 (m, 1H), 1.39 (s, 3H), 1.66
(m, 2H), 2.02 (m, 6H), 2.33 (m, 4H), 2.49 (m, 4H), 2.83 (m,
2H), 3.16 (m, 2H), 3.36 (m, 1H), 3.52 (m, 2H), 4.29 (m, 4H),
4.78 (s, 1H), 4.83 (d, J ) 17.6 Hz, 1H), 5.16 (d, J ) 17.4 Hz,
1H), 5.47 (s, 1H), 5.92 (s, 1H), 6.16 (d, J ) 10 and 1.6 Hz, 1H),
7.32 (dd, J ) 10 Hz, 1H). Anal. Calcd for (C33H43F6N3O13): C,
H, N.
P r ed n isolon e 21-[4-(3-Nitr ooxyp r op yl)p ip er a zin -1-yl]-
a ceta te Bistr iflu or oa cetic Sa lt (9). By following the same
procedure reported for the synthesis of 8 but with 42 in place
of 41, compound 9 was isolated as a white solid (yield 47%).
P r ed n isolon e 21-[4′-(Hyd r oxym eth yl)ben zoa te] (10).
To a solution of prednisolone (1) (14.8 g, 41 mmol) in tetra-
hydrofuran (400 mL) were added triethylamine (5.7 mL, 41
mmol) and 4-hydroxymethylbenzoyl chloride (2.8 g, 16 mmol).
The reaction was stirred for 1 h at room temperature and the
solvent was evaporated under vacuum. The residue was
dissolved in chloroform and water. The organic phase was
washed with a saturated aqueous solution of NaHCO3 and
with brine and dried (Na2SO4), and the solvent was removed
under reduced pressure. The crude product was purified by
silica gel chromatography (n-hexane/chloroform/tetrahydro-
furan 5/3/2 v/v/v) to afford 10: yield 12%; white solid, mp )
228-235 °C. 1H NMR (DMSO-d6) δ 0.80 (s, 3H), 0.85-1.15
Aliquots of blood samples (500 µL) were taken at fixed times
and anaerobically transferred into EPR quartz tubes (3 mm
i.d.). To increase both sensitivity and reproducibility, the
plasma erythrocytes were directly separated by tube centrifu-

Synthesis of Nitro Esters of Prednisolone
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 719
(12) Egbertson, M. S.; Chang, C. T.; Duggan, M. E.; Gould, R. J .;
Halczenko, W.; Hartman, G. D.; Laswell, W. L.; Lynch, J . J .,
J r.; Lynch, R. J .; Manno, P. D.; Naylor, A. M.; Prugh, J . D.;
Ramjit, D. R.; Sitko, G. R.; Smith, R. S.; Turchi, L. M.; Zhang,
G. Non-peptide fibrinogen receptor antagonists. 2. Optimization
of a tyrosine template as a mimic for Arg-Gly-Asp. J . Med. Chem.
1994, 37, 2537-2551.
(13) Brundish, D.; Bull, A.; Donovan, V.; Fullerton, J . D.; Garman,
S. M.; Hayler, J . F.; J anus, D.; Kane, P. D.; McDonnell, M.;
Smith, G. P.; Wakeford, R.; Walker, C. V.; Howarth, G.; Hoyle,
W.; Allen, M. C.; Ambler, J .; Butler, K.; Talbot, M. D. Design
and synthesis of thrombin inhibitors: analogues of MD-805 with
reduced stereogenicity and improved potency. J . Med. Chem.
1999, 42, 4584-4603.
(14) Siegel, M. G.; Chaney, M. O.; Bruns, R. F.; Clay, M. P.; Schober,
D. A.; Van Abbema, A. M.; J ohnson, D. W.; Cantrell, B. E.; Hahn,
P. J .; Hunden, D. C.; Gehlert, D. R.; Zarrinmayeh, H.; Ornstein,
P. L.; Zimmerman, D. M.; Koppel, G. A. Rapid parallel synthesis
applied to the optimization of a series of potent nonpeptide
neuropeptide Y-1 receptor antagonists. Tetrahedron 1999, 55,
11619-11639.
gation at 4 °C (3200 rpm, 10 min) for erythrocyte nitrosyl-
hemoglobin analysis.
EPR measurements were carried out with a Bruker EMX
spectrometer (X band) equipped with a high-sensitivity cylin-
drical cavity (ER4119HS; Bruker). EPR samples were stored
in liquid nitrogen (77 K) and analyzed at 100 K. The spec-
trometer was operated at a microwave frequency of 9.33 GHz,
microwave power of 20.1 mW, modulation frequency of 100
kHz, modulation amplitude of 5.0 G, scan number of 20,
resolution of 1024 points, conversion time of 20.48 ms, time
constant of 10.24 ms, sweep time of 20.97 s, center field of
3300 G, and sweep width of 1200 G. Recorded EPR data were
manipulated and plotted with the EPR software (Bruker
WINEPR system, version 2.11) and Origin for Windows,
version 7.0 (Microcal, Northampton, MA).
The concentration of HbFe(II)NO, expressed as micromolar,
was determined by double integration of the signal with
CuSO4-ethylenediaminetetraacetic acid (EDTA) as reference
standard.26 All spectra were subtracted for the vehicle and the
final concentration of nitrosylhemoglobin in whole blood was
determined by improvement for the plasma.
(15) Hromatka, O.; Engel, E. Triethylenediamine (1,4-diazabicyclo-
[2.2.2]octane) II. Chem. Ber. 1943, 76B, 712-717.
(16) Schliemann, W.; Buege, A.; Reppel, L. Studies on the synthesis
of some 1-(p-fluorophenyl)-1-pyrid-2′-ylbutylamines. Pharmazie
1980, 35, 69-72.
(17) Xue, C. B.; Roderick, J .; J ackson, S.; Rafalski, M.; Rockwell, A.;
Mousa, S.; Olson, R. E.; DeGrado, W. F. Design, synthesis, and
in vitro activities of benzamide-core glycoprotein IIb/IIIa an-
tagonists: 2,3-diaminopropionic acid derivatives as surrogates
of aspartic acid. Bioorg. Med. Chem. 1997, 5, 693-705.
(18) Boschi, D.; Di Stilo, A.; Fruttero, R.; Medana, C.; Sorba, G.;
Gasco, A. Alpha 1-adrenoceptor blocking activity of some ring-
open analogues of prazosin. Arch. Pharm. (Weinheim, Germany)
1994, 327, 661-667.
Refer en ces
(1) Schimmer, B. P.; Parker, K. L. Adrenocorticotropic hormone;
adrenocortical steroids and their synthetic analogues; inhibitors
of the synthesis and actions of adrenocortical hormones. In The
Pharmacological Basis of Therapeutics, 10th ed.; Gilman, A. G.,
Goodman, L. S., Rall, T. W., Murad, F., Eds.; Macmillan
Publishing Co.: New York, 2001; pp 1649-1677.
(2) Scha¨cke, H.; Do¨cke, W. D.; Asadullah, K. Mechanisms involved
in the side effects of glucocorticoids. Pharmacol. Ther. 2002, 96,
23-43.
(3) Necela, B. M.; Cidlowski, J . A. Crystallization of the human
glucocorticoid receptor ligand binding domain: a step towards
selective glucocorticoids. Trends Pharmacol. Sci. 2003, 24, 58-
61.
(4) Coghlan, M. J .; Elmore, S. W.; Kym P. R.; Kort M. E. Selective
Glucocorticoid Receptor Modulators. Annu. Rep. Med. Chem.
2002, 37, 167-176.
(5) Wallace, J . L.; Soldato, P. D. The therapeutic potential of NO-
NSAIDs. Fundam. Clin. Pharmacol. 2003, 17, 11-20.
(6) Keeble, J . E.; Moore, P. K. Pharmacology and potential thera-
peutic applications of nitric oxide-releasing nonsteroidal anti-
inflammatory and related nitric oxide-donating drugs. Br. J .
Pharmacol. 2002, 137, 295-310.
(7) Chiroli, V.; Benedini, F.; Ongini, E.; Del Soldato, P. Nitric oxide-
donating nonsteroidal antiinflammatory drugs: the case of
nitroderivatives of aspirin. Eur. J . Med. Chem. 2003, 38, 441-
446.
(8) Moncada, S.; Palmer, R. M.; Higgs, E. A. Nitric oxide: physiol-
ogy, pathophysiology, and pharmacology. Pharmacol. Rev. 1991,
43, 109-142.
(9) Wallace, J . L.; Ignarro, L. J .; Fiorucci, S. Potential cardiopro-
tective actions of no-releasing aspirin. Nat. Rev. Drug. Discovery
2002, 1, 375-382.
(19) Vallance, P.; Leiper, J .; Blocking NO synthesis: how, where and
why? Nat. Rev. Drug Discovery 2002, 1, 939-950.
(20) Carini, M.; Aldini, G.; Stefani, R.; Orioli, M.; Facino, R. M.
Nitrosylhemoglobin, an unequivocal index of nitric oxide release
from nitroaspirin: in vitro and in vivo studies in the rat by ESR
spectroscopy. J . Pharm. Biomed. Anal. 2001, 26, 509-518.
(21) Casini, A.; Scozzafava, A.; Mincione, F.; Menabuoni, L.; Ilies,
M. A.; Supuran, C. T. Carbonic anhydrase inhibitors: water-
soluble 4-sulfamoylphenylthioureas as topical intraocular pres-
sure-lowering agents with long-lasting effects. J . Med. Chem.
2000, 43, 4884-4892.
(22) Paul-Clark, M.; Del Soldato, P.; Fiorucci, S.; Flower, R. J .;
Perretti, M. 21-NO-prednisolone is a novel nitric oxide-releasing
derivative of prednisolone with enhanced antiinflammatory
properties. Br. J . Pharmacol. 2000, 131, 1345-1354.
(23) Fiorucci, S.; Antonelli, E.; Distrutti, E.; Del Soldato, P.; Flower,
R. J .; Clark, M. J .; Morelli, A.; Perretti, M.; Ignarro, L. J . NCX-
1015, a nitric-oxide derivative of prednisolone, enhances regula-
tory T cells in the lamina propria and protects against 2,4,6-
trinitrobenzenesulfonic acid-induced colitis in mice. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 15770-15775.
(24) Paul-Clark, M.; Mancini, L.; Del Soldato, P.; Flower, R. J .;
Perretti, M. Potent antiarthritic properties of a glucocorticoid
derivative, NCX-1015, in an experimental model of arthritis.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1677-1682.
(25) Peskar, B.; Maricic, N.; Ehrlich, K.; Swaka, N. NCX-1015, a novel
nitric oxide-releasing derivative of prednisolone, combines potent
antiinflammatory activity and low toxicity in the rat stomach
subjected to ischemia-reperfusion. Gastroenterology 2002, 229
(Suppl. A), M-891 (abstr.)
(10) Hinton, I. G.; Mann, F. G.; Vanterpool, A. Interaction of
o-chloromethylphenacyl chloride and primary amines: a novel
formation of the isoindoline system. J . Chem. Soc. 1959, 610-
614.
(11) Ambler, J .; Brown, L.; Cockcroft, X. L.; Grutter, M.; Hayler, J .;
J anus, D.; J ones, D.; Kane, P.; Menear, K.; Priestle, J .; Smith,
G.; Talbot, M.; Walker, C. V.; Wathey, B. Optimisation of the
P2 pharmacophore in a series of thrombin inhibitors: ion-dipole
interactions with lysine 60G. Bioorg. Med. Chem. Lett. 1999, 9,
1317-1322.
(26) Kosaka, H.; Sawai, Y.; Sakaguchi, H.; Kumura, E.; Harada, N.;
Watanabe, M.; Shiga, T. ESR spectral transition by arterio-
venous cycle in nitric oxide hemoglobin of cytokine-treated rats.
Am. J . Physiol. 1994, 266, C1400-C1405.
J M030964B