Synthesis and anti-inflammatory activity of a series of N-substituted naproxen glycolamides: Nitric oxide-donor naproxen prodrugs

Article abstract of DOI:10.1016/j.bmc.2005.11.040

A series of glycolamide naproxen prodrugs containing a nitrate group as a nitric oxide (NO) donor moiety has been synthesized. These compounds were evaluated for their anti-inflammatory activity, naproxen release, and gastric tolerance. Compounds 4a, 4b,

Full text of DOI:10.1016/j.bmc.2005.11.040

Bioorganic & Medicinal Chemistry 14 (2006) 2589–2599
Synthesis and anti-inflammatory activity of a series
of N-substituted naproxen glycolamides: Nitric
oxide-donor naproxen prodrugsq
Ramani R. Ranatunge,*,ꢀ Michael E. Augustyniak, Vijay Dhawan, James L. Ellis,
David S. Garvey, David R. Janero, L. Gordon Letts, Stewart K. Richardson,
Mathew J. Shumway, A. Mark Trocha, Delano V. Young and Irina S. Zemtseva
NitroMed, Inc., 125 Spring St., Lexington, MA 02421, USA
Received 22 October 2005; revised 17 November 2005; accepted 21 November 2005
Available online 13 December 2005
Abstract—A series of glycolamide naproxen prodrugs containing a nitrate group as a nitric oxide (NO) donor moiety has been syn-
thesized. These compounds were evaluated for their anti-inflammatory activity, naproxen release, and gastric tolerance. Compounds
4a, 4b, 5a, 5b, 7b, and 7c exhibited anti-inflammatory activity equivalent to that of the parent NSAID, naproxen-Na, in the rat car-
rageenan paw edema model. At equimolar doses relative to naproxen-Na, the NO-donor glycolamide derivatives 4a, 4b, 5a, 5b, 7b,
and 7c were gastro-sparing in the rat. Naproxen formation from these NO-donor glycolamides varied among the structures exam-
ined, with the N-substituent on the amide group having a particular influence, and demonstrated their prodrug nature. Compound
7b was selected for exemplary demonstration that the glycolamide nitrates can be bioactivated to release NO. These data open the
possibility that naproxen glycolamide nitrates may represent a safer alternative to naproxen as anti-inflammatory medicines.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
number of patients concomitantly taking low-dose aspi-
rin for cardiovascular prophylaxis8, coxibs can exacer-
bate aspirinꢁs GI toxicity. An apparent class-effect
cardiovascular risk that predisposes patients to heart at-
tack and stroke with chronic coxib use has recently been
uncovered.9 Thus, there remains compelling need for
effective NSAIDs with improved safety. As an alternative
to COX-2 selective inhibitors, we and others have deve-
loped COX-inhibiting nitric oxide (NO) donors (CIN-
ODs), a new class of anti-inflammatory agents utilizing
the gastroprotective properties of NO to reduce the
GI damage associated with nonselective COX
inhibition.10–19 Herein, we report the synthesis and phar-
macological profiles of a series of glycolamide prodrugs of
naproxen (II–IV) containing a nitrate group as a NO-
donor moiety. These compounds exhibit analgesic and
anti-inflammatory potencies similar to those of naprox-
en-Na and the prototypic CINOD AZD358219a in rodent
models, but with a significantly less gastric damage.
Conventional nonsteroidal anti-inflammatory drugs
(NSAIDs) that inhibit nonselectively both major cyclo-
oxygenase (COX) isoforms are effective therapies
against the signs and symptoms of inflammation.1,2
Naproxen (AleveTM, NaprosynTM, AnaproxTM, and
NaprelanTM) (I) is one of the most popular NSAIDs for
relieving arthritic pain, although its use invites gastroin-
testinal (GI) complications ranging from stomach irrita-
tion to life-threatening GI ulceration and bleeding.3–5
Selective COX-2 inhibitors (coxibs) possess the salutary
analgesic and anti-inflammatory properties of NSAIDs
with generally enhanced, but still not absolute, GI toler-
ance.6 Yet in both healthy subjects7 and the increasing
Keywords: Non-steroidal anti-inflammatory drug (NSAID); Nitric
oxide; Naproxen; CINOD; Anit-inflammatory; Cyclo-oxygenase.
q
A portion of this work was presented in a poster at the 228th ACS
National Meeting (MEDI # 247), Philadelphia, PA, USA, August
22–28, 2004.
2. Chemistry
*
Corresponding author. Tel.: +1 781 266 4139; fax: +1 781 275
8083; e-mail: ramani@nitromed.com
Following the corresponding author, co-authors appear in
ꢀ
Recent recognition of a cardiovascular liability associated
with coxib therapy in moderate to high-risk patients
alphabetical order.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.040

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R. R. Ranatunge et al. / Bioorg. Med. Chem. 14 (2006) 2589–2599
O
R1
O
OH
N
O
O
O
O
ONO2
II
I
O
O
CH3
O
O
N
N
O
R2
O
O
X
ONO2
O
O
n
III
IV
O
ONO2
O
O
AZD3582
has refocused considerable attention on existing
NSAIDs with the least clinical side effects such as
naproxen.10–19 Accordingly, we sought to design a
naproxen NSAID derivative with an intrinsic NO-do-
nating capability that would leverage NOꢁs gastropro-
tective properties to promote GI safety. To facilitate
covalent incorporation of an NO-donor group, the
naproxen core structure should contain an appropriate
functionality that can be derivatized easily to a NO-do-
nating prodrug. Many types of bioreversible derivatives
for diverse functional groups have been explored in
prodrug design.20–22 Glycolamide esters appeared par-
ticularly suitable for our purpose, since they are rapidly
cleaved in human plasma.23 Herein, we have prepared a
series of NO-donating, N-substituted glycolamide esters
of naproxen and have determined their biological (i.e.,
anti-inflammatory) activity and acute gastric tolerance.
The synthesis of the N-methyl glycolamide ester of
naproxen, 6, is illustrated in Scheme 2. Compounds 7a
and 7b were synthesized from 6 in a manner similar to
those of 4 and 5 (Scheme 2).
3. Results and discussion
A series of NO-donor naproxen glycolamides was syn-
thesized and evaluated for oral anti-inflammatory acti-
vity, gastric tolerance, and naproxen release relative to
the sodium salt of the parent drug, naproxen (I). Com-
pounds 4c–4d and 7d were not evaluated biologically
due to their inherent instability at ambient temperature.
The anti-inflammatory activities and gastric irritancies
of naproxen-Na, AZD3582, and selected NO-donor
naproxen glycolamide derivatives (4a–4b, 5a–5d, and
7a–7c) are given in Table 1. The rat paw-edema test
was performed according to the standard method of
Winter et al.24 All compounds (4a–4b, 5a–5b and
7b–7c) showed significant anti-inflammatory activity in
this test at an equimolar dose relative to 30 mg/kg
naproxen-Na. Compound 7c was the most potent anti-
inflammatory agent in this series containing a sarcosine
glycolamide linker with a three-carbon aliphatic nitrate
ester group; its anti-inflammatory potency was compa-
rable to that of AZD3582.19a Compound 5d, a piperizine
glycolamide, elicited a 44% reduction of paw edema,
while the other naproxen glycolamides appreciably
reduced inflammation by ꢀ44% as well. The in vivo gas-
tric tolerance of these compounds was characterized as
described by Kitagawa et al.25 and Al-Ghamdi et al.26
Naproxen sodium at a high oral dose (30 mg/kg) caused
significant stomach lesions, whereas at equimolar oral
doses 4a–4b, 5a–5b, and 7a–7c caused comparatively lit-
tle gastric damage (Table 1), exhibiting a level of gastric
tolerance comparable to that of AZD3582. Compounds
The structures and synthesis of naproxen derivatives 2–5
are presented in Scheme 1. The naproxen glycolic ester 3
was prepared by esterification of 1 with tert-butylbro-
moacetate in the presence of NaHCO3 in DMF at room
temperature followed by hydrolysis of the tert-butyl es-
ter with a mixture of TFA/CH2Cl2. Compounds (4a–4d,
5a–5d) were prepared by direct amidation of the glycolic
acid group of 3 with an appropriate amine nitrate salt
(4a-I–4d-I, 5a-I–5c-I in Scheme 1) using either EDAC
or DCC in the presence of 1–2 equiv of DMAP in
CH2Cl2 or DMF (Scheme 1). It has been found and
characterized that compound 4d slowly decomposes into
compound 4d-II at ambient temperature.ꢂ
ꢂ
This could be attributed to both the electronic and steric properties
of substitution on N-glycolamide. With bulky and more lipophilic
branched alkyl groups, iso-propyl- and cylcohexyl-substituted com-
pounds 4c and 4d are unstable at room temperature compared to the
methyl- and ethyl-substituted derivatives 4a and 4b.

R. R. Ranatunge et al. / Bioorg. Med. Chem. 14 (2006) 2589–2599
2591
O
OH
a
O
O
O
O
O
O
O
1
2
b
O
X
O
R1
ONO2
.HNO3
O
R1
N
H
OH
N
O
O
O
c
O
ONO2
3
4
4a-I R1 = -CH3
ONO2
HN
n
4b-I R1 = -CH2CH3
4c-I R1 = -CH(CH3)CH3
4d-I R1 = -cyclohexyl
4a R1 = -CH3
4b R1 = -CH2CH3
4c R1 = -CH(CH3)CH3
4d R1 = -cyclohexyl
5a-I X = C, n = 1
c
.HNO3
5b-I X = C, n = 2
5c-I X = N, n = 2
O
O
N
O
O
O
X
ONO2
O
O
n
rt
5
4d
O
5a X = C, n = 1
5b X = C, n = 2
5c X = N, n = 2
O
N
H
4d-II
.HNO3
5d X = N, n = 2, HCl salt
Scheme 1. Reagents: (a) tert-Butyl bromoacetate/NaHCO3/DMF; (b) TFA/CH2Cl2; (c) EDAC/DMAP/CH2Cl2.
O
CH3
O
a
N
3
O
O
O
OH
6
b
O
CH3
O
7a R2 = -NHCH2CH2ONO2
7b R2 = -OCH2CH2ONO2
7c R2 = -OCH2CH2CH2ONO2
7d R2 = -N(CH3)CH2CH2ONO2
N
O
O
7
O
R2
Scheme 2. Reagents: (a) (i) Sarcosine tert-butyl ester hydrochloride/TEA/DMAP/DCC/CH2Cl2; (ii) TFA/CH2Cl2; (b) nitrate tether salt/EDAC or
DCC/DMAP/CH2Cl2.
5c and 5d were less damaging to the stomach than
naproxen-Na, but less well tolerated than most other
compounds (4a–4b, 5a–5b, and 7b–7c) in this series
and were accordingly not studied further. Based upon
the combined criteria of in vivo oral anti-inflammatory
activity and gastric safety, compounds 5b, 7b, and 7c
exhibited the best overall pharmacological profiles.
lead anti-inflammatory, gastrosparing naproxen glycol-
amides was examined in vitro by incubating the com-
pounds in human whole blood at
a
nominal
concentration of 40 lM and quantifying the naproxen
formed therein by LC. Naproxen itself was catabolized
only slightly (by <20%) over 120 min of the incubation
(Table 2). Production of naproxen from all NO-donor
glycolamides tested demonstrates the prodrug nature
of this compound class, whereby the ester bond of
the NO-donor naproxen glycolamide derivative is
hydrolyzed in human blood to liberate naproxen.
The prototypic CINOD AZD3582 is known to be
metabolized as a prodrug, resulting in naproxen forma-
tion.19a The potential for naproxen generation from the

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R. R. Ranatunge et al. / Bioorg. Med. Chem. 14 (2006) 2589–2599
Table 1. Anti-inflammatory activity and gastric toxicity of NO-donating naproxen glycolamides in rats
O
O
O
CH3
R1
O
O
O
N
N
N
O
O
O
O
X
ONO2
O
O
O
ONO2
n
4
5
R2
7
5a X = C, n = 1
5b X = C, n = 2
5c X = N, n = 2
4a R1 = -CH3
4b R1 = -CH2CH3
7a R2 = -NHCH2CH2ONO2
7b R2 = -OCH2CH2ONO2
7c R2 = -OCH2CH2CH2ONO2
5d X = N, n = 2, HCl salt
Compounda
Anti-inflammatory activity (% paw swelling reduction)b
Gastric toxicity (% lesion reduction)c
0
Vehicle
Naproxen
AZD3582
0
52 0.5
52 0.05
38 0.06
40 0.04
40 0.07
0
6.2
88 1.9
77 1.5
74 3.7
70 0.5
94 0.5
44 6.2
28 4.5
74 0.5
97 0.5
81 7.7
4a
4b
5a
5b
5c
5d
7a
7b
7c
39 0.05
d
44 0.08
d
37 0.05
49 0.08
All values are means SEM and are significantly different from vehicle control (P < 0.01 by ANOVA followed by Dunnettꢁs Multiple Comparisons
Test).
a Equimolar doses relative to naproxen-Na 30 mg/kg.
b Relative to vehicle.
c Relative to naproxen.
d Not determined.
Table 2. Naproxen production from NO-donating naproxen glycolamides in human whole blood
O
O
O
CH3
R1
O
O
O
N
N
N
O
O
O
O
X
ONO2
O
O
O
ONO2
n
4
5
R2
7
5a X = C, n = 1
5b X = C, n = 2
4a R1 = -CH3
4b R1 = -CH2CH3
7b R2 = -OCH2CH2ONO2
Time (min)
Plasma naproxen concentration (lM)
Naproxen-Na
4a
4b
5a
5b
7b
0
5
0
0
9.9
0
2.9
0
0
0
a
a
32.3
33.1
31.9
32.1
12.9
19
20
40
31.2
29
23.3
35.1
37.9
14.4
23.9
36.2
9.9
11.8
21.4
25.3
25.4
24.7
34.3
60
33
120
240
32.1
a
33.2
32.8
37.9
a
37.5
a
28.1
a
a Not determined.
Compounds 4a, 4b and 5a were also rapidly metabolized
to naproxen over 120-min incubation with similar kine-
tics. Production of naproxen from compounds 5b and 7b
was somewhat slower than from the other glycolamides
tested, yet reached comparable level in the case of 7b by
240 min.
2 h with cultured human liver parenchymal cells (hepa-
tocytes) under physiological conditions. This test system
was chosen because of the high intrinsic capacity of
mammalian liver to biotransform organic nitrate vasodi-
lators to NO.27 By 1 h incubation with the hepatocytes,
7b released 60% of the available NO-equivalents as ni-
trate and nitrite products of NO oxidative decomposi-
tion. Since in living systems NO and NO-derived
metabolites participate in other reactions not necessarily
To illustrate the potential of this series of nitrated
naproxens to donate NO, 7b was incubated for up to

R. R. Ranatunge et al. / Bioorg. Med. Chem. 14 (2006) 2589–2599
2593
leading to nitrate/nitrite formation,27 this figure most
5.3. Gastric irritancy
likely underestimates the extent of acute bioactivation
of 7b to NO, but nonetheless shows that an exemplary
nitrated naproxen glycolamide can be catabolized to
NO and thus acts as a NO donor.
The rat gastric lesion test, described by Kitagawa et al.25
and Al-Ghamdi et al.26, was used to evaluate the gastro-
sparing activity of test compounds. Male Sprague–Daw-
ley rats (Charles River Laboratories, Wilmington, MA)
weighing 230–250 g were used for the experiments.
Twelve animals per treatment group were used. The rats
were fasted for 24 h with free access to water and then
dosed by oral gavage with vehicle (PEG 400/Methocel)
or test compound given at a dosing volume of 1.0 mL/
kg. Food was withheld after dosing. The rats were
euthanized by CO2 asphyxiation 3 h after dosing. Their
stomachs were extirpated, dissected along the greater
curvature, washed with 0.9% saline, pinned open, and
digitally photographed. The images were analyzed for
visible hemorrhagic lesions using Image-J software
(National Institute of Health, Bethesda, MD) by inves-
tigators blinded to treatment. The total length of all le-
sions for each stomach was summed to obtain a mean
total lesion score, which was expressed as a percent
lesion reduction relative to the lesion score from naprox-
en-Na SEM.
4. Conclusion
We have synthesized a series of N-substituted glycol-
amides of naproxen that contain a nitrate functionality
as a NO-donor group. Compounds of this class were
demonstrated to be NO-donating, naproxen-releasing
prodrugs. Compounds 4a, 4b, 5a, 5b 7b, and 7c pos-
sessed oral anti-inflammatory activity equivalent to that
of the conventional NSAID, naproxen-Na, in the rat
carrageenan paw edema model. Naproxen generation
from these NO-donor glycolamides varied, with the N-
substituent of the glycolamide group having a particular
influence. N-Methyl glycolamides appeared to be the
best prodrug substituents in this series. At equimolar
doses relative to naproxen-Na, compounds 4a, 4b, 5a,
5b, 7b, and 7c were gastrosparing in rats. Our data sug-
gest that these naproxen glycolamide nitrate prodrugs
may afford a safer alternative to naproxen (I) for the
treatment of inflammatory disease and pain.
5.4. NO-donating capability
Compound 7b was incubated at a final concentration of
70 lM in 0.4 mL of medium with a monolayer of
3.9 · 105 viable human hepatocytes cultured under stan-
dard conditions prescribed by the supplier (Clonetics,
San Diego, CA). After 1 h, the culture medium was
recovered and analyzed for net nitrate/nitrite formation
relative to control incubations without test compound
by using a diazotization-based colorimetric assay (Cay-
man, Ann Arbor, MD).
5. Experimental
5.1. Naproxen formation in human blood
Freshly obtained human whole blood was aliquoted
(1 mL/well) into 24-well plates and pre-equilibrated
to 37 ꢁC. To each well, test compound in DMSO
was added to a final concentration of 40 lM, and
the samples were incubated at 37 ꢁC for varying peri-
ods of time up to 240 min. At the end of each incuba-
tion, blood was de-proteinated by mixing it with
acetonitrile in a 1:1 volume ratio and centrifuging
the mixture at 1500 rpm for 10 min at 4 ꢁC. The
resulting supernatant was recovered, filtered, and ana-
lyzed by LC for its naproxen content.16
5.4.1. General procedures. All reagents and anhydrous
solvents were generally used as received from the com-
mercial supplier. Reactions were routinely performed
under a nitrogen atmosphere in oven-dried glassware.
Melting points were determined with an electrothermal
heating block (Metler) and are uncorrected. 1H and
13C spectra were recorded on Varian 300 and
75 MHz, respectively. NMR spectra were recorded in
CDCl3 unless otherwise specified, and chemical shifts
are reported relative to tetramethylsilane as an inter-
nal standard. Low resolution mass spectra were ob-
tained using atmospheric pressure-turbo ion spray
ionization. Elemental analyses were performed by
Robertson Microlit Laboratories Inc., Madison, N.J.
Gravity and flash column chromatographies were per-
formed using EM Science silica gel 60 (230–400 mesh).
TLC was performed on 250 lm precoated EM Science
silica gel 60 F254 aluminum sheets. Preparative TLC
was performed using 20 · 20 cm 1000 lm precoated
silica gel plates (Whatman). Spots were visualized un-
der 254 nm light or after staining with phosphomolyb-
dic acid.
5.2. Anti-inflammatory activity
The rat paw edema test was used to assess anti-inflam-
matory activity.24 Male Sprague–Dawley rats (Charles
River Laboratories, Wilmington, MA) weighing 180–
220 g were fasted overnight with water ad libitum.
Twelve animals per treatment group were used. Test
compound or vehicle (PEG 400/Methocel) was adminis-
tered by oral gavage 1 h prior to carrageenan (Sigma, St.
Louis, MO) injection (50 lL of 1% carrageenan in sa-
line) into the subplantar region of right hind paw at a
dose volume of 1 mL/kg. Paw volume was determined
using the water-displacement method as the average of
two measurements differing by less than 0.2 mL. Paw
volume measurements were assessed immediately as pri-
or to and 3 h post carrageenan injection. Results are
expressed as the mean % inhibition of the change in
paw volume produced by carrageenan injection relative
to vehicle standard error of the mean (SEM).
5.4.2. ((tert-Butyl)oxycarbonyl)methyl (2S)-2-(6-meth-
oxy(2-naphthyl))propanoate (2). A mixture of (2S)-2-(6-
methoxy(2-naphthyl))propanoic acid (naproxen, 3 g,
13.0 mmol), NaHCO3 (1.4 g, 16.7 mmol), and tert-butyl

2594
R. R. Ranatunge et al. / Bioorg. Med. Chem. 14 (2006) 2589–2599
bromoacetate (5.08 g, 26.0 mmol) in DMF (30 mL) was
stirred at room temperature for 2 days, and the solvent
was evaporated in high vacuo. The residue was recrys-
tallized from a mixture of CH2Cl2/EtOAc/hexane to give
2 as a white solid in quantitative yield. Mp 85–86 ꢁC. 1H
NMR (300 MHz, CDCl3) d 7.69–7.70 (m, 3H), 7.43 (dd,
J = 1.6 and 8.6 Hz, 1H), 7.10–7.15 (m, 2H), 4.47 (ABq,
JAB = 15.7 Hz, DmAB = 40.7 Hz, 2H), 3.97 (q,
J = 7.2 Hz, 1H), 3.90 (s, 3H), 1.63 (d, J = 7.2 Hz, 3H),
1.42 (s, 9H). 13C NMR (75 MHz, CDCl3) d 174.2,
166.9, 157.8, 135.4, 133.9, 129.5, 129.1, 127.3, 126.5,
126.3, 119.1, 105.8, 82.5, 61.7, 55.5, 45.3, 28.1, 18.8.
MS (API-TIS) m/z 345 (MH+), 362 (MNH4+).
(s, 3H), 1.65 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz,
CDCl3) d 174.4, 167.1, 157.8, 135.3, 133.9, 129.4,
129.0, 127.3, 126.4, 126.3, 119.1, 105.7, 71.3, 61.7,
55.4, 46.3, 45.3, 36.0, 18.7. MS (API-TIS) m/z 391
(MH+), 408 (MNH4+). Anal. Calcd for C19H22N2O7:
C, 58.46; H, 5.68; N, 7.18. Found: C, 58.32; H, 5.55;
N, 6.94.
5.4.5. (N-Ethyl-N-(2-(nitrooxy)ethyl)carbamoyl)methyl
(2S)-2-(6-methoxy(2-naphthyl))propanoate (4b)
5.4.5.1. Ethyl(2-(nitroxy)ethyl)ammonium nitrate (4b-I).
Ethyl(2-(hydroxy)ethyl)amine (5 g, 56 mmol) in EtOAc
(60 mL) was added dropwise to a mixture of fuming
HNO3 (17.7 g, 11.8 mL, 280 mmol) and Ac2O (45.8 g,
42.3 mL, 448 mmol) at À10 ꢁC. The reaction mixture
was stirred at À10 ꢁC for 30 min and diluted with EtOAc
and hexane. The oil layer was separated and dried in
high vacuo to give 4b-I (8.4 g, 76%) as a low-melting
5.4.3. 2-((2S)-2-(6-Methoxy(2-naphthyl))propanoyloxy)-
acetic acid (3). Trifluoroacetic acid (8.4 mL) was added
dropwise to a solution of the product of 2 (4.8 g,
14 mmol) in CH2Cl2 (20 mL). The reaction mixture
was stirred at room temperature for 2 days, and the vol-
atile material was removed in vacuo. The residue after
evaporation was recrystallized from EtOAc/hexane to
give 3 (2.7 g, 66%) as a white solid. Mp 122–123 ꢁC.
1H NMR (300 MHz, CDCl3) d 7.67–7.73 (m, 3H), 7.42
(d, J = 9.1 Hz, 1H), 7.12–7.17 (m, 2H), 4.65 (ABq,
JAB = 16.4 Hz, DmAB = 34.3 Hz, 2H), 3.98 (q,
J = 7.1 Hz, 1H), 3.92 (s, 3H), 1.63 (d, J = 7.1 Hz, 3H).
13C NMR (75 MHz, CDCl3) d 174.2, 173.0, 157.9,
135.1, 133.9, 129.5, 129.1, 127.4, 126.4, 126.3, 119.2,
105.8, 60.5, 55.5, 45.2, 18.7. MS (API-TIS) m/z 289
(MH+), 306 (MNH4+).
1
solid. H NMR (300 MHz, DMSO-d6) d 8.60–8.95 (br
s, 2H), 4.84 (m, 2H), 3.37–3.49 (m, 2H), 3.02–3.16 (m,
2H), 1.24 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz,
DMSO-d6) d 69.1, 43.6, 42.6, 11.0. MS (API-TIS) m/z
135 (MH+).
5.4.5.2. (N-Ethyl-N-(2-(nitrooxy)ethyl)carbamoyl)meth-
yl(2S)-2-(6-methoxy(2-naphthyl))propanoate (4b).
A
mixture of 3 (1.0 g, 3.47 mmol), 4b-I (0.75 g, 3.82
mmol), and DMAP (0.42 g, 3.47 mmol) in CH2Cl2
(20 mL) at 0 ꢁC was treated with 1-(3-(dimethylami-
no)propyl)-3-ethylcarbodiimide hydrochloride (0.73 g,
3.82 mmol). The reaction mixture was stirred at 0 ꢁC
for 3 h, diluted with more CH2Cl2, washed with water,
brine, and dried over Na2SO4. The residue after filtra-
tion and evaporation was chromatographed on silica
gel eluting with 1:2 to 1:1 EtOAc/hexane to give 4b
5.4.4. (N-Methyl-N-(2-(nitrooxy)ethyl)carbamoyl)methyl
(2S)-2-(6-methoxy(2-naphthyl))propanoate (4a)
5.4.4.1. Methyl(2-(nitrooxy)ethyl)ammonium nitrate
(4a-I). Methyl(2-(hydroxy)ethyl)amine (1.5 g, 20 mmol)
in EtOAc (65 mL) was added dropwise to a mixture of
fuming HNO3 (6.3 g, 4.2 mL, 100 mmol) and Ac2O
(16.3 g, 15.1 mL, 160 mmol) at À10 ꢁC. The reaction
mixture was stirred at À10 ꢁC for 30 min and diluted
with EtOAc and hexane. The precipitate was collected
by filtration and washed with hexane to give 4a-I
1
(0.4 g, 29%) as a white solid. Mp 60–61 ꢁC. H NMR
(300 MHz, CDCl3) d 7.65–7.74 (m, 3H), 7.44 (dd,
J = 1.5 and 8.5 Hz, 1H), 7.06–7.17 (m, 2H), 4.70
(ABq, JAB = 14.4 Hz, DmAB = 64.9 Hz, 2H), 4.54–4.65
(m, 2H), 4.01 (q, J = 7.1 Hz, 1H), 3.91 (s, 3H), 3.50–
3.72 (m, 2H), 3.26 (q, J = 7.2 Hz, 2H), 1.64 (d,
J = 7.2 Hz, 3H), 1.17 (t, J = 7.1 Hz, 3H). 13C NMR
(75 MHz, CDCl3) d 174.5, 166.9, 157.8, 135.3, 133.9,
129.4, 129.0, 127.3, 126.4, 126.3, 119.1, 105.7, 71.0,
61.5, 55.4, 45.3, 43.9, 43.3, 18.7, 14.0. MS (API-TIS)
m/z 405 (MH+), 422 (MNH4+). Anal. Calcd for
C20H24N2O7: C, 59.40; H, 5.98; N, 6.93. Found: C,
59.28; H, 5.81; N, 6.70.
1
(2.79 g, 82%) as an off-white solid. Mp 58–62 ꢁC. H
NMR (300 MHz, DMSO-d6) d 4.59–4.67 (m, 2H),
4.64–4.73 (m, 2H), 2.61 (br s, 3H). 13C NMR
(75 MHz, DMSO-d6) d 59.8, 38.7, 24.4. MS (API-TIS)
m/z 121 (MH+).
5.4.4.2. (N-Methyl-N-(2-(nitrooxy)ethyl)carbamoyl)meth-
yl(2S)-2-(6-methoxy(2-naphthyl))propanoate (4a). A mix-
ture of 3 (1.03 g, 3.57 mmol), 4a-I (0.72 g, 3.93 mmol),
and DMAP (0.44 g, 3.57 mmol) in CH2Cl2 (20 mL) at
0 ꢁC was treated with 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride (0.75 g, 3.93 mmol).
The reaction mixture was stirred at 0 ꢁC for 3 h, diluted
with more CH2Cl2, washed with water, brine, and dried
over Na2SO4. The residue after filtration and evapora-
tion was chromatographed on silica gel eluting with
1:3 to 1:2 to 1:1 EtOAc/hexane to give 4a (0.71 g,
51%). Mp 80–81 ꢁC. 1H NMR (300 MHz, CDCl3) d
7.67–7.74 (m, 3H), 7.45 (dd, J = 1.5 and 8.5 Hz, 1H),
7.08–7.18 (m, 2H), 4.70 (ABq, JAB = 14.5 Hz,
DmAB = 58.2 Hz, 2H), 4.53–4.67 (m, 2H), 4.02 (q,
J = 7.1 Hz, 1H), 3.92 (s, 3H), 3.56–3.77 (m, 2H), 2.97
5.4.6. (N-i-Propyl-N-(2-(nitrooxy)ethyl)carbamoyl)meth-
yl(2S)-2-(6-methoxy(2-naphthyl))propanoate (4c)
5.4.6.1. i-Propyl(2-(nitroxy)ethyl)ammonium nitrate
(4c-I). i-Propyl(2-(hydroxy)ethyl)amine (5 g, 48 mmol)
in EtOAc (60 mL) was added dropwise to a mixture of
fuming HNO3 (15.2 g, 10.1 mL, 242 mmol) and Ac2O
(39.6 g, 36.6 mL, 387.7 mmol) at À10 ꢁC. The reaction
mixture was stirred at À10 ꢁC for 30 min and diluted
with EtOAc and hexane. The oil layer was separated
and triturated with ice-cold EtOAc. The solid was fil-
tered and dried in high vacuo to give 4c-I (2.4 g, 23%)
1
as a white solid. Mp 102–105 ꢁC. H NMR (300 MHz,
DMSO-d6) d 8.50-8.72 (br s, 2H), 4.77 (t, J = 4.9 Hz,
2H), 3.32–3.40 (m, 3H), 1.23 (d, J = 6.5 Hz, 6H).

R. R. Ranatunge et al. / Bioorg. Med. Chem. 14 (2006) 2589–2599
2595
13C NMR (75 MHz, DMSO-d6) d 69.2, 50.2, 41.4, 18.5.
MS (API-TIS) m/z 149 (MH+).
(75 MHz, CDCl3) d 174.4, 167.0, 157.7, 135.3, 133.9,
129.4, 129.0, 127.3, 126.4, 126.2, 119.1, 105.7, 70.3,
62.0, 56.5, 55.4, 45.2, 39.4, 31.6, 25.8, 25.0, 18.7. MS
(API-TIS) m/z 459 (MH+), 476 (MNH4+).
5.4.6.2. (N-i-Propyl-N-(2-(nitrooxy)ethyl)carbamoyl)-
methyl(2S)-2-(6-methoxy(2-naphthyl))propanoate (4c). A
mixture of 3 (1.5 g, 5.2 mmol), 4c-I (1.1 g, 5.2 mmol),
and DMAP (0.64 g, 5.2 mmol) in CH2Cl2 (10 mL) at
0 ꢁC was treated with 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride (1 g, 5.2 mmol). The
reaction mixture was stirred at 0 ꢁC for 3 h, diluted with
more CH2Cl2, washed with water, brine, and dried over
Na2SO4. The residue after filtration and evaporation
was chromatographed on silica gel eluting with 1:3
EtOAc/hexane to give 4c (0.49 g, 22%) as a colorless
5.5. {[2-(Cyclohexylamino)ethyl]oxycarbonyl}methyl
(2S)-2-(6-methoxy(2-naphthyl))propanoate, nitric acid
salt 4d-II
1
Mp 135–137 ꢁC. H NMR (300 MHz, CDCl3) d 8.50–
8.75 (br s, 2H), 7.70 (d, J = 8.7 Hz, 3H), 7.40 (dd,
J = 1.7 and 8.4 Hz, 1H), 7.06–7.17 (m, 2H), 4.63 (ABq,
JAB = 16.1 Hz, DmAB = 18.9 Hz, 2H), 4.27–4.43 (m,
2H), 3.96 (q, J = 7.2 Hz, 1H), 3.90 (s, 3H), 3.04–3.20
(m, 2H), 2.82–2.98 (m, 1H), 1.78–2.02 (m, 2H), 1.68–
1.80 (m, 2H), 1.59 (d, J = 7.2 Hz, 3H), 1.00–1.47 (m,
6H). 13C NMR (75 MHz, CDCl3) d 174.4, 167.6,
157.8, 135.1, 133.8, 129.4, 129.0, 127.3, 126.4, 126.2,
119.2, 105.7, 60.8, 60.4, 58.0, 55.4, 45.1, 43.4, 29.0,
24.8, 24.5, 18.6. MS (API-TIS) m/z 414 (MH+). Anal.
Calcd for C24H32N2O8: C, 60.49; H, 6.77; N, 5.88.
Found: C, 60.47; H, 6.54; N, 5.78.
1
oil. H NMR (300 MHz, CDCl3) d 7.72–7.69 (m, 3H),
7.44 (dd, J = 1.6 and 8.5 Hz, 1H), 7.11–7.16 (m, 2H),
4.74 (ABq, JAB = 14.3 Hz, DmAB = 65.4 Hz, 2H), 4.54–
4.65 (m, 2H), 4.01 (q, J = 7.2 Hz, 1H), 3.91 (s, 3H),
3.75–3.82 (m, 1H), 3.43–3.52 (m, 2H), 1.64 (d,
J = 7.1 Hz, 3H), 1.15 (d, J = 6.7 Hz, 3H), 1.14 (d,
J = 6.7 Hz, 3H). 13C NMR (75 MHz, CDCl3) d 174.4,
166.9, 157.7, 135.3, 133.8, 129.4, 129.0, 127.3, 126.4,
126.2, 119.1, 105.6, 70.1, 61.9, 55.4, 47.7, 45.2, 38.5,
21.1, 18.7. MS (API-TIS) m/z 419 (MH+), 436
(MNH4+). LCMS 97%.
5.5.1. 2-(4-((Nitrooxy)methyl)piperidyl)-2-oxoethyl (2S)-
2-(6-methoxy(2-naphthyl))propanoate (5a)
5.5.1.1. Nitrooxy(4-piperidylmethyl)hydrogen nitrate
(5a-I). 4-Piperidylmethan-1-ol (2.5 g, 21.7 mmol) in a
mixture of EtOAc (25 mL) and CH2Cl2 (5 mL) was add-
ed dropwise to a mixture of fuming HNO3 (6.83 g,
4.6 mL, 109 mmol) and Ac2O (17.7 g, 16.3 mL,
174 mmol) at À10 ꢁC. The reaction mixture was stirred
at À10 ꢁC for 30 min and diluted with EtOAc and hex-
ane. The precipitate was collected by filtration and
washed with hexane to give 5a-I (2.5 g, 52%) as a pale
green solid. Mp 51–53 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 8.45–8.75 (br s, 1H), 8.10–8.40 (br s,
1H), 4.44 (d, J = 6.4 Hz, 2H), 3.22–3.35 (m, 2H), 2.80–
2.98 (m, 2H), 1.96–2.15 (m, 1H), 1.75–1.89 (m, 2H),
1.40–1.52 (m, 2H). 13C NMR (75 MHz, DMSO-d6) d
76.4, 42.7, 31.2, 24.9. MS (API-TIS) m/z 161 (MH+).
Anal. Calcd for C6H13N3O6: C, 32.29; H, 5.87; N,
18.83. Found: C, 32.03; H, 5.78; N, 18.73.
5.4.7. (N-Cyclohexyl-N-(2-(nitrooxy)ethyl)carbamoyl)-
methyl(2S)-2-(6-methoxy(2-naphthyl))propanoate (4d)
5.4.7.1.
i-Cyclohexyl(2-(nitroxy)ethyl)ammonium
nitrate (4d-I). i-Cyclohexyl(2-(hydroxy)ethyl)amine
(25 g, 174 mmol) in EtOAc (200 mL) was added drop-
wise to a mixture of fuming HNO3 (55 g, 36.7 mL,
0.87 mol) and Ac2O (143 g, 131.7 mL, 1.4 mol) at
À10 ꢁC. The reaction mixture was stirred at À10 ꢁC
for 30 min and diluted with EtOAc and hexane. The sol-
id was filtered, washed with EtOAc, and dried in high
vacuo to give 4d-I (27 g, 62%) as a white solid. Mp
127–129 ꢁC. 1H NMR (300 MHz, DMSO-d6) d 8.50–
8.60 (br s, 2H), 4.77 (t, J = 4.9 Hz, 2H), 3.32–3.45 (m,
2H), 2.48–3.14 (m, 1H), 1.90–2.05 (m, 2H), 1.65–1.80
(m, 2H), 1.50–1.61 (m, 1H), 1.00–1.30 (m, 5H). 13C
NMR (75 MHz, DMSO-d6) d 69.2, 50.2, 41.4, 18.5.
MS (API-TIS) m/z 149 (MH+). Anal. Calcd for
C8H17N3O6: C, 38.25; H, 6.82; N, 16.73. Found: C,
38.47; H, 7.05; N, 16.76.
5.5.1.2. 2-(4-((Nitrooxy)methyl)piperidyl)-2-oxoethyl
(2S)-2-(6-methoxy(2-naphthyl))propanoate (5a). A mix-
ture of 3 (1.5 g, 5.21 mmol), 5a-I (1.16 g, 5.21 mmol),
and DMAP (0.63 g, 5.21 mmol) in CH2Cl2 (30 mL) at
0 ꢁC was treated with 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride (1.0 g, 5.21 mmol).
The reaction mixture was stirred at 0 ꢁC for 3 h, diluted
with CH2Cl2, washed with water, brine, and dried over
Na2SO4. The residue after filtration and evaporation
was chromatographed on silica gel eluted with 1:2 to
1:1 EtOAc/hexane to give the title compound (1.74 g,
78%) as a white solid. Mp 94–95 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 7.75–7.82 (m, 3H), 7.44 (dd,
J = 1.3 and 8.5 Hz, 1H), 7.30 (d, J = 2.5 Hz, 1H), 7.16
(dd, J = 2.5 and 8.9 Hz, 1H), 4.65–4.89 (m, 2H), 4.35
(bd, J = 6.4 Hz, 2H), 4.21–4.32 (m, 1H), 4.01 (q,
J = 7.1 Hz, 1H), 3.86 (s, 3H), 3.55–3.73 (m, 1H),
2.81–3.02 (m, 1H), 2.45–2.63 (m, 1H), 1.87–2.03 (m,
1H), 1.55–1.72 (m, 2H), 1.51 (d, J = 7.1 Hz, 3H),
0.90–1.22 (m, 2H). 13C NMR (75 MHz, DMSO-d6) d
5.4.7.2. (N-Cyclohexyl-N-(2-(nitrooxy)ethyl)carbamo-
yl)methyl(2S)-2-(6-methoxy(2-naphthyl))propanoate (4d).
A mixture of 3 (1.5 g, 5.2 mmol), 4d-I (1.3 g, 5.2 mmol),
and DMAP (0.64 g, 5.2 mmol) in CH2Cl2 (10 mL) at
0 ꢁC was treated with 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride (1 g, 5.2 mmol). The
reaction mixture was stirred at 0 ꢁC for 3 h, diluted with
more CH2Cl2, washed with water, brine, and dried over
Na2SO4. The residue after filtration and evaporation
was chromatographed on silica gel eluting with 1:3
EtOAc/hexane to give 4d (1 g, 42%) as a colorless oil.
1H NMR (300 MHz, CDCl3) d 7.66–7.74 (m, 3H), 7.44
(dd, J = 1.7 and 8.6 Hz, 1H), 7.08–7.17 (m, 2H), 4.74
(ABq, JAB = 14.3 Hz, DmAB = 68.5 Hz, 2H), 4.52–4.63
(m, 2H), 4.01 (q, J = 7.2 Hz, 1H), 3.91 (s, 3H), 3.41–
3.62 (m, 2H), 3.18–3.32 (m, 1H), 1.65–1.88 (m, 5H),
1.64 (d, J = 7.2 Hz, 3H), 0.90–1.48 (m, 5H). 13C NMR

2596
R. R. Ranatunge et al. / Bioorg. Med. Chem. 14 (2006) 2589–2599
173.5, 164.3, 157.2, 135.5, 133.3, 129.1, 128.4, 126.9,
126.5, 125.8, 118.7, 105.6, 76.9, 61.8, 55.1, 44.3, 43.2,
33.2, 28.2, 27.5, 18.6. MS (API-TIS) m/z 431 (MH+),
448 (MNH4+). Anal. Calcd for C22H26N2O7: C, 61.39;
H, 6.09; N, 6.51. Found: C, 61.24; H, 5.98; N, 6.40.
product was used without further purification. Mass
spectrum (API-TIS) m/z 176 (MH+).
5.5.3.2. 2-(4-(2-(Nitrooxy)ethyl)piperazinyl)-2-oxoethyl
(2S)-2-(6-methoxy(2-naphthyl))propanoate (5c). A mix-
ture of the product of 3 (2.67 g, 9.27 mmol), 5c-I
(2.2 g, 9.27 mmol), and DMAP (3.39 g, 27.8 mmol) in
CH2Cl2 (20 mL) and DMF (9.3 mL) at 0 ꢁC was treated
with 1-(3-(dimethylamino) propyl)-3-ethylcarbodiimide
hydrochloride (1.78 g, 9.27 mmol). The reaction mixture
was stirred at 0 ꢁC for 3 h, diluted with CH2Cl2, washed
with saturated NaHCO3, and dried over Na2SO4. The
residue after filtration and evaporation was chromato-
graphed on silica gel eluted with MeOH/EtOAc/CH2Cl2
(1:25:25) to give 5c (1.2 g, 29% yield) as an oil. 1H NMR
(300 MHz, DMSO-d6) d 7.65–7.72 (m, 3H), 7.43 (dd,
J = 1.6 and 8.5 Hz, 1H), 7.07–7.16 (m, 2H), 4.66 (ABq,
JAB = 14.0 Hz, DmAB = 28.0 Hz, 2H), 4.43 (t,J = 5.5 Hz,
2H), 3.98 (q, J = 7.1 Hz, 1H), 3.89 (s, 3H), 3.46–3.61
(m, 2H), 3.14–3.23 (m, 2H), 2.49–2.56 (m, 2H), 2.32–
2.44 (m, 2H), 2.12–2.20 (m, 2H), 1.63 (d, J = 7.2 Hz,
3H). 13C NMR (75 MHz, DMSO-d6) d 174.2, 172.2,
164.9, 157.8, 135.4, 135.8, 129.4, 129.0, 127.3, 126.4,
126.3, 119.1, 105.7, 70.1, 62.1, 55.4, 54.8, 52.9, 45.3,
44.6, 41.9, 18.7. Mass spectrum (API-TIS) m/z 446
(MH+). Anal. Calcd for C22H27N3O7: C, 59.32; H,
6.11; N, 9.43. Found: C, 59.11; H, 5.86; N, 9.24.
5.5.2. 2-(4-(2-(Nitrooxy)ethyl)piperidyl)-2-oxoethyl (2S)-
2-(6-methoxy(2-naphthyl))propanoate (5b)
5.5.2.1. Nitrooxy(2-(4-piperidyl)ethyl)hydrogennitrate
(5b-I). 4-Piperidylethan-1-ol (10 g, 77.3 mmol) in a
mixture of EtOAc (90 mL) and CH2Cl2 (5 mL) was
added dropwise to
a mixture of fuming HNO3
(24.4 g, 16.3 mL, 387 mmol) and Ac2O (63.2 g,
58.4 mL, 619 mmol) at À10 ꢁC. The reaction mixture
was stirred at À10 ꢁC for 30 min and then diluted
with EtOAc and hexane. The precipitate was collected
by filtration and washed with hexane to give 5b-I
1
(7.5 g, 41% yield) as a white solid. Mp 86–88 ꢁC. H
NMR (300 MHz, DMSO-d6) d 8.20–8.30 (br s, 1H),
8.05–8.20 (br s, 1H), 4.57 (t, J = 6.2 Hz, 2H), 3.15–
3.30 (m, 2H), 2.70–2.90 (m, 2H), 1.75–1.90 (m, 2H),
1.55–1.72 (m, 3H), 1.19–1.37 (m, 2H). 13C NMR
(75 MHz, DMSO-d6) d 71.6, 43.2, 31.9, 30.0, 28.1.
Mass spectrum (API-TIS) m/z 175 (MH+). Anal.
Calcd for C7H15N3O6: C, 35.44; H, 6.37; N, 17.71.
Found: C, 35.62; H, 6.39; N, 17.65.
5.5.2.2. 2-(4-(2-(Nitrooxy)ethyl)piperidyl)-2-oxoethyl
(2S)-2-(6-methoxy(2-naphthyl))propanoate (5b). A mix-
ture of 3 (1.5 g, 5.21 mmol), 5b-I (1.2 g, 5.21 mmol),
and DMAP (0.63 g, 5.21 mmol) in CH2Cl2 (30 mL) at
0 ꢁC was treated with 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride (1.0 g, 5.21 mmol).
The reaction mixture was stirred at 0 ꢁC for 3 h, diluted
with CH2Cl2, washed with water, brine, and dried over
Na2SO4. The residue after filtration and evaporation
was chromatographed on silica gel eluted with EtOAc/
hexane (1:3 to 1:2 to 1:1) to give 5b (1.0 g, 43% yield)
5.5.4. 2-(4-(2-(Nitrooxy)ethyl)piperazinyl)-2-oxoethyl(2-
S)-2-(6-methoxy(2-naphthyl))propanoate hydrogen chlo-
ride (5d). To a solution of 5c (0.3 g, 0.67 mmol) in EtOAc
(4.3 mL) at 0 ꢁC was added dropwise a solution of HCl
gas in Et2O (24 mg, 0.33 mL, 2 M solution, 0.67 mmol).
The cloudy solution was dissolved in excess EtOAc and
hexane was added. The solvent was evaporated to a small
volume. The precipitate was filtered and washed with
hexane to give 5d (0.3 g, 92% yield) as a white solid.
Mp 129–131 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
11.65–12.00 (br s, 1H), 7.73–7.82 (m, 3H), 7.44 (dd,
J = 1.4 and 8.6 Hz, 1H), 7.28–7.32 (m, 1H), 7.16 (dd,
J = 2.5 and 8.9 Hz, 1H), 4.78–4.97 (m, 4H), 4.22–4.45
(m, 1H), 4.03 (q, J = 7.1 Hz, 1H), 3.87 (s, 3H), 3.80–
4.05 (m, 1H), 3.35–3.67 (m, 5H), 2.90–3.30 (m, 3H),
1.52 (d, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-
d6) d 173.5, 165.0, 157.2, 135.5, 133.3, 129.1, 128.4,
126.9, 126.5, 125.8, 118.7, 105.7, 67.4, 61.4, 55.2, 50.8,
18.7. Mass spectrum (API-TIS) m/z 446 (MH+). Anal.
Calcd for C22H28ClN3O7: C, 54.83; H, 5.86; N, 8.72.
Found: C, 54.78; H, 5.89; N, 8.65.
1
as a white solid. Mp 83–85 ꢁC. H NMR (300 MHz,
DMSO-d6) d 7.65–7.75 (m, 3H), 7.44 (d, J = 8.5 Hz,
1H), 7.07–7.18 (m, 2H), 4.46–4.81 (m, 3H), 4.30–4.44
(m, 2H), 3.99 (q, J = 6.9 Hz, 1H), 3.91 (s, 3H), 3.50–
3.59 (m, 1H), 2.75–2.93 (m, 1H), 2.41–2.60 (m, 1H),
1.63 (d, J = 7.9 Hz, 3H), 1.32–1.74 (m, 5H), 0.69–1.20
(m, 2H). 13C NMR (75 MHz, DMSO-d6) d 174.3,
164.8, 157.8, 135.5, 133.9, 129.4, 129.0, 127.3, 126.5,
126.4, 119.1, 105.7, 70.7, 62.3, 55.4, 45.4, 44.8, 42.2,
33.0, 32.6, 32.0, 31.4, 18.4. Mass spectrum (API-TIS)
m/z 445 (MH+), 462 (MNH4+). Anal. Calcd for
C23H28N2O7: C, 62.15; H, 6.35; N, 6.30. Found: C,
62.21; H, 6.42; N, 6.26.
5.5.5. 2-(2-((2S)-2-(6-Methoxy(2-naphthyl))propanoyl-
oxy)-N-methylacetylamino)acetic acid (6)
5.5.3. 2-(4-(2-(Nitrooxy)ethyl)piperazinyl)-2-oxoethyl
(2S)-2-(6-methoxy(2-naphthyl))propanoate (5c)
5.5.5.1. (N-(((tert-Butyl)oxycarbonyl)methyl)-N-meth-
ylcarbamoyl)methyl (2S)-2-(6-methoxy(2-naphthyl))pro-
panoate (6a). A mixture of 3 (10 g, 34.7 mmol),
sarcosine tert-butyl ester hydrochloride (6.3 g,
34.7 mmol), and DMAP (4.24 g, 34.7 mmol) in CH2Cl2
(125 mL) at 0 ꢁC was treated with 1-(3-(dimethylami-
no)propyl)-3-ethylcarbodiimide hydrochloride (6.7 g,
34.7 mmol). The reaction mixture was stirred at room
temperature for 16 h, diluted with CH2Cl2, washed with
water, brine, and dried over Na2SO4. The residue after
filtration and evaporation was chromatographed on
5.5.3.1. Nitrooxy(2-piperazinylethyl)dihydrogennitrate
(5c-I). 2-Piperazinylethan-1-ol (15 g, 115 mmol) in a
mixture of EtOAc (132 mL) and CH2Cl2 (5 mL) was
added dropwise to a mixture of fuming HNO3 (36.3 g,
24.2 mL, 576 mmol) and Ac2O (94.1 g, 87 mL,
921 mmol) at À10 ꢁC. The reaction mixture was stirred
at À10 ꢁC for 30 min and diluted with EtOAc and hex-
ane. The oil was separated and dried under vacuum to
give 5c-I (5.1 g, 19% yield) as a sticky oil. The crude

R. R. Ranatunge et al. / Bioorg. Med. Chem. 14 (2006) 2589–2599
2597
silica gel eluted with 1:9 to 1:1 EtOAc/hexane to give 6a
(11.8 g, 82% yield) as a white solid. Mp 103–104 ꢁC. H
(m, 1H), 7.07–7.17 (m, 2H), 6.53–6.60 (br s, 1H), 4.68
(ABq, JAB = 14.3 Hz, DmAB = 39.6 Hz, 2H), 4.47 (t,
J = 5.3 HZ, 2H), 3.93–4.07 (m, 3H), 3.91 (s, 3H), 3.46–
3.58 (m, 2H), 3.01 (br s, 3H), 1.63 (d, J = 7.1 Hz, 3H).
13C NMR (75 MHz, CDCl3) d 175.0, 168.9, 167.9, 157.9,
135.2, 133.9, 129.4, 129.0, 127.3, 126.4, 126.3, 119.2,
105.8, 71.3, 61.6, 55.4, 52.3, 45.2, 37.0, 35.8, 18.6. Mass
spectrum (API-TIS) m/z 448 (MH+), 465 (MNH4).
1
NMR (300 MHz, CDCl3) d 7.65–7.77 (m, 3H), 7.44 (dd,
J = 1.3 and 8.9 Hz, 1H), 7.03–7.18 (m, 2H), 4.74 (ABq,
JAB = 14.5 Hz, DmAB = 64.7 Hz, 2H), 3.72–4.19 (m,
3H), 3.90 (s, 3H), 2.93 (s, 3H), 1.63 (d, J = 7.1 Hz,
3H), 1.45 (s, 9H). 13C NMR (75 MHz, CDCl3) d
174.3, 168.0, 167.1, 157.8, 135.5, 133.9, 129.5, 129.1,
127.3, 126.5, 126.3, 119.0, 105.8, 82.2, 61.5, 55.4, 51.6,
45.3, 35.3, 28.2, 18.8. Mass spectrum (API-TIS) m/z
416 (MH+), 433 (MNH4+), 438 (MNa+).
5.5.7. (N-Methyl-N-(((2-(nitrooxy)ethyl)oxycarbonyl)meth-
yl)carbamoyl)methyl (2S)-2-(6-methoxy(2-naphthyl))pro-
panoate (7b)
5.5.5.2. 2-(2-((2S)-2-(6-Methoxy(2-naphthyl))propa-
noyloxy)-N-methylacetylamino)acetic acid (6). Trifluoro-
acetic acid (5 mL) was added dropwise to a solution of
the product of 6a (3.54 g, 8.5 mmol) in CH2Cl2
(15 mL). The reaction mixture was stirred at room tem-
perature for 24 h, and the volatile material was removed
in vacuo. The residue, after evaporation, was recrystal-
lized from CH2Cl2/EtOAc/hexane to give 6 (2.55 g,
5.5.7.1. (N-(((2-Hydroxyethyl)oxycarbonyl)methyl)-N-
methylcarbamoyl)methyl
(2S)-2-(6-methoxy(2-naph-
thyl))propanoate (7b-I). To a solution of 6 (0.5 g,
1.4 mmol), DMAP (84.9 mg, 0.69 mmol), and ethylene
glycol (1.6 g, 27.8 mmol) in CH2Cl2 (4 mL) was added
dropwise a solution of DCC (0.36 g, 1.7 mmol) in
CH2Cl2 (2 mL). The reaction mixture was stirred at
room temperature for 16 h. The precipitate was filtered.
The residue after evaporation of the solvent was redis-
solved in EtOAc, washed with water, brine, and dried
over Na2SO4. The residue after filtration and evapora-
tion was chromatographed on silica gel eluted with
1:1:0.1 EtOAc/hexane/MeOH to give 7b-I (0.4 g, 71%
yield) as a white solid. Mp 113–114 ꢁC. 1H NMR
(300 MHz, CDCl3) d 7.67–7.73 (m, 3H), 7.44 (dd,
J = 1.6 and 8.6 Hz, 1H), 7.11–7.15 (m, 2H), 4.74 (ABq,
JAB = 14.6 Hz, DmAB = 55.9 Hz, 2H), 4.22–4.31 (m,
2H), 4.09–4.12 (br s, 2H), 4.01 (q, J = 7.2 Hz, 1H),
3.90 (s, 3H), 3.74–3.83 (m, 2H), 2.99 (s, 3H), 2.40 (br
s, 1H), 1.64 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz,
CDCl3) d 174.6, 168.9, 167.6, 157.8, 135.3, 133.9,
129.4, 129.0, 127.3, 126.5, 126.3, 119.1, 105.8, 67.1,
61.6, 60.9, 55.4, 50.2, 45.3, 35.5, 18.6. Mass spectrum
(API-TIS) m/z 404 (MH+), 421 (MNH4+). Anal. Calcd
for C21H25NO7Æ1 mol H2O: C, 59.85; H, 6.46; N, 3.32.
Found: C, 59.94; H, 6.15; N, 3.39.
1
83% yield) as a white solid. Mp 174–175 ꢁC. H NMR
(300 MHz, DMSO-d6) d 7.76–7.80 (m, 3H), 7.42–7.45
(m, 1H), 7.27–7.29 (m, 1H), 7.15 (dd, J = 2.5 and
8.5 Hz, 1H), 4.84 (ABq, JAB = 15.1 Hz, DmAB = 26.9 Hz,
2H), 3.93–4.12 (m, 3H), 3.87 (s, 3H), 2.94 (s, 3H), 1.51
(d, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) d
173.4, 170.5, 166.6, 157.2, 135.5, 133.3, 129.2, 128.4,
126.9, 126.5, 125.8, 118.7, 105.8, 61.4, 55.2, 48.9, 44.3,
34.5, 18.7. Mass spectrum (API-TIS) m/z 360 (MH+),
377 (MNH4+), 382 (MNa+). Anal. Calcd for
C19H21NO6: C, 63.50; H, 5.89; N, 3.90. Found: C,
63.33; H, 5.90; N, 3.89.
5.5.6. (N-Methyl-N-((N-(2-(nitrooxy)ethyl)carbamoyl)meth-
yl)carbamoyl)methyl(2S)-2-(6-methoxy(2-naphthyl))pro-
panoate (7a)
5.5.6.1. 2-(Nitrooxy)ethylammonium nitrate (7a-I). 2-
Hydroxyethylamine (5 g, 81.9 mmol) in EtOAc
(40 mL) was added dropwise to a mixture of fuming
HNO3 (18 g, 12 mL, 0.29 mol) and Ac2O (54 g, 50 mL,
0.53 mol) at À10 ꢁC. The reaction mixture was stirred
at 0 ꢁC for 10 min and at room temperature for
10 min. The solvent was evaporated in high vacuo at
40 ꢁC. The residue was sonicated with ether. The solid
was filtered and washed with hexane to give 7a-I as a
white solid. Mp 92–94 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 7.71–8.25 (br s, 3H), 4.72 (t, J = 5.0 Hz,
2H), 3.23 (d, J = 5.0 Hz, 2H). 13C NMR (75 MHz,
DMSO-d6) d 70.9, 37.4.
5.5.7.2. (N-Methyl-N-(((2-(nitrooxy)ethyl)oxycarbon-
yl)methyl)carbamoyl)methyl(2S)-2-(6-methoxy(2-naphthyl))-
propanoate (7b).
A
suspension of 7b-I (0.26 g,
0.64 mmol) in EtOAc (0.28 mL) was added dropwise
to a mixture of fuming HNO3 (61.3 mg, 41 lL,
0.97 mmol) and Ac2O (148 mg, 137 lL, 1.45 mmol) at
0 ꢁC. The reaction mixture was stirred at 0 ꢁC for 3 h.
Then an additional ice-cold mixture of fuming HNO3
(61.3 mg, 41 lL, 0.97 mmol) and Ac2O (148 mg,
137 lL, 1.45 mmol) was added dropwise to the reaction
mixture at 0 ꢁC. The stirring was continued for another
2 h. The reaction mixture was diluted with EtOAc,
washed with ice-cold saturated NaHCO3, water, and
dried over Na2SO4. The residue after filtration and evap-
oration was chromatographed on silica gel eluted with
1:1:0.1 EtOAc/hexane/MeOH to give 7b (0.2 g, 69%
yield) as a white solid. Mp 71–73 ꢁC. 1H NMR
(300 MHz, CDCl3) d 7.66–7.75 (m, 3H), 7.44 (dd,
J = 1.6 and 8.5 Hz, 1H), 7.08–7.17 (m, 2H), 4.76 (ABq,
JAB = 14.6 Hz, DmAB = 59.7 Hz, 2H), 4.64–4.69 (m,
2H), 4.39–4.42 (m, 2H), 4.14 (ABq, JAB = 17.4 Hz,
DmAB = 32.0 Hz, 2H), 3.93–4.07 (m, 1H), 3.91 (s, 3H),
2.98 (s, 3H), 1.64 (d, J = 7.2 Hz, 3H). 13C NMR
(75 MHz, CDCl3) d 174.3, 168.6, 167.4, 157.8, 135.4,
5.5.6.2. (N-Methyl-N-((N-(2-(nitrooxy)ethyl)carbamo-
yl)methyl)carbamoyl)methyl(2S)-2-(6-methoxy(2-naphthyl))-
propanoate (7a). A mixture of 6 (0.5 g, 1.39 mmol), 7a-I
(0.26 g, 1.53 mmol), and DMAP (0.17 g, 1.39 mmol) in
CH2Cl2 (8 mL) at 0 ꢁC was treated with 1-(3-(dimethyla-
mino)propyl)-3-ethylcarbodiimide hydrochloride (0.29 g,
1.53 mmol). The reaction mixture was stirred at 0 ꢁC for
3 h, diluted with CH2Cl2, washed with water, brine, and
dried over Na2SO4. The residue after filtration and evapo-
ration was chromatographed on silica gel eluted with 1:2
EtOAc/hexane to 1:1:0.1 EtOAc/hexane/MeOH to give
1
7a (0.61 g, 89% yield) as a white solid. Mp 79–81 ꢁC. H
NMR (300 MHz, CDCl3) d 7.67–7.75 (m, 3H), 7.35–7.45

2598
R. R. Ranatunge et al. / Bioorg. Med. Chem. 14 (2006) 2589–2599
133.9, 129.5, 129.0, 127.3, 126.5, 126.3, 119.1, 105.7,
70.2, 61.5, 61.1, 55.4, 49.5, 45.3, 35.3, 18.7. Mass spec-
trum (API-TIS) m/z 449 (MH+), 466 (MNH4+). Anal.
Calcd for C21H24N2O9: C, 56.25; H, 5.39; N, 6.25.
Found: C, 56.00; H, 5.17; N, 6.09.
49.7, 45.3, 35.3, 26.4, 18.7. Mass spectrum (API-TIS)
m/z 463 (MH+), 480 (MNH4+). Anal. Calcd for
C22H26N2O9: C, 57.14; H, 5.67; N, 6.06. Found: C,
56.91; H, 5.86; N, 6.02.
5.5.9. [N-Methyl-N-({N-methyl-N-[2-(nitrooxy)ethyl]car-
bamoyl}methyl)carbamoyl]methyl(2S)-2-(6-methoxy(2-
naphthyl))propanoate (7d). A mixture of 6 (1.5 g,
4.2 mmol), 4a-I (0.71 g, 4.2 mmol), and DMAP
(0.51 g, 4.2 mmol) in CH2Cl2 (24 mL) at 0 ꢁC was
treated with 1-(3-(dimethylamino)propyl)-3-ethylcarbo-
diimide hydrochloride (0.88 g, 4.5 mmol). The reaction
mixture was stirred at 0 ꢁC for 3 h, diluted with
CH2Cl2, washed with water, brine, and dried over
Na2SO4. The residue after filtration and evaporation
was chromatographed on silica gel eluted with 1:2
EtOAc/hexane to 1:1:0.1 EtOAc/hexane/MeOH to give
5.5.8. (N-Methyl-N-(((3-(nitrooxy)propyl)oxycarbonyl)meth-
yl)carbamoyl)methyl (2S)-2-(6-methoxy(2-naphthyl))pro-
panoate (7c)
5.5.8.1. (N-(((3-Hydroxypropyl)oxycarbonyl)methyl)-
N-methylcarbamoyl)methyl(2S)-2-(6-methoxy(2-naph-
thyl))propanoate (7c-I). To a solution of 6 (0.5 g,
1.4 mmol), DMAP (84.9 mg, 0.69 mmol), and 1,3-pro-
panediol (2.1 g, 27.8 mmol) in CH2Cl2 (4 mL) was add-
ed dropwise a solution of DCC (0.36 g, 1.7 mmol) in
CH2Cl2 (2 mL). The reaction mixture was stirred at
room temperature for 16 h. The precipitate was filtered.
The residue after evaporation of the solvent was redis-
solved in EtOAc, washed with water, brine, and dried
over Na2SO4, filtered, and evaporated. The residue
was chromatographed on silica gel eluted with 1:1:0.1
EtOAc/hexane/MeOH to give 7c-I (0.35 g, 60% yield)
1
7d (0.8 g, 42% yield) as a sticky white solid. H NMR
(300 MHz, CDCl3) d 7.67–7.75 (m, 3H), 7.35–7.45 (m,
1H), 7.11–7.17 (m, 2H), 4.78 (ABq, JAB = 14.6 Hz,
DmAB = 59.5 Hz, 2H), 4.59 (t, J = 5.2 HZ, 2H), 4.18
(ABq, JAB = 16.2 Hz, DmAB = 51.5 Hz, 2H), 3.96–4.05
(m, 1H), 3.92 (s, 3H), 3.65–3.69 (m, 2H), 3.06 (br s,
3H), 2.98 (br s, 3H), 1.65 (d, J = 7.2 Hz, 3H). 13C
NMR (75 MHz, CDCl3) d 174.4, 168.2, 167.3, 157.7,
135.4, 133.8, 129.4, 129.0, 127.3, 126.5, 126.3, 119.1,
105.7, 71.1, 61.6, 55.4, 49.4, 46.3, 45.3, 36.3, 35.3,
18.6. Mass spectrum (API-TIS) m/z 462 (MH+), 479
(MNH4+). LCMS 99.4%.
1
as a white solid. Mp 80–82 ꢁC. H NMR (300 MHz,
CDCl3) d 7.40–7.73 (m, 3H), 7.43 (dd, J = 1.5 and
8.6 Hz, 1H), 7.07–7.15 (m, 2H), 4.75 (ABq,
JAB = 14.6 Hz,
DmAB = 57.7 Hz,
2H),
4.28
(t,
J = 5.9 Hz, 2H), 3.95–4.20 (m, 3H), 3.90 (s, 3H),
3.61–3.72 (m, 2H), 2.97 (s, 3H), 1.78–2.00 (m, 3H),
1.63 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d 174.4, 169.1, 167.3, 157.8, 135.3, 133.8, 129.4,
129.0, 127.2, 126.4, 126.2, 119.0, 105.7, 62.4, 61.5,
59.0, 55.4, 49.8, 45.2, 35.3, 31.6, 18.7. Mass spectrum
(API-TIS) m/z 418 (MH+), 435 (MNH4+), 440
(MNa+). Anal. Calcd for C22H27NO7Æ1/4 mol H2O: C,
62.62; H, 6.58; N, 3.32. Found: C, 62.35; H, 6.47; N,
3.44.
Acknowledgment
The authors thank Dr. Radha Iyengar for helpful com-
ments and suggestions.
5.5.8.2.
bonyl)methyl)carbamoyl)methyl
naphthyl))propanoate (7c).
(N-Methyl-N-(((3-(nitrooxy)propyl)oxycar-
References and notes
(2S)-2-(6-methoxy(2-
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A
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