Pyrrolidinobenzoic acid inhibitors of influenza virus neuraminidase: Modifications of essential pyrrolidinone ring substituents

Article abstract of DOI:10.1016/S0968-0896(03)00271-2

We recently reported the first benzoic acid, 1-[4-carboxy-2-(3-pentylamino)phenyl]-5,5-bis(hydroxymethyl)pyrrolidin-2-one (8), that is a potent inhibitor of avian influenza A neuraminidase (N9) and, unlike other reported potent neuraminidase inhibitors, d

Full text of DOI:10.1016/S0968-0896(03)00271-2

Bioorganic & Medicinal Chemistry 11 (2003) 2739–2749
Pyrrolidinobenzoic Acid Inhibitors of Influenza Virus Neuraminidase:
Modifications of Essential Pyrrolidinone Ring Substituents
Wayne J. Brouillette,a,b,* Saroj N. Bajpai,a Shoukath M. Ali,a
Sadanandan E. Velu,b Venkatram R. Atigadda,a Barbara S. Lommer,c
James B. Finley,c Ming Luob,c and Gillian M. Aird
aDepartment of Chemistry, 901 14th Street South, CHEM 201, The University of Alabama at Birmingham, Birmingham, AL 35294, USA
bCenter for Biophysical Sciences and Engineering, The University of Alabama at Birmingham, Birmingham, AL 35294, USA
cDepartment of Microbiology, The University of Alabama at Birmingham, Birmingham, AL 35294, USA
dDepartment of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA
Received 12 August 2002; accepted 2 April 2003
Abstract—We recently reported the first benzoic acid, 1-[4-carboxy-2-(3-pentylamino)phenyl]-5,5-bis(hydroxymethyl)pyrrolidin-2-
one (8), that is a potent inhibitor of avian influenza A neuraminidase (N9) and, unlike other reported potent neuraminidase inhi-
bitors, does not contain a basic aliphatic amine or guanidine nor a simple N-acetyl grouping. However, 8 was a poor inhibitor of
influenza B neuraminidase. In the present study we further evaluated 8 as an inhibitor of human influenza A NA isolates, and it was
effective against N2 NA but found to be 160-fold less active against N1 NA. We also synthesized analogues of 8 involving moderate
modifications of essential substituents on the pyrrolidinone ring. Specifically, the aminomethyl (9), hydroxyethyl (10), and ami-
noethyl (11) analogues were prepared. Only the most conservative change (compound 9) resulted in continued effective inhibition of
influenza A, in addition to a noteworthy increase in the activity of 9 for N1 NA. The effectiveness of 9 against influenza B neur-
aminidase was furthermore improved 10-fold relative to 8, but this activity remained 50-fold poorer than for type A NA.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
A new viral target has recently been exploited to
develop clinically useful anti-influenza drugs. This tar-
get, the neuraminidase (NA, also called sialidase), is one
of two major surface glycoproteins of influenza viruses;
the second is the hemagglutinin (HA). Both play
important roles in the infectious and antigenic proper-
ties of the virus. HA is required for attachment of the
virus to host cells through interaction with sialic acid
residues. NA catalyses the cleavage of a-glycosidic
bonds to the terminal sialic acid residues to facilitate
spread of newly-budded virus.2
Influenza virus causes major respiratory tract disease in
humans resulting every year in excess morbidity and
mortality. Epidemics of influenza occur every winter
and are responsible for approximately 20,000 deaths per
year in the United States.1 It is particularly dangerous
to the very young, elderly, and to those who have sup-
pressed immune systems. Vaccination has been the main
preventive measure and has provided limited control,
but vaccines must be reformulated each year in response
to antigenic drift and may be ineffective against new
variants of influenza viruses. While influenza drugs are
an attractive alternative to vaccines, until recently the
only effective drugs were amantidine and rimantidine,
which have limited use since they are selective for influ-
enza A viruses and susceptible strains rapidly become
resistant.
Neuraminidase is a tetrameric 240 kDA protein and
appears as mushroom-shaped heads on thin stalks pro-
jecting out of the virion membrane. Based on their
antigenic properties, nine subtypes (N1–N9) of neur-
aminidase have been found in influenza A viruses, while
no distinct subtypes of neuraminidase have been descri-
bed in influenza B viruses. Despite the fact that the
protein sequence identity among different strains is only
about 30%, the catalytic site for all influenza A and B
virus neuraminidases is completely conserved, which is
*Corresponding author. Tel.: +1-205-934–8288; fax: +1-205-934-
2543; e-mail: wbrou@uab.edu
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00271-2

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one reason that NA has been considered an attractive
target for antiviral drug development.
as a template because it provides planarity near the
carboxylate group that mimics the transition state-like
structure of Neu5Ac2en and its derivatives. Further, the
benzene ring has no stereogenic centers at substitution
sites, and such inhibitors potentially offered a more
economical synthesis of anti-influenza drugs. Our lead
benzoic acid inhibitor9 was BANA 113 (7, Ki 2.5 mM).
Optimization of BANA 113 resulted in the potent ben-
zoic acid inhibitor 8, which contained a substituted
pyrrolidinone ring in place of the N-acetyl functionality
and exhibited an IC50 of 48 nm against influenza A NA
(N9, an avian strain).10 Thus compound 8 satisfied both
of the above goals, since it contains no chiral carbons
and also lacks a strongly basic nitrogen. However,
compound 8 is nevertheless a chiral molecule resulting
from atropisomerism due to restricted rotation about
the aromatic C to pyrrolidinone N bond, and this com-
pound was prepared and evaluated as the racemic mix-
ture. This compound class is unique among known NA
inhibitors.
Influenza neuraminidase contains 18 conserved amino
acid residues in the active site. Mutations of these con-
served residues generally result in enzyme inactiva-
tion,2d suggesting that the virus may not easily escape
sialidase-targeted drug therapy. Sialic acid (1, Neu5Ac,
NANA), the product of the enzyme reaction, is a weak
inhibitor with a Ki of about 1 mM. The first moderately
effective inhibitor to be described (over 25 years ago!) is
the dehydrated analogue of sialic acid, 2-deoxy-2,3-
didehydro-N-acetylneuraminic acid (2, Neu5Ac2en,
DANA, Ki 10 mM).3 In recent years, several very potent
inhibitors of both influenza A and B NA have been
developed that are either in human clinical trials or
have been marketed as anti-influenza drugs. Zanami-
vir4 (GG167, 4-Guan-Neu5Ac2en) (3) is a potent
inhibitor (IC50 5 nm) of both influenza A and B virus
neuraminidases. Zanamivir, although orally inactive
and rapidly excreted from the body, is effective in
both the prevention and the treatment of influenza
when administered directly into the lungs or nose.5
The first orally active neuraminidase inhibitor to be
marketed was based upon GS 4071 (4), a potent inhi-
bitor of both influenza A and B NA.6a Its ethyl ester
serves as the orally active prodrug form, GS 4104
(5).6b A second orally active influenza neuraminidase
inhibitor, BCX-1812 (6),7 also a potent inhibitor of
both influenza A and B NA, recently entered Phase
III human clinical trials. Other pharmaceutical com-
panies continue to develop new influenza neur-
aminidase inhibitors.8
Although the structures of the catalytic sites in type A
and B neuraminidases appear to be identical, compound
8 is highly selective for influenza virus A NA and was
much less effective against influenza virus B NA. This
observation is consistent with earlier reports for NA
inhibitors that induce a conformational change in
Glu278 upon ligand binding,11,12 since this conforma-
tional change in type B NA is energetically unfavor-
able.11 This alteration in binding site structure is
required to accomodate many ligands that present a
hydrophobic moiety in the Glu278 binding pocket.
However, some of the most potent inhibitors with broad
spectrum activity against both types A and B NA also
contain this hydrophobic moiety (as in compounds 5
and 6), creating uncertainties regarding the optimization
of type A-selective inhibitors to include type B activity.
In addition to the carboxylate and N-acetyl groupings,
two common structural features of the clinically useful
neuraminidase inhibitors are (1) considerable stereo-
chemical complexity and (2) a basic functionality (ali-
phatic amine or guanidine). We have been interested in
developing neuraminidase inhibitors that are chemically
simpler and, if possible, avoid the basic functionality.
Toward this end, we have pursued benzoic acids as
neuraminidase inhibitors. The benzene ring was selected
Here, we continued the evaluation of 8 as an inhibitor
of influenza A NA, targeting the human isolates N1 and
N2. Additionally, in order to begin exploring structure–
activity relationships for the unique pyrrolidinone ring
in 8, we proposed two types of modifications to the

W. J. Brouillette et al. / Bioorg. Med. Chem. 11 (2003) 2739–2749
2741
essential pyrrolidinone side chains: (1) the introduction
of one basic aliphatic amino functionality in place of a
hydroxy group (target 9), and (2) conversion of the
hydroxymethyl and aminomethyl groups on 8 and 9,
respectively, to their one carbon larger homologues
(targets 10 and 11).
assuming that minor repositioning of the benzoic acid
template is permitted. This modification was achieved
by preparing the hydroxyethyl (10) and aminoethyl (11)
analogues, while maintaining one hydroxymethyl group
in each case.
The synthesis of the 5-(hydroxyethyl)-5-(hydroxy-
methyl)lactam 10 was accomplished as summarized in
Scheme 2. Aniline 20 was reacted with diethyl bromo-
malonate to give 21. Malonate 21 was alkylated to pro-
vide 22, which upon treatment with NaOMe underwent
cyclization and decarboxylation to yield lactone 23. The
Michael reaction of 23 with ethyl acrylate gave the spiro
compound 24, and subsequent nitration with nitronium
tetrafluoroborate proceeded in greater than 90% yield
to provide 25. Reduction of the lactone with NaBH4
gave the diol 26. Catalytic hydrogenation of the nitro
functionality gave the corresponding amine (27).
Reductive coupling of 27 with 3-pentanone in the pres-
ence of NaCNBH3 and acetic acid gave 28, which on
saponification yielded the target compound 10.
Chemistry
We first pursued the synthesis of analogue 9, which is
essentially isosteric with 8 but contains a basic aliphatic
amino functionality. As shown in Scheme 1, diester
1210a was reduced with sodium borohydride to provide
the bis-(hydroxymethyl) derivative 13. This was reacted
with benzaldehyde in the presence of TsOH at reflux to
give a mixture of separable diastereomeric products 14a
and 14b. Ring opening of the acetal mixture 14a, 14b
was done with NBS in benzene to give bromo com-
pound 15 as a diastereomeric mixture (the same mixture
was produced from reacting either 14a or 14b sepa-
rately). Displacement of the bromine with azide was
accomplished using sodium azide in DMF to give 16.
The nitro group in compound 16 was reduced to the
amine 17 using sodium dithionite, and 17 was subjected
to reductive amination with 3-pentanone and sodium
cyanoborohydride to give product 18. Removal of the
benzoate group and ester hydrolysis was done in one
step using 1 N sodium hydroxide. Acidification and
recrystallization of the precipitate gave the pure azide
19. Reduction of the azide to give amine 9 was accom-
plished using catalytic hydrogenation in methanol.
As summarized in Scheme 3, compound 11 was pre-
pared in an analogous but regioselective manner start-
ing with intermediate diol 26. In this procedure, the
seven-membered benzylidene acetal 29 was obtained in
high yields from 26 using benzaldehyde as solvent
(neat). Under standard reaction conditions in organic
solvent we obtained poor yields. Ring opening of the
acetal with NBS occurred regioselectively on the least
hindered carbon to give the desired bromoester 30 in
good yield (85%). Substitution with azide to provide the
azidobenzoate 31 was followed by selective reduction of
the nitro functionality using sodium dithionate to give
32. The reductive amination of 3-pentanone gave 33,
which underwent saponification to give 34. The catalytic
reduction of azide 34 using H2/Pd/C gave the final tar-
get 11.
We also proposed analogues of 8 and 9 containing
chain-extended substituents. X-ray structural studies10a
of 8 in complex with NA revealed that a hydroxy group
in 8 replaces an ordered water in the C4 subsite. How-
ever, the alcohol of 8 in the enzyme complex does not
quite reach the original location of the ordered water.
Modeling suggested that lengthening the side chain by
one carbon atom might further enhance this interaction,
As similarly discussed earlier for 8, it should be noted
that compounds 9–11 in Schemes 1–3, while containing
Scheme 1. Reagents: (a) NaBH4, THF–MeOH; (b) PhCHO, TsOH, PhCH3; (c) NBS, benzene; (d) NaN3, DMF, heat; (e) Na2S2O4, THF–H2O;
(f) 3-pentanone, NaCNBH3, HOAc; (g) NaOH; (h) H2/Pd/C, MeOH.

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W. J. Brouillette et al. / Bioorg. Med. Chem. 11 (2003) 2739–2749
Scheme 2. Reagents: (a) diethyl bromomalonate; (b) NaH, DMF, 2-bromoethyl acetate; (c) NaOMe, MeOH; (d) LDA, THF, À78 ꢀC, ethyl acry-
late; (e) NO+2 BF4À, CH2Cl2; (f) NaBH4, THF–MeOH; (g) H2/Pd/C, MeOH; (h) 3-pentanone, NaCNBH3, AcOH, C2H4Cl2; (i) 1 N NaOH.
Scheme 3. Reagents: (a) benzaldehyde, TsOH; (b) NBS, benzene, 38 ꢀC; (c) NaN3, DMF, 75 ꢀC; (d) Na2S2O4, THF–H2O; (e) 3-pentanone,
NaCNBH3, HOAc; (f) NaOH, MeOH; (g) H2/Pd/C, MeOH.
only one chiral carbon, exist as diastereomers due to
atropisomerism resulting from restricted rotation about
the aromatic C to pyrrolidinone N single bond. In these
cases, the diastereomeric mixtures for intermediates and
products were not separable by silica chromatography,
and targets 9, 10, and 11 were all isolated and evaluated
as the approximately 1:1 diastereomeric mixture.
via a thiobarbiturate assay,13 and IC50 values were
determined from a plot of inhibitor concentration ver-
sus fractional enzyme activity. The results are contained
in Table 1.
The most effective new compound, 9, was further eval-
uated for its ability to inhibit viral replication in Madine
Darby canine kidney (MDCK) cells using A/Udorn/72
(see Table 1).
Biological Assays
All compounds were evaluated for in vitro inhibitory
activity of NA using intact virions of influenza virus A
(N1 and N2 subtypes), and influenza virus B. Colori-
metric detection of the enzyme product was employed
Results and Discussion
X-ray crystallographic studies of the early NA inhibitor,
Neu5Ac2en (DANA, 2), in complex with NA clearly

W. J. Brouillette et al. / Bioorg. Med. Chem. 11 (2003) 2739–2749
2743
Table 1. In vitro inhibitory activities of benzoic acid analogues for
influenza virus A and B neuraminidases
120. The acetamido group fits into the hydrophobic
pocket (C5-subsite) formed by Trp 180, Ile 224, and Arg
226. The C8 and C9 hydroxyl groups of the glycerol side
chain undergo interactions with the C6-subsite contain-
ing Glu 278.
Compd
IC50 values (mM)
Type of influenza virus neuraminidase
N1
N2
B/Lee
The structure of the bis-(hydroxymethyl) compound 8
in complex with the N9 neuraminidase10a (Fig. 1)
revealed that one of the hydroxymethyl groups hydro-
gen bonds to an ordered water located in the C5-subsite,
whereas the other hydroxymethyl group replaces an
ordered water located in the C4-subsite. The carbox-
ylate makes salt bridges with Arg 119, 294, and 372, and
the pyrrolidinone ring sits in the hydrophobic pocket
created by Trp 180 and Ile 224. The 3-pentylamino
moiety occupies the glycerol subsite of Neu5Ac2en,
which is in this case a hydrophobic pocket created by
the rotation of Glu 278 (Glu 275 using type B number-
ing) to form an intramolecular salt-bridge interaction
with Arg 226. This movement in the catalytic site is
required to create the hydrophobic pocket, and it has
been suggested11 that rotation of Glu 275 in type B NA
is energetically more costly, resulting in a poorer inhi-
bition constant (IC50=271 mM for 8) for many com-
pounds that contain such hydrophobic groups.
However, GS 4071, which is a potent inhibitor of both
type A and type B NA, contains a similar lipophilic
grouping in the glycerol pocket but undergoes tight
binding to type B NA, possibly due to differences in the
extent of rotation induced for Glu 275. Apparently,
3 (GG167)
0.006
79d
0.015
7a
8
271
26
8b,c
9b,c
0.49
0.52
e
0.88
(EC50=4Æ1 mM)
10
11
19
>1000f
59
41
>1000f
>1000f
>670f
180
268
aRef 11a.
bCompound 8 was evaluated as its racemic mixture, and compounds
9–11 and 19 as an approximately 1:1 mixture of diastereomers.
cCompounds 8 and 9 were measured side by side against all virus strains.
dIn order to estimate the accuracy of these results, this IC50 determi-
nation was repeated in four separate experiments. The mean (ÆSEM)
IC50=73Æ13 mM.
eThe concentration that reduced viral yield by 50% in a viral replica-
tion assay using A/Udorn/72 virus and MDCK cells.
fNo significant inhibition was observed at the highest tested concen-
tration, which is given in the table.
defined a compact catalytic site containing four binding
subsites with which all reported NA inhibitors interact
(see Fig. 1; N9 numbering). The carboxylate makes tight
salt-bridge interactions with an arginine triad consisting
of Arg 119, 294, and 372 (C2-subsite). The 4-hydroxyl
group occupies the C4-subsite near Asp 152 and Glu
Figure 1. Depiction of the interactions for compound 8 in the active site of influenza neuraminidase.

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W. J. Brouillette et al. / Bioorg. Med. Chem. 11 (2003) 2739–2749
subtle structural changes not yet clearly defined con-
tribute to the potency of inhibition and type A selectiv-
ity seen for hydrophobic inhibitors. We therefore
proposed to evaluate the effects of modest changes in
the essential pyrrolidinone side chains for compound 8.
eggs as described.14 IC50 determinations were carried
out using the thiobarbiturate colorimetric assay descri-
bed by Aymard-Henry et al.13 but adapted to 96-well
plates. The enzymatic reaction was carried out in a
200-mLsolution containing 0.125 mM CaCl 2, 0.4 mM
MgCl2, 75 mM NaCl, 0.25% NaN3, 50 mM phosphate
buffer (pH 5.9), and 0.5% DMSO. The reactions inclu-
ded neuraminidase in the form of intact virions in
allantoic fluid, 10 mg/mLof the substrate fetuin
(Sigma), and inhibitor at various dilutions. The reaction
mixtures were incubated for 1 h at 37 ꢀC, after which
formation and extraction of the chromophore were car-
ried out as described.13 The data was analyzed by plot-
ting the enzyme fractional activity against the inhibitor
concentration, and the IC50 was determined from the
linear region of the dose–response curve.
As shown in Table 1, we first evaluated 8 as an inhibitor
of NA from two human influenza isolates, N1 and N2.
Interestingly, compound 8 was highly selective for
N2 NA over N1 NA; in fact, the activity against N1 NA
was only slightly poorer than that for B/Lee NA.
We also evaluated the newly synthesized analogues of 8
against the same viral strains of NA. The most con-
servative modification to 8 resulted in the aminomethyl
analogue 9. As shown in Table 1, compound 9 exhibited
potent inhibitory activity, comparable to 8, against
N2 NA. However compound 9 was also approximately
100-fold more active than 8 against N1 NA, revealing a
broader spectrum activity against the two most impor-
tant human strains of influenza A NA. Thus the spec-
trum of activity for 9 makes it more attractive than
compound 8 for further development into inhibitors of
influenza virus A infections in humans. Its synthetic pre-
cursor, the azide 19, exhibits an IC50 of only 268 mM
against N1 NA, revealing the importance of the amino
grouping. While compound 9 was 10-fold more effective
than 8 against influenza B NA, it remains 50-fold selec-
tive for the inhibition of type A over type B NA.
Inhibition of viral replication assay
MDCK cell monolayers in 24-well plates were washed
free of serum and then infected with 100 TCID (tissue
culture infectious dose) of A/Udorn/72 virus. After
virus was adsorbed to the cells for 30 min, 0.5-mLsolu-
tion of serum-free infection medium15 containing tryp-
sin (1 mg/mL) and 10-fold dilutions of NA inhibitor 9
was added to each well, and the plates were incubated at
37 ꢀC in 5% CO2 for 48 h. The yield of virus in each well
was measured by hemagglutination titration and com-
pared to wells with no inhibitor.16 The EC50 was calcu-
lated from
a narrow-range experiment in which
Interestingly, the homologues of 8 and 9, compounds 10
and 11, each exhibited relatively poor activity against
NA from both influenza viruses A and B. This result
suggests that these side chains are too large for the C4
and C5 subsites, or that the rigidity of the catalytic site
prevents small adjustments in the position of the ben-
zoic acid core. This further emphasizes the sensitivity of
the NA catalytic site to relatively modest changes in
inhibitor structure.
doubling dilutions of inhibitor were used. The EC50 is
defined as the inhibitor concentration at which virus
output is reduced to 50%, averaged from duplicate
experiments.
Chemistry
NMR data was recorded on a Bruker ARX 300 MHz
spectrometer. IR spectra were obtained on a Bruker FT/
OPUS spectrophotometer. Mass spectra were obtained
using electrospray ionization on a MicroMass Platform
LCZ LC/MS with HP1100HPLC/autoinjector and
diode array detector. Flash column chromatography
was carried out using silica gel (40 mm, Mallinckrodt,
Inc.). TLC was performed using silica gel (250 mM layer,
Whatman), and spots were visualized by irradiation
with UV light (UV-254 nm). Melting points were deter-
mined on a Mel-Temp electrothermal melting point
apparatus (Laboratory Devices) and are uncorrected.
Anhydrous reactions were carried out in oven-dried
glassware under a nitrogen atmosphere. Compound 12
(Scheme 1) was prepared as previously described.10a We
also previously described10a a synthesis for 13, but here
we describe an improved procedure. GG167 was a gift
from BioCryst Pharmaceuticals.
To confirm that the inhibition of NA by compound 9
would result in antiviral actions, the EC50 for reduction
of viral yield in MDCK cells was determined using
influenza virus A/Udorn/72. As shown in Table 1,
compound 9 was highly effective in this assay with an
EC50 value of 4 mM.
In summary, properly substituted pyrrolidinobenzoic
acids can serve as novel structural templates for potent
inhibitors of influenza A NA. We were able to improve
the cross-reactivity against type B influenza, but, as for
many other reported NA inhibitors containing hydro-
phobic substituents, the activity of examples prepared
thus far shows at least 50-fold selectivity for influenza A
over type B NA.
Note that pyrrolidino-benzenes containing one or more
chiral carbons were synthesized as diastereomeric mix-
tures due to atropisomerism via restricted rotation
about the pyrrolidinone N to benzene C bond. In all
cases the diastereomeric atropisomers were not separable
by silica chromatography, and spectral and biological
data are reported for the mixture.
Experimental
Neuraminidase inhibition assays
Influenza virus strains A/PR/8/34 (N1), A/Udorn/72
(N2), and B/Lee/40 were grown in embryonated chicken

W. J. Brouillette et al. / Bioorg. Med. Chem. 11 (2003) 2739–2749
2745
1-(4-Methoxycarbonyl-2-nitrophenyl)-5,5-bis-(hydroxy-
methyl)pyrrolidin-2-one (13). A solution of nitrodiester
1210a (10.0 g, 2.45 mmol) was dissolved in a THF–
MeOH mixture (1:1, 135 mL) and cooled to zero degrees
under a nitrogen atmosphere. Sodium borohydride
(5.14 g, 15.2 mmol) was added in small amounts over a
period of 1.5 h at 0 ꢀC, and the mixture was stirred for
another 2.5 h at 7–10 ꢀC. The reaction mixture was
quenched with ice cold 6 N HCl (31.5 mL) while stirring
and then concentrated to dryness under vacuum. The
yellow solid residue was dissolved in chloroform
(75 mL) and washed with water (60 mL) and brine
(40 mL). The combined aqueous layers were further
extracted with chloroform (5 Â 60 mL). The combined
organic layers were dried (Na2SO4) and concentrated to
give a pale yellow fluffy solid, which was triturated with
ether and filtered to give 13 (6.2 g, 78%) as a pale yellow
solid: mp 143–144 ꢀC (ethanol–ether). MS (ES) m/z 325
column (5% methanol–chloroform) to give 15 (1.52 g,
72.3%) as a solid: mp 187–188 ꢀC (ethyl acetate–hex-
ane). MS (ES) m/z 491 (M+H); IR (KBr) 1723,
1704 cmÀ1 1H NMR (CDCl3) d 2.27–2.53 (m, 2H),
;
2.66–2.87 (m, 2H), 3.63–3.89 (m, 2H), 3.99 (s, 3H),
4.59–4.92 (m, 2H), 7.24–8.71 (m, 8H). Anal. calcd for
C21H19N2O7Br: C, 51.43; H, 3.88; N, 5.71. Found: C,
51.49; H, 3.83; N, 5.57.
1-(4-Methoxycarbonyl-2-nitrophenyl)-5-azidomethyl-5-
(benzoyloxymethyl)-pyrrolidin-2-one (16). The bromo
compound 15 (1.10 g, 2.24 mmol) was dissolved in DMF
(5 mL), sodium azide (650 mg, 10.0 mmol) was added,
and the mixture was heated at 90 ꢀC for 20 h. The reac-
tion mixture was diluted with ethyl acetate (120 mL),
washed with water (2 Â 25 mL) and brine (10 mL), dried
(Na2SO4), and evaporated to dryness to give 16 (880 mg,
87.1%) as a white solid: mp 214–215 ꢀC (ethyl acetate–
hexane). MS (ES) m/z 454 (M+H); IR (KBr) 2108,
1
(M+H); IR (KBr): 3419, 1729, 1664 cmÀ1; H NMR
(CD3OD) d 2.00–2.30 (m, 2H), 2.47–2.68 (m, 2H), 3.54–
3.92 (m, 4H), 3.97 (s, 3H), 7.75 (d, 1H), 8.31 (dd, 1H),
8.60 (d, 1H). Compound 13 was identical to that pre-
viously described.10a
1726, 1699 cmÀ1 1H NMR (CDCl3) d 2.21–2.40 (m,
;
2H), 2.63–2.80 (m, 2H), 3.70–3.90 (m, 2H), 3.97 (s, 3H),
4.45–4.71 (m, 2H), 7.26–7.50 (m, 5H), 7.97 (d, 1H), 8.30
(d, 1H), 8.58 (d, 1H). Anal. calcd for
.
C21H19N5O7 0.5H2O: C, 54.54; H, 4.11; N, 15.15.
Found: C, 54.85; H, 4.29; N, 14.84.
(1-(4-Methoxycarbonyl-2-nitrophenyl)-5,5-spiro(3,5-di-
oxa-4-phenylcyclohexyl)-pyrrolidin-2-one (14a and 14b).
The diol 13 (2.00 g, 6.17 mmol) was dissolved in dry
toluene (80 mL), benzaldehyde (2.00 mL, 19.6 mmol)
and pTsOH (25 mg) were added, and the mixture was
heated at reflux using a Dean–Stark trap for 24 h. The
solution was concentrated to dryness, and the residue
was dissolved in ethyl acetate (150 mL) and washed with
5% sodium bicarbonate (2 Â 10 mL), water (10 mL),
and brine (10 mL), and dried (Na2SO4). The organic
layer was concentrated to dryness and the residue was
purified on a flash silica gel column (9:1 ether–ethanol).
The two diastereomeric products 14a and 14b (total
yield 1.93 g, 76.2%) were isolated. For isomer 14a
(1.1 g): mp 155–156 ꢀC; Rf 0.76 (9:1 ether–ethanol); MS
1-((2-Amino-4-methoxycarbonyl)phenyl)-5-azidomethyl-
5-(benzoyloxymethyl)-pyrrolidin-2-one (17). The nitro
derivative 16 (800 mg, 1.76 mmol) was dissolved in THF
(20 mL) and water (8 mL) was added. To this stirring
mixture was added sodium dithionite (650 mg,
3.73 mmol), and this was stirred at room temperature
for 2 h. The solution was warmed to 40 ꢀC for 10 min
and then allowed to cool to room temperature. Another
portion of sodium dithionite (150 mg) was added and
stirring was continued for another 3 h. The reaction
mixture was concentrated to dryness under vacuum, the
solid residue was triturated with ethyl acetate (3 Â
10 mL), and the suspension was filtered. The filtrate was
concentrated to dryness and the oily residue was pur-
ified by flash chromatography (5% methanol in chloro-
form) to yield 17 (540 mg, 72.3%) as a solid: mp 61–
62 ꢀC (ether–methanol). MS (ES) m/z 424 (M+H); IR
(KBr) 3361, 2109, 1723 cmÀ1; 1H NMR (CDCl3) d 2.21–
2.43 (m, 2H), 2.67–2.88 (m, 2H), 3.44–3.81 (m, 2H), 3.88
(s, 3H), 4.26–4.52 (m, 2H), 7.03–7.66 (m, 7H), 7.92 (d,
1
(ES) m/z 413 (M+H); IR (KBr) 1734, 1698 cmÀ1; H
NMR (CDCl3) d 2.64 (br m, 4H), 3.69 (d, 1H), 4.00 (s,
3H), 4.13 (d, 1H), 4.28 (m, 2H), 5.33 (s, 1H), 7.25–7.44
(m, 6H), 8.34 (d, 1H), 8.70 (br s, 1H). For isomer 14b
(836 mg): mp 203–204 ꢀC; Rf 0.31 (9:1 ether–ethanol);
MS (ES) m/z 413 (M+1); IR (KBr) 1728, 1696 cmÀ1
;
1H NMR (CDCl3) d 1.88–2.05 (m, 2H), 2.59–2.68 (m,
2H), 3.70–3.82 (m, 2H), 3.95 (s, 3H), 4.56 (m, 2H), 5.45
(s, 1H), 7.20–7.30 (m, 5H), 7.76 (d, 1H), 8.15 (dd, 1H),
8.65 (d, 1H). Anal. calcd for C21H20N2O7: C, 61.16; H,
4.85; N, 6.80. Found: C, 61.12; H, 4.90; N, 6.81.
.
1H). Anal. calcd for C21H21N5O5 0.5 CH3OH: C, 58.76;
H, 5.23; N, 15.94. Found: C, 58.37; H, 4.97; N, 15.51.
1-(4-Methoxycarbonyl-2-(3-pentylamino)phenyl)-5-azido-
methyl-5-(benzoyloxymethyl)-pyrrolidin-2-one (18). In a
flame-dried flask, aniline 17 (1.40 g, 3.31 mmol), 3-pen-
tanone (32 mL, 31.7 mmol) and AcOH (9.30 mL) were
stirred under nitrogen for 36 h. Sodium cyanoborohy-
dride (1.91 g, 30.4 mmol) was added and the mixture
stirred for 6 h. An additional portion of sodium cyano-
borohydride (1.91 g, 30.4 mmol) was added and the
mixture stirred for 6 more hours. The reaction was
quenched with saturated NaHCO3 solution (10 mL),
extracted with EtOAc (3Â25 mL), and the organic
extracts were washed with brine (20 mL) and dried
(Na2SO4). The organic layer was concentrated under
vacuum, and the crude oily residue (2.50 g) was dissolved
1-(4-Methoxycarbonyl-2-nitrophenyl)-5-bromomethyl-5-
(benzoyloxymethyl)-pyrrolidin-2-one (15). The benzyli-
dene acetal 14 (1.80 g, 4.36 mmol) (either 14a, 14b, or a
mixture) was dried under vacuum and dissolved in
anhydrous benzene (150 mL). Dry NBS (1.20 g,
6.70 mmol) was added, and the mixture was stirred
under nitrogen at room temperature for 30 h. The solu-
tion was concentrated to dryness, the residue was dis-
solved in ethyl acetate (150 mL), and this was washed
with water (5 Â 60 mL) followed by brine (50 mL). The
organic layer was dried (Na2SO4) and evaporated to
dryness. The residue was purified on a flash silica gel

2746
W. J. Brouillette et al. / Bioorg. Med. Chem. 11 (2003) 2739–2749
in hexane (100 mL) and concentrated to help remove the
side product, 3-pentanol, as an azeotrope. The crude
mixture was purified by flash chromatography (silica)
using stepwise elution with hexane (500 mL), then 20%
ether in hexane (500 mL), and finally 40% ether in hex-
ane (250 mL) to afford 18 (0.90 g, 55%) as an oil. Start-
ing material (0.23 g) and a cyclized byproduct (0.2 g)
were also recovered. MS (ES) m/z 494 (M+H); IR
1H), 4.80 (d, 1H), 4.31 (q, 4H), 3.82 (s, 3H), 1.31 (t, 6H).
Anal. calcd for C15H19NO6: C, 56.52; H, 4.35; N, 10.15.
Found: C, 56.55; H, 4.40; N, 10.13.
Diethyl 2-(2-Acetyloxyethyl)-2-[(4-methoxycarbonyl)phe-
nylamino]malonate (22). To a suspension of 60% NaH
in mineral oil (0.52 g, 13 mmol) in 12 mLof anydrous
DMF was added amine 21 (4.0 g, 13 mmol). The result-
ing mixture bubbled, was stirred at room temperature
for 15 min, and 2-bromoethyl acetate (2.4 g, 14 mmol)
was added. The mixture was stirred for 36 h at room
temperature. The reaction was quenched with 1 N HCl
(10 mL) and the product was extracted into ethyl acetate
(3Â50 mL). The combined organic extracts were washed
with H2O (25 mL), dried (Na2SO4) and evaporated to
give an oil (5.1 g) that was purified by flash chromato-
graphy (silica gel, 5 Â 40 cm, 2:1 ether/hexane) to give
an oil (4.5 g, 88%) (Rf 0.60). The oil was crystallized
from ether to give 22 (4.1 g, 80%) as white crystals: mp
88–89 ꢀC. MS (ES) m/z 396 (M+H); 1H NMR (CDCl3)
d 7.83 (d, 2H), 6.58 (d, 2H), 5.70 (s, 1H), 4.33–4.17 (m,
4H), 4.05 (t, 2H), 3.85 (s, 3H), 2.77 (t, 2H), 1.93 (s, 3H),
1.22 (t, 6H). Anal. calcd for C19H26NO8: C, 57.71; H,
6.37; N, 3.54 Found: C, 57.77; H, 6.30; N, 3.51.
1
(CHCl3) 2110, 1720, 1630 cmÀ1; H NMR (CDCl3) d
0.92 (t, 3H), 0.99 (t, 3H), 1.27 (m, 2H), 1.52 (m, 2H);
2.29 (m, 2H); 2.65 (m, 2H), 3.16–3.41 (m, 1H), 3.51–3.74
(m, 3H), 3.87, 3.90 (s, 3H), 4.25–4.52 (m, 2H), 6.98–7.67
(m, 7H), 7.89 (d, 1H). Anal. calcd for
.
C26H31N5O5 0.5H2O: C, 62.14; H, 6.42; N, 13.93.
Found: C, 62.59; H, 6.54; N, 13.03.
1-(4-Carboxy-2-(3-pentylamino)phenyl)-5-azidomethyl-5-
hydroxymethyl-pyrrolidin-2-one (19). The ester 18
(370 mg, 0.750 mmol) was dissolved in methanol (1 mL),
1 N NaOH (2.5 mL, 2.5 mmol) was added, and the
mixture was stirred overnight at room temperature. The
solution was concentrated to dryness, and the residue
was dissolved in water (1.5 mL) and acidified to pH 1
using 1 N HCl at zero degrees. After further cooling in
an ice bath the mixture was filtered. The solid material
was triturated with ether (2 Â 10 mL) and chloroform
(5 mL), and then filtered. The collected solid was puri-
fied on a flash silica gel column (9:1 ether–ethanol) to
give 19 (126 mg, 44.8%) as a solid: mp 157–158 ꢀC
(methanol). MS (ES) m/z 376 (M+H); IR (KBr) 3387,
2 - [(4 - Methoxycarbonylphenyl)amino] - ꢀ - butyrolactone
(23). To an ice cold suspension of 22 (1.00 g,
2.53 mmol) in dry methanol (12 mL) was added freshly
cut Na (0.250 g, 10.9 mmol). The mixture was stirred for
1 h at 0 ꢀC and then quenched with 1 N HCl (10 mL).
The methanol was removed under vacuum, the aqueous
residue was extracted with ethyl acetate (3Â25 mL), and
the organic layers were dried (Na2SO4) and con-
centrated to give an oil (0.550 g). This was purified by
flash chromatography (silica gel, 2 Â 40 cm, ether) to give
23 (0.500 g, 84.0%) as a white solid: mp 136–138 ꢀC. MS
(ES) m/z 236 (M+H); 1H NMR (CDCl3) d 7.90 (d, 2H),
6.64 (d, 2H), 4.70 (br s, 1H), 4.53 (t, 1H), 4.40–4.32 (m,
1H), 4.23 (dd, 1H), 3.87 (s, 3H), 2.96–2.90 (m, 1H), 2.23–
2.15 (m, 1H). Anal. calcd for C12H13NO4: C, 61.27; H,
5.57; N, 5.95. Found: C, 61.19; H, 5.65; N, 5.89.
1
2109, 1710, 1632 cmÀ1; H NMR (CD3OD) d 0.92 (t,
6H), 1.57 (m, 4H), 2.50 (m, 4H), 2.93–3.72 (m, 5H),
6.90–7.51 (m, 3H). Anal. calcd for C18H25N5O4: C,
57.59; H, 6.71; N, 18.65. Found: C, 57.55; H, 6.71; N,
18.37.
1-(4-Carboxy-2-(3-pentylamino)phenyl)-5-aminomethyl-
5-hydroxymethyl-pyrrolidin-2-one (9). The azide 19
(80 mg, 0.21 mmol) was dissolved in methanol (15 mL),
10% Pd/C (40 mg) was added, and the mixture was
hydrogenated at 40 psi for 2.5 h. The solution was fil-
tered and the filter washed with methanol. The filtrate
and the washings were combined and concentrated
under vacuum to give a solid residue, which was tritu-
rated three times with ether and filtered to give 9 (62 mg,
84%) as a solid: mp >162 ꢀC (dec) (methanol). MS (ES)
Spiro 2-(ꢀ-butyrolactone)-5-[N-(4-methoxycarbonylphe-
nyl)pyrrolidin-2-one] (24). A solution of 23 (0.050 g,
0.21 mmol) in dry THF (1 mL) was cooled to À78 ꢀC
and lithium diisopropylamide (0.30 g, 0.28 mmol) was
added. After 20 min of stirring, ethyl acrylate (0.030 g,
0.30 mmol) was added and the stirring continued for 4 h
at À70 to À60 ꢀC. The reaction mixture was quenched
with 1 N HCl (1 mL), the product was extracted into
ethyl acetate (3Â15 mL), and the organic extracts were
dried (Na2SO4) and concentrated to give an oil (0.50 g,
82%). This was purified by flash chromatography (silica
gel, 1.5 Â 36 cm, 6.4% ethanol in ether, Rf=0.50) to
give 24 (0.040 g, 67%) as a white solid: mp 102–104 ꢀC.
1
m/z 350 (M+H), IR (KBr) 3375, 1694, 1587 cmÀ1; H
NMR (CD3OD) d 0.94 (m, 6H), 1.57 (m, 4H), 2.50 (m,
4H), 2.93–3.72 (m, 5H), 6.90–7.51 (m, 3H). Anal. calcd
.
for C18H27N3O4 0.75H2O: C, 59.57; H, 7.49; N, 11.57.
Found: C, 59.32; H, 7.52; N, 11.27.
Diethyl 2 - [4 - (Methoxycarbonyl)phenylamino]malonate
(21). A mixture of methyl p-aminobenzoate (20) (3.10 g,
20.5 mmol) and diethyl bromomalonate (2.45 g,
10.2 mmol) was heated at 112 ꢀC for 15 h in an oven.
The reaction mixture was cooled, suspended in ether
(70 mL), and then washed with 2 N HCl (3Â15 mL),
water (15 mL) and brine (10 mL). The organic layer was
dried (Na2SO4) and concentrated to give a solid, which
was crystallized from ether/hexane to give 21 (2.8 g,
86%): mp. 67–69 ꢀC. MS (ES) m/z 310 (M+H); 1H
NMR (CDCl3) d 7.90 (d, 2H), 6.60 (d, 2H), 5.21 (br s,
1
MS (ES) m/z 290 (M+H); H NMR (CDCl3) d 8.11–
8.07 (m, 2H), 7.33–7.27 (m, 2H), 4.37–4.20 (m, 2H), 3.92
(s, 3H), 2.96–2.84 (m, 1H), 2.68–2.55 (m, 3H), 2.29–2.22
(m, 1H). Anal. calcd for C15H15NO5: C, 62.28; H, 5.23;
N, 4.84. Found: C, 62.31; H, 5.18; N, 4.82.
Spiro 2-(ꢀ-butyrolactone)-5-[N-(4-methoxycarbonyl-2-ni-
trophenyl)pyrrolidin-2-one] (25). An ice-cold solution of

W. J. Brouillette et al. / Bioorg. Med. Chem. 11 (2003) 2739–2749
2747
24 (0.080 g, 0.28 mmol) in methylene chloride (2 mL)
was treated with nitronium tetrafluoroborate (0.19 g,
1.4 mmol). The reaction mixture was stirred at 0 ꢀC for
6 h and at room temperature for 10 h. The reaction
mixture was quenched with H2O (2 mL) and the CH2Cl2
was removed under vacuum. The aqueous residue was
extracted with ethyl acetate (5Â15 mL), and the com-
bined extracts were washed with brine (5 mL), dried
(Na2SO4) and concentrated to give a crude yellow solid
(0.090 g). This was purified by flash chromatography
(silica gel, 1.5Â40 cm, 5% ethanol in ether, Rf=0.60) to
give 25 (0.080 g, 87%) as a white solid: mp 147–150 ꢀC.
MS (ES) m/z 335 (M+H); 1H NMR (CDCl3) d 8.65 (d,
1H), 8.35 (dd, 1H), 7.53 (d, 1H), 4.33–4.38 (m, 2H), 3.99
(s, 3H), 2.91–2.79 (m, 1H), 2.70–2.60 (m, 2H), 2.52–2.43
(m, 3H). Anal. calcd for C15H14N2O7: C, 53.89; H, 4.22;
N, 8.37. Found: C, 53.49; H, 4.26; N, 8.23.
extracted with ethyl acetate (5Â20 mL). The combined
organic layers were dried (Na2SO4) and concentrated to
give an oil (90 mg, 82%). This was purified by flash
chromatography (silica gel, 1.5Â40 cm, 8% ethanol in
ether, Rf=0.60) to give 28 (78 mg, 71%) as a white
solid: mp 141–143 C. MS (ES) m/z 379 (M+H); H
NMR (CDCl3) d 7.55 (d, 1H), 7.50 (d, 1H), 7.48 (d,
1H), 4.25 (br s, 2H), 3.92 (s, 3H), 3.89–3.82 (m, 2H),
3.35–3.29 (m, 2H), 3.17 (d, 1H), 2.70–2.45 (m, 3H),
2.18–2.09 (m, 1H), 1.95–1.84 (m, 2H), 1.81–1.47 (m,
4H), 0.98–0.92 (m, 6H). Anal. calcd for C20H30N2O5: C,
63.47; H, 7.98; N, 7.40. Found: C, 62.57; H, 7.86; N,
7.32.
ꢀ
1
N-[4-Carboxy-2-(3-pentylamino)phenyl]-5-(2-hydroxy-
ethyl)-5-(hydroxymethyl)pyrrolidin-2-one (10). A suspen-
sion of 28 (0.060 g, 0.16 mmol) in 1 N NaOH (1.0 mL,
1.0 mmol) was stirred at room temperature for 17 h. The
reaction mixture was neutralized with acetic acid and
evaporated to dryness under high vacuum. The resulting
solid was dissolved in H2O (3 mL) and extracted with
ethyl acetate (7Â15 mL). The combined organic layers
were dried (Na2SO4) and concentrated to give a semi-
solid, which was triturated with ether and filtered to
give 10 (40 mg, 70%) as white crystals: mp 188–190 ꢀC.
MS (ES) m/z 365 (M+H); 1H NMR (CDCl3/CD3OD) d
7.50 (br s, 1H), 7.48–7.40 (m, 1H), 7.08–7.00 (d, 1H),
3.82–3.69 (m, 2H), 3.44 (d, 1H), 3.35–3.27 (m, 1H), 3.25
(d, 1H), 2.79–2.68 (m, 1H), 2.60–2.39 (m, 2H), 2.22–2.10
(m, 1H), 1.93–1.42 (m, 6H), 1.00–0.88 (m, 6H). Anal.
calcd for C19H28N2O5: C, 62.61; H, 7.74; N, 7.68.
Found: C, 62.33; H, 7.76; N, 7.63.
5-(2-Hydroxyethyl)-5-hydroxymethyl-N-(4-methoxy-
carbonyl-2-nitrophenyl)pyrrolidin-2-one (26). To an ice
cold solution of 25 (0.040 g, 0.12 mmol) in a mixture of
dry methanol (0.50 mL) and dry THF (0.50 mL) was
added NaBH4 (0.20 g, 0.50 mmol) in small portions over
25–30 min. The reaction mixture was stirred at 0–5 ꢀC
for 4 h and quenched with 1 N HCl (0.75 mL). The sol-
vent was removed under vacuum and the aqueous resi-
due was extracted with ethyl acetate (4Â15 mL). The
combined extracts were dried (Na2SO4) and con-
centrated to give a crude semisolid (0.040 g). This was
purified by flash chromatography (silica gel, 1.5 Â
25 cm, 6.4% ethanol in ether, Rf=0.40) to give 26
(0.030 g, 81%) as yellow crystals: mp 151–153 ꢀC. MS
1
(ES) m/z 339 (M+H); H NMR (CD3OD) d 8.59 (s,
1H), 8.33 (d, 1H), 7.77 (d, 1H), 3.98 (s, 3H), 3.87–3.73
(dd, 2H), 3.62 (t, 2H), 2.76–2.60 (m, 1H), 2.47–2.31 (m,
3H), 1.94–1.91 (m, 1H), 1.74–1.71 (m, 1H). Anal. calcd
for C15H18N2O7: C, 53.25; H, 5.36; N, 8.28. Found: C,
53.14; H, 5.44; N, 8.22.
Spiro 5-[N-(4-Methoxycarbonyl-2-nitrophenyl)pyrroli-
din-2-one]-5-(1,3-dioxa-2-phenylcycloheptane) (29).
A
suspension of 26 (3.00 g, 9.73 mmol), benzaldehyde
(120 mL, 1.18 mol), and 5–10 mg of p-toluene sulphonic
acid was stirred at room temperature for 24 h. To the
reaction mixture was added saturated NaHCO3 (1 mL)
followed by water (25 mL), and this was extracted with
EtOAc (4 Â 25 mL). The combined organic layers were
washed with water (50 mL), brine (50 mL), and dried
(MgSO4). The organic layer was concentrated under
vacuum to give an oil (130 mg). The crude oil was puri-
fied by silica column chromatography, using stepwise
elution, first with 20% EtOAc in hexane (300 mL), and
subsequently with 80% EtOAc in hexane to give the
desired compound 29 (3.2 g, 77%; combined weight of
isomers A and B).
N-(2-Amino-4-methoxycarbonylphenyl)-5-(2-hydroxye-
thyl)-5-(hydroxymethyl)pyrrolidin-2-one (27). A mixture
of 26 (0.050 g, 0.15 mmol) and 10% Pd/C (0.05 g) in
methanol (10 mL) was hydrogenated on a Parr shaker at
38 psi for 1 h. The reaction mixture was diluted with
methanol (10 mL), filtered, the filter was washed with
additional methanol (10 mL), and the filtrate was con-
centrated to dryness to give 27 (0.045 g, 99%) as an oil.
1
MS (ES) m/z 309 (M+H); H NMR (CD3OD) d 7.65–
6.98 (m, 3H), 3.87 (s, 3H), 3.78–3.51 (m, 3H), 3.28–3.39
(m, 1H), 2.74–2.65 (m, 1H), 2.52–2.19 (m, 3H), 1.95–
1.62 (m, 2H). Anal. calcd for C15H20N2O5: C, 58.43; H,
6.53; N, 9.09. Found: C, 58.65; H, 6.55; N, 9.31.
Isomer A. MS (ES) m/z 427 (M+H); 1H NMR
(CDCl3) d 8.70 (d, 1H), 8.30–8.31 (d, 1H), 7.46–7.36 (m,
6H), 5.78 (s, 0.6H), 5.47 (s, 0.4H), 4.08–4.01 (m, 1H),
3.99 (s, 3H), 3.63–3.45 (m, 4H), 2.73–2.51 (m, 3H),
2.11–2.07 (m, 1H). Anal. calcd for C22H22N2O7: C,
61.97; H, 5.20; N, 6.57. Found: C, 61.62; H, 5.38; N,
6.36.
5-(2-Hydroxyethyl)-5-hydroxymethyl-N-[4-methoxy-
carbonyl-2-(3-pentylamino)phenyl]pyrrolidin-2-one (28).
A solution of 27 (0.090 g, 0.29 mmol) in 1,2-dichlor-
oethane (1 mL) and acetic acid (0.5 mL) was treated
with 3-pentanone (0.18 g, 2.0 mmol) and NaCNBH3
(68 mg, 1.1 mmol). The resulting mixture was stirred at
room temperature for 18 h. Additional 3-pentanone
(0.18 g, 2.0 mmol) and NaCNBH3 (68 mg, 1.1 mmol)
were added and the stirring continued for 17 h. To the
reaction mixture was added satd NaHCO3, and this was
Isomer B. MS (ES) m/z 427 (M+H);1H NMR
(CDCl3) d 8.61 (d, 1H,), 8.18–8.15 (dd, 1H), 7.37–7.22
(m, 6H), 5.72 (s, 1H), 4.08–3.99 (m, 1H), 3.95 (s, 3H),

2748
W. J. Brouillette et al. / Bioorg. Med. Chem. 11 (2003) 2739–2749
3.81–3.70 (m, 4H), 2.66–2.52 (m, 3H), 2.20–2.13 (m, 1H)
and 1.90–1.70 (m, 1H).
2.72–2.63 (m, 2H), 2.44–2.00 (m, 4H), 2.04–2.00. Anal.
calcd for C22H23N5O5: C, 60.44; H, 5.30; N, 16.00.
Found: C, 60.56; H, 5.18; N, 15.91.
5-Benzoyloxymethyl-5-(2-bromoethyl)-N-(4-methoxy-
carbonyl-2-nitrophenyl)pyrrolidin-2-one (30). A solution
of 29 (isomers A and B combined, 2.75 g, 6.45 mmol) in
anhydrous benzene (117mL) was treated with N-bromo-
succinimide (1.60 g, 8.99 mmol) at ambient temperature
and stirred under nitrogen for 23 h. Benzene (63 mL)
was added, and stirring was continued for an additional
3 h. The reaction mixture was concentrated under
vacuum, the residue was suspended in water (50 mL)
and the product was extracted into chloroform (3 Â
50 mL). The combined organic layers were dried
(MgSO4) and concentrated to give an oily residue. This
was placed on a flash chromatography column (ether)
to give 30 (2.35 g, 72.3%) as a white powder: mp 69–
5-(2-Azidoethyl)-5-benzoyloxymethyl-N-[4-methoxy-
carbonyl-2-(3-pentylamino)phenyl]pyrrolidin-2-one (33).
A solution of 32 (0.19 g, 0.43 mmol) in 2 mLof 1,2-
dichloroethane was treated with 1 mLof acetic acid and
stirred under nitrogen. After 10 min, 3-pentanone
(0.39 g, 4.5 mmol) and NaCNBH3 (0.14 g, 2.2 mmol)
were added, and stirring was continued for 5 h. Another
portion of 3-pentanone (0.39 g, 4.5 mmol) and
NaBH3CN (0.14 g, 2.2 mmol) was added, followed by
an additional five portions, each added every 5 h. No
further progress in reaction was observed. To the reac-
tion mixture was added saturated NaHCO3 (8 mL), and
this was concentrated under high vacuum. The white
residue was triturated with ether (5 Â 25 mL). The
combined ether washes were shaken with H2O (10 Â
20 mL), brine (20 mL) and dried (MgSO4). This was
concentrated to give a green oil (180 mg), which was
placed on a flash chromatography column (ether) to
give recovered 32 (100 mg) and the desired product 33
(70 mg, 70% conversion) as an oil. This material was
carried forward without additional purification. MS
71 ꢀC. MS (ES) m/z 505 (M+H); H NMR (CDCl3) d
1
8.68 (s, 0.6H), 8.58 (s, 0.4H), 8.32–8.26 (m, 1H), 7.98–
7.96 (d, 1H), 7.66–7.26 (m, 5H), 4.73–4.41 (m, 2H), 3.98
(s, 3H), 3.51–3.46 (m, 1H), 3.36–3.33 (m, 1H), 2.71–2.24
(m, 6H). Anal. calcd for C22H21N2O7Br: C, 52.33; H,
4.19; N, 5.55. Found: C, 52.24; H, 4.31; N, 5.39.
5-(2-Azidoethyl)-5-benzoyloxymethyl-N-(4-methoxy-
carbonyl-2-nitrophenyl)pyrrolidin-2-one (31). Compound
30 (1.90 g, 3.76 mmol) was dissolved in DMF (5.7 mL),
NaN3 (0.300 g, 4.61 mmol) was added, and the mixture
was stirred at 70–75 ꢀC for 16 h. The DMF was removed
under vacuum at 40 ꢀC, the residue was diluted with
EtOAc (25 mL), and the mixture was washed with water
(4Â15 mL) followed by brine (15 mL). The organic layer
was dried (MgSO4) and concentrated. The residue was
purified by flash chromatography (ether) to give 31
(1.48 g, 85.0%) as pale yellow needles: mp 46–48 ꢀC. MS
(ES) m/z 468 (M+H); 1H NMR (CDCl3) d 8.66 (s, 1H),
8.28–8.25 (d, 1H), 7.98–7.95 (d, 1H), 7.64–7.22 (m, 5H),
4.72–4.40 (m, 2H), 3.97 (s, 3H), 3.61–3.57 (t, 1H), 3.45–
3.40 (t, 1H), 2.77–2.08 (m, 5H) and 1.94–1.84 (m, 1H).
Anal. calcd for C22H21N5O7: C, 56.53; H, 4.53; N,
14.98. Found: C, 56.32; H, 4.53; N, 14.75.
1
(ES) m/z 508 (M+H); H NMR (CDCl3) d 8.02–7.99
(d, 1H), 7.78–7.76 (d, 1H), 7.63–7.56 (m, 1H), 7.50–7.27
(m, 4H), 7.03–6.99 (m, 1H), 4.66–4.62 (d, 0.5H), 4.53–
4.49 (d, 0.5H), 4.38 (s, 1H), 4.28–4.18 (m, 1H),
4.01–3.98 (d, 0.5H), 3.90 (s, 3H), 3.85–3.82 (d, 0.5H),
3.52–3.29 (m, 2H), 2.86–2.57 (m, 2H), 2.34–2.27 (m,
2H), 2.10–2.07 (t, 1H), 1.98–1.90 (t, 1H), 1.66–1.40 (m,
4H), 1.01–0.95 (m, 3H), 0.83–0.77 (m, 3H).
5-(2-Azidoethyl)-N-[4-carboxy-2-(3-pentylamino)phenyl]-
5-(hydroxymethyl)pyrrolidin-2-one (34). A solution of 33
(0.047 g, 0.093 mmol) in 1 N NaOH (1 mL) and metha-
nol (1 mL) was stirred at ambient temperature for 12 h.
The mixture was acidified to pH 3 with acetic acid and
concentrated to dryness under high vacuum. The white
solid residue was triturated with EtOAc (2Â10 mL), and
the combined organic washes were extracted with water
(10 mL) and brine (10 mL). The organic layer was con-
centrated to give a white solid, which was triturated
with ether (5 mL) followed by hexane (5 mL). The inso-
luble residue was purified by flash chromatography (4%
ethanol in ether) to give 34 (25 mg, 70%) as a solid: mp
164–166 ꢀC; MS (ES) m/z 390 (M+H); 1H NMR
(CH3OD) d 7.39 (d, 0.4H), 7.33–7.32 (d, 0.6H), 7.30–
7.27 (m, 0.4H), 7.23–7.19 (dd, 1H), 6.99–6.96 (d, 0.6H),
3.64–3.60 (t, 1H), 3.52–3.35 (m, 3H), 3.27–3.16 (m, 1H),
2.81–2.64 (m, 1H), 2.57–2.29 (m, 2H), 2.27–2.12 (m,
1H), 1.99–1.88 (m, 1H), 1.82–1.73 (m, 1H), 1.68–1.41
(m, 4H), 0.99–0.88 (m, 6H). Anal. calcd for
C19H27N5O5: C, 57.27; H, 7.08; N, 17.57. Found: C,
57.60; H, 6.96; N, 17.22.
N-(2-Amino-4-methoxycarbonylphenyl)-5-(2-azidoethyl)-
5-(benzoyloxymethyl)pyrrolidin-2-one (32). Compound
31 (1.50 g, 3.21 mmol) was dissolved in THF (22 mL)
and water (9.6 mL), and to this was added Na2S2O7
(2.14 g, 12.2 mmol). After stirring under nitrogen for
20 h, more Na2S2O7 (0.197 mg, 1.13 mmol) was added,
and the mixture was stirred for an additional 5 h at
room temperature. The THF was removed under
vacuum and the aqueous residue was extracted with
EtOAc (5 Â 25 mL). The aqueous layer was con-
centrated to dryness and the residue was triturated with
EtOAc (25 mL) and filtered. The combined organic lay-
ers were dried (MgSO4) and concentrated to give an oil
(1.31 g). This was purified by flash chromatography (8%
ethanol in ether) to give 32 (1.20 g, 86.0%) as a white
solid: mp 65–68 ꢀC; MS (ES) m/z 438 (M+H); 1H
NMR (CD3OD) d 8.01–7.98 (m, 1H), 7.68–7.63 (m,
0.5H), 7.54–7.45 (m, 3H), 7.39–7.36 (m, 0.5H), 7.31–
7.24 (m, 2H), 7.13–7.10 (d, 0.5H), 7.02–7.00 (d, 0.5H),
4.72–4.56 (dd, 1H), 4.36–4.35 (dd, 1H), 3.90 (s, 3H),
3.88–3.87 (d, 1H), 3.66–3.48 (m, 1H), 3.46–3.39 (q, 1H),
5-(2-Aminoethyl)-N-[4-carboxy-2-(3-pentylamino)phe-
nyl]-5-(hydroxymethyl)pyrrolidin-2-one (11). A suspen-
sion of 34 (0.065 g, 0.167 mmol), 10% Pd/C (0 073g), and
methanol (15 mL) was reacted on a Parr shaker for 3 h at
40 psi H2. The reaction mixture was diluted with MeOH
(50 mL), and the mixture was filtered and concentrated to

W. J. Brouillette et al. / Bioorg. Med. Chem. 11 (2003) 2739–2749
2749
give an off-white solid residue. The solid was suspended
in ether and filteꢀred to give 11 (0.060 g, 96%) as a pow-
der: mp 203–205 C. MS (ES) m/z 364 (M+H); 1H NMR
(35 ꢀC, DMSO-d6) d 7.23–7.01 (m, 1.8H), 6.98–6.95 (d,
0.6H), 6.83–6.81 (d, 0.6H), 5.18 (bs, 2H), 4.1 (bs, 1H),
3.49–3.47 (d, 2H), 3.23–3.19 (d, 2H), 2.77–2.62 (m, 1H),
2.29–2.21 (m, 2H), 2.12–1.97 (m, 1H), 1.88–1.62 (m, 2H),
1.58–1.43 (m, 3.4H), 1.42–1.29 (m, 1.6H), 0.93–0.78 (m,
6H). Anal. calcd for C19H29N3O4: C, 61.34; H, 8.12; N,
11.29. Found: C, 61.19; H, 8.01; N, 11.02.
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Acknowledgements
This work was supported in part by NIH grant AI31888
and by a research grant from BioCryst Pharmaceuticals,
Inc. We thank Eric Johnson for technical assistance.
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