Pyrrolidinobenzoic acid inhibitors of influenza virus neuraminidase: The hydrophobic side chain influences type A subtype selectivity

Article abstract of DOI:10.1016/j.bmc.2012.05.001

Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A series of influenza neuraminidase inhibitors with the pyrrolidinobenzoic acid scaffold containing lipophilic side chains at the C3 position have been synthesized and evaluated for influenza neuraminidase inhibitory activity. The size and geometry of the C3 side chains have been modified in order to investigate structure-activity relationships. The results indicated that size and geometry of the C3-side chain are important for selectivity of inhibition against N1 versus N2 NA, important type A influenza variants that infect man, including the highly lethal avian influenza.

Full text of DOI:10.1016/j.bmc.2012.05.001

Bioorganic & Medicinal Chemistry 20 (2012) 4582–4589
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrrolidinobenzoic acid inhibitors of influenza virus neuraminidase:
The hydrophobic side chain influences type A subtype selectivity
Yanwu Li a, Arundutt Silamkoti a, Gundurao Kolavi a, Liyuan Mou a, Shelly Gulati b, Gillian M. Air b,
Wayne J. Brouillette a,
⇑
a Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA
b Department of Biochemistry, University of Oklahoma Health Science Center, Oklahoma, OK 73190, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new ther-
apeutic agents. A series of influenza neuraminidase inhibitors with the pyrrolidinobenzoic acid scaffold
containing lipophilic side chains at the C3 position have been synthesized and evaluated for influenza
neuraminidase inhibitory activity. The size and geometry of the C3 side chains have been modified in
order to investigate structure–activity relationships. The results indicated that size and geometry of
the C3-side chain are important for selectivity of inhibition against N1 versus N2 NA, important type A
influenza variants that infect man, including the highly lethal avian influenza.
Received 14 March 2012
Revised 28 April 2012
Accepted 2 May 2012
Available online 17 May 2012
Keywords:
Neuraminidase
Avian influenza
Pyrrolidinobenzoic acid
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
RapiactaÒ (1e, peramivir), and all have been marketed as
anti-influenza drugs. These drugs have in common a nonaromatic
Annual epidemics of influenza virus are responsible for consid-
erable morbidity and mortality,1 and pandemics are particularly
devastating.2–4 Infection is especially dangerous to the very young,
elderly, and to those who have suppressed immune systems.5–7
Vaccination has been the main preventive measure and has pro-
vided limited control, but vaccines must be reformulated each year
in response to antigenic drift and may be ineffective against new
variants of influenza viruses.2,8 The replicative cycle of influenza
virus offers several virus-specific events which could be considered
as targets for chemotherapeutic intervention.9 One of these is
influenza neuraminidase (NA), which catalyzes the cleavage of
the terminal sialic acid attached to host cell glycoproteins and gly-
colipids.10,11 This process is believed to be necessary for the release
of newly formed virus from infected cells and for efficient spread of
virus in the respiratory tract.
central ring containing a carboxylate and several side chains with
defined stereochemistry, including the N-acetyl moiety, a strongly
basic nitrogen, and (except for RelenzaÒ) a hydrophobic group. The
requirement for multiple chiral centers in some cases has resulted
in high manufacturing costs.
We have developed a number of NA inhibitors containing the
benzoic acid ring scaffold.20–24 The benzene ring was selected as
a template because it provides planarity near the carboxylate as
found in structures 1a–1d and, further, the benzene ring has no
stereogenic centers at substitution sites. Such inhibitors thus
potentially offered a more economical synthesis of anti-influenza
drugs. As shown in Figure 1 Our lead benzoic acid inhibitor20
was 4-(N-acetylamino)-3-guanidinobenzoic acid (2) (BANA 113,
Ki 2.5
lM). Further optimization of 2 resulted in the potent benzoic
acid inhibitor
3
(1-[4-carboxy-2-(3-pentylamino)phenyl]-5,5-
In recent years several very potent inhibitors of both influenza A
and B NA have been developed (Fig. 1).12–18 Early studies on NA
inhibition revealed that 2,3-didehydro-2-deoxy-N-acetylneurami-
nic acid (1a) (Neu5Ac2en), an analog of sialic acid, is a modest
bis(hydroxymethyl)pyrrolidin-2-one), which is unique among po-
tent inhibitors in that it contains a substituted pyrrolidinone ring
in place of the N-acetyl functionality. Note that compound 3 is also
unique in that it contains no chiral carbons and lacks a strongly ba-
sic nitrogen. Compound 3 exhibited an IC50 of 48 nM against influ-
enza A NA (N9, an avian strain),22 but was less effective against
other type A NAs (see Table 1).
Here we describe the synthesis and evaluation of additional
inhibitors related to 3, with special emphasis on determining ef-
fects on the N1 and N2 NAs of human viruses. Given the impor-
tance of the hydrophobic side chain in potent inhibitors 1c, 1e,
and 3, we proposed for this study modifications to the hydrophobic
inhibitor with a Ki of 4 lM, and is believed to be a transition state
analog due to planarity near the carboxylate. Solving the crystal
structure of 1a in complex with NA was crucial in that it provided
for the structure-based drug design19 of RelenzaÒ (1b), which was
followed by TamifluÒ (the prodrug 1d; 1c is the active form), and
⇑
Corresponding author.
E-mail address: wbrou@uab.edu (W.J. Brouillette).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.001

Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4582–4589
4583
O
OH
O
OH
O
O
OH
O
OR
O
OH
HO
NH2
NH
OH
NH
N
R
O
O
NH2
N
HN
H
O
H
O
HO
HO
HN
OH OH HN
HN
N
O
NH2
NHAc
1e
1a R = OH
2
1c R = H
1d R = Et
3
1b
R = NH(C=NH)NH2
Figure 1. Structures of clinically marketed neuraminidase inhibitors compared to benzoic acid inhibitors.
side chain at the C3 position of 3 by incorporating alkyl or ether
moieties.
one ‘pot’), only the purified intermediates are shown in Scheme
2: the first step was the introduction of the pyrrolidinone ring,
which was accomplished by reductive amination of the aniline
with diethyl ketomalonate, N-alkylation with 3-bromopionic aid
in the presence of PCl3, and cyclization in the presence of NaH.
We found that overall yields were highest when these reactions
were carried out without purifying the intermediates. The alkenes
12a and 12b were purified at this stage, while alkane 12c was ob-
tained after further hydrogenation in the presence of Pd/C. Final
compounds 13a and 13c were prepared by catalytic hydrogenation
of the alkene, selective reduction of the malonate esters to diols
using NaBH4, and saponification of the benzoates to the final acids.
Compounds 13a and 13c were synthesized following the same se-
quence except that the intermediate were purified at different
stage. Compound 13b was prepared in a similar fashion, but with-
out the catalytic hydrogenation step, to allow the alkene to be pre-
served in the larger side chain of final product 13b, and the
catalytic hydrogenation of 13b then afforded 14b.
2. Chemistry
We first pursued the synthesis of analog 10a–e, which intro-
duced lipophilic ether side chains (similar to 1c) at C3. These were
prepared as shown in Scheme 1. 3-Methyl-4-nitrobenzoic acid was
esterified with MeOH to give 4, and reduced with Zn/HCl to afford
methyl 3-methyl-4-aminobenzoate (5). Intermediate 5 was re-
acted with diethyl bromomalonate to give 6, which was N-acylated
with 3-bromopropionic acid and PCl3 to provide 7. This underwent
cyclization in the presence of NaH to yield lactam 8, which was
treated with NBS to produce the bromo compound 9 in 18% overall
yield. There were three steps involved in the conversion of com-
pound 9 to the final acids 10a–e and intermediates were not puri-
fied. The first step was the alkylation with the appropriate alkoxide
to form the corresponding ether, In this step, the methyl benzoate
of 9 was partially transesterified by the alkoxide to provide a mix-
ture of methyl benzoate and/or alkyl benzoate (eg., 2-propyl, etc.).
The ethers containing mixed benzoate esters were used for the
next steps without further purification. The second step was selec-
tive reduction of the ethyl malonate esters on the pyrrolidinone
ring to form the diols. The third step was hydrolysis of the mixed
benzoate esters using aqueous NaOH to afford the final acids
10a–e.
3. Biological assays
All compounds were evaluated for in vitro inhibitory activity
against NA using intact purified virions of influenza virus A (cur-
rent vaccine strains of H1N1 and H3N2 subtypes) and influenza
virus B using the fluorogenic substrate 20-(4-methylumbelliferyl)-
a-D
-N-acetylneuraminic acid,32 and IC50 values were determined
using Prism software. The results are contained in Table 1.
Next, compounds 13a, 13c, and 14b were designed as analogs of
3 that contain an alkyl side chain in place of the aliphatic amino
side chain at the C3 position. The preparations of 13a, 14b, and
13c are described in Scheme 2. Compounds 11a–c were synthe-
sized through a palladium-catalyzed cross-coupling reaction be-
tween methyl 4-amino-3-iodobenzoate and the corresponding
vinyl bromide (a–c). While the syntheses of intermediates 12a–c
from the intermediate 11a–c each involved three major steps (in
4. Results and discussion
The numerous crystal structures that have been reported for a
variety of type A and B neuraminidases (NAs) demonstrate that
this site is highly conserved and, essentially, structurally identical
irregardless of the source. Thus it is not surprising to find that
Table 1
In vitro inhibitory effects of benzoic acid analogs on influenza A and B neuraminidases
Inhibition of Neuraminidase, IC50
(lM)
Compounds
H3N2
H1N1
B
A/Uruguay/716/07
A/Brisbane/59/07
A/Solomon Islands/3/06
B/Malaysia/5206/04
3a
0.49
79
271
N/A
80 30
30 1.4
N/A
10a
10b
10c
10d
10e
4000 15
0.20 0.19
0.50 0.10
40 8.1
>10000
16 2.1
1.0 0.21
420 45
N/A
N/Ab
11 4.4
0.80 0.07
400 25
1600 255
200 95
N/A
H3N2
H1N1
B
A/Wisconsin/05
A/Brisbane/59/07
A/OK 3052/09
B/Malaysia/2506/04
13a
13b
13c
14b
0.50 0.15
0.41 0.070
3.0 0.90c
0.20 0.090
N/A
N/A
3.4 1.2
N/A
190 10
120 16
140 17
32 3.4
2.4 0.9
70 7.8
1.6 0.6
3.2 1.3
a
b
c
Data from Ref. 24 using A/PR/8/34 (N1), A/Udorn/72 (N2), and B/Lee/40.
N/A = data not available.
A/Uruguay/716/07.

4584
Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4582–4589
COOMe
COOMe
COOMe
COOH
NO2
COOMe
iii
ii
iv
i
EtOOC
NH
EtOOC
EtOOC
N
O
NO2
NH2
COOEt
4
5
6
7
Br
v
COOH
COOMe
COOMe
R
a. 2-propyl
b. 2-butyl
c. 3-pentyl
d. phenyl
e. benzyl
vii
vi
O
Br
R
EtOOC
EtOOC
N
O
HO
HO
N
O
EtO2C
EtO2C
N
O
8
10a-e
9
Scheme 1. Reagents and conditions: (i) MeOH, H2SO4, reflux, 15 h; (ii) Zn/HCl, EtOAc, 0–20 °C 15 h; (iii) diethyl bromomalonate, 120 °C, 22 h; (iv) 3-bromopropionic acid,
PCl3, toluene, 100 °C, 22 h; (v) NaH, 0–20 °C, 4 h; (vi) CCl4, NBS, AIBN, 80 °C, 3 h; (vii) (a) BuLi, ROH, ꢀ10–20 °C 15 h; (b) NaBH4, MeOH, THF, ꢀ21 h; (c) NaOH, MeOH, 20 °C,
1–2 h.
COOMe
COOH
COOMe
COOMe
For 11a,b
For 12a
Br
R
iii+iv
ii
R
R
i
+
I
R
R
R
a-c
NH2
NH2
EtO2C
EtO2C
N
O
HO
HO
N
O
11a-c
12a-b
R = a. propyl
b. ethyl
13a
For 11c
ii+iii
For 12b
iv
c. methyl
COOH
COOH
COOMe
COOH
iii
iv
HO
HO
N
O
EtO2C
EtO2C
N
O
HO
HO
N
O
HO
HO
N
O
14b
13c
12c
13b
Scheme 2. Reagents and conditions: (i) (a) Vinyl bromide, dioxane, Et3N, Pd(PPh3)4, pinacolborane, 80 °C, 1 h; (b) methyl 4-amino-3-iodobenzoate, Ba(OH)2ꢁ8H2O, water,
90 °C, 8 h; (ii) (a) p-TSA, toluene, diethyl ketomalonate, reflux, 19 h; (b) NaBH4, DEC, AcOH, 15 h; (c) 3-bromopropionic acid, PCl3, toluene, 100 °C, 20 h; (d) NaH, 0–20 °C, 4 h;
(iii) Pd/C, H2, MeOH, 25 °C; (iv) (a) NaBH4, MeOH/THF, 2–5 h; (b) NaOH, MeOH, 20 °C, 1–2 h.
inhibitors often show similar effects for all subtypes. For example,
with oseltamivir carboxylate (1c; the active form of TamifluÒ),
inhibition (IC50) of N1 NAs ranged from 0.30 to 0.80 nM, N2 NAs
ranged from 0.68 to 1.78 nM, and type B NAs ranged from 5.00
to 24.3 nM.28 One recent exception to this structural conservation
among NA binding sites is the observation that type A NAs can be
grouped into two structural classes. The Group 1 NAs include N1,
which in some crystal structures (but not all) shows an additional
cavity next to the active site that is not present in N2 (a member of
Group 2 NAs). The cavity is created by movement of the ‘150 loop’
(residues 147–152) and has been suggested to provide a new tar-
get for drug design.29 While, as mentioned previously, selectivity
of inhibition for N1 versus N2 NAs is not observed for the potent
clinically used inhibitors, any observed selectivity might be ratio-
nalized based on this observation.
disease in humans and is the major target of clinically useful NA
inhibitors. Since pyrrolidinone 3 exhibited good activity against
N2 NA (relatively rigid binding site), but poor activity against N1
NA (may contain extra binding cavity), here we proposed to deter-
mine if smaller or larger side chains of different presentation might
improve N1 activity without decreasing N2 activity. Particular
choices for these side chains were limited by synthetic accessibility
and resources but attempted to vary side chain length, presenta-
tion, and branching size. For this study we chose to measure inhi-
bition against NAs of recently circulating H3N2 and H1N1 viruses,
using the current vaccine strains in most cases.
In evaluating the analogs of 3 described in Table 1, we were
therefore interested in two trends-changes in potency of inhibition
and changes in selectivity for N1 versus N2 NA. Analogs of 3 in-
cluded in this study were those containing aliphatic side chains
with the same branching position(13a–c, and 14b), relative to
the benzene ring, as found in 3, and those containing ether side
chains branched one atom further from the benzene ring (10a–e).
For the latter series, the most effective inhibitors against N1 and
N2 NA are 10b and 10c. If the hydrophobic chain is too small (10a)
or too large (10d–e) potency is lost. Further, 10c is less selective
than 10b for N2 versus N1 NA. While 10b and 10c exhibit nearly
As shown in Table 1, the parent compound for the present
study, 25,26 pyrrolidinone 3, indeed exhibited significant selectivity
for N2 versus N1 NA. The much poorer inhibition seen for type B
NA is commonly observed for certain inhibitors containing a
26,27
hydrophobic side chain,
and has been rationalized based on
an energetically unfavorable conformational change in Glu276
upon ligand binding.30,31 Type A influenza causes more serious

Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4582–4589
4585
the same IC50 against N2, 10c is much more effective against N1,
and is the most potent N1 NA inhibitor of this series, with dramat-
ically improved (80- to 100-fold) N1 activity compared to the par-
ent 3. The activities of 10b and 10c for N2 NA are similar to that for
the parent 3.
determined on a Mel-Temp electrothermal melting point appara-
tus (Laboratory Devices) and are uncorrected.
27
6.2.1. Methyl 3-methyl-4-nitrobenzoate (4)
To
a
solution of 3-methyl-4-nitrobenzoic acid (25.0 g,
For the former series (13a–c, 14b), all but 13c are nM inhibitors
against N2 NA that exhibit similar potency to each other and to the
parent 3. Within this series, compounds 13b and 14b are also the
least selective for N2 versus N1 NA and exhibit a 30- to 40-fold in-
crease in N1 NA inhibition compared to the parent 3.
138 mmol) in anhydrous methanol (400 mL) was added concd
H2SO4 (2 mL), at room temperature. The resulting solution was re-
fluxed for 15 h. The reaction mixture was concentrated. The solid
was filtered and washed with cold methanol and dried to obtain
the ester 4 (24 g, 89%) as a colorless solid. mp 78–80 °C.
As discussed above, for the series of compounds presented, N2
activity is always better than N1 activity, suggesting that any
involvement of the 150-loop for binding of these inhibitors results
in a penalty in all but the most potent N1 inhibitors such as 10c.
27
6.2.2. Methyl 4-amino-3-methylbenzoate (5)
Methyl 3-methyl-4-nitrobenzoate (23 g, 117.84 mmol) was dis-
solved in ethyl acetate (400 mL) and cond HCl (58 mL). Then the
solution was cooled to 0 °C and Zn powder (46.2 g, 707 mmol)
was added in portions over 20 min with stirring. The mixture
was allowed to warm up to room temperature and stirring was
continued for 15 h. The reaction mixture was passed through a
bed of Celite and washed with ethyl acetate (100 mL). The filtrate
was washed with satd NaHCO3 solution (4 ꢂ 100 mL), water
(3 ꢂ 100 mL) and brine (2 ꢂ 100 mL). The organic layer was dried
over anhydrous Na2SO4, filtered and concentrated to dryness to ob-
tain the amino compound 5 (17.4 g, 90.0%) as a colorless solid. mp
111–113 °C; 1H NMR (300 MHz, CDCl3) d 2.18 (s, 3H), 3.85 (s, 3H),
4.02 (s, br, 2H), 6.65 (d, J = 8.1 Hz, 1H), 7.76–7.72 (m, 2H); 13C NMR
(75 MHz, CDCl3) d 17.2, 51.7, 113.8, 119.5, 121.1, 129.4, 132.3,
149.5, 167.6; MS (ES) m/z 166 (M+1).
5. Conclusions
In summary, none of these analogs resulted in significantly en-
hanced potency, relative to 3, against N2 NA. However, relatively
subtle changes in the nature of the hydrophobic group did yield
up to 100-fold improvements against N1 NA. In this regard the best
compounds (10c, 13b, and 14b) all contain the same 3-pentyl
grouping, but this was contained in different structures with either
a one or two atom spacer to the benzene ring. It is interesting that
this spacer length made little difference in activity.
It is important to note that 3, and the analogs in Table 1, can
only undergo three subsite interactions with NA, unlike the more
potent inhibitors 1b, 1c, and 1e, which all contain four interactions
(eg., 1c contains substituents that occupy subsites for the carbox-
ylate, N-acetyl, amino, and hydrophobic groups). Thus we antici-
pate that introduction of the missing basic group to provide a
fourth interaction on pyrrolidinobenoic acids should also provide
highly potent inhibitors. Current studies are pursuing such targets.
6.2.3. Diethyl 2-(4-methoxycarbonyl-2-methyl-
phenylamino)malonate (6)
A mixture of methyl 4-amino-3-methylbenzoate 5 (10.0 g,
60.5 mmol) and diethylbromomalonate (7.90 g, 33.3 mmol) was
heated at 120 °C for 22 h. The mixture was then cooled to room
temperature and triturated with ethyl acetate (100 mL). The sepa-
rated solid was filtered and washed with ethyl acetate (50 mL). The
filtrate was concentrated and the crude product was purified by
flash silica chromatography (75% hexane in EtOAc) to give a pale
yellow solid (5.53 g, 56.0%). mp 40–42 °C; 1H NMR (300 MHz,
CDCl3) d 1.30 (t, J = 7.1 Hz, 6H), 2.27 (s, 3H), 3.85 (s, 3H), 4.30 (q,
J = 7.1 Hz, 4H), 4.82 (d, J = 6.8 Hz, 1H), 5.17 (d, J = 6.8 Hz, 1H),
6.50 (d, J = 8.3 Hz, 1H), 7.77–7.81 (m, 2H); 13C NMR (100 MHz, Ace-
tone-d6) d 14.3, 17.2, 51.6, 60.6, 63.0, 110.3, 120.3, 122.8, 130.1,
132.3, 148.7, 167.1, 168.0; MS (ES) m/z 324 (M+1).
6. Experimental section
6.1. Neuraminidase inhibition assays
Influenza virus strains A/Uruguay/716/07 (H3N2), A/Brisbane/
59/07 (H1N1), A/Solomon Islands/3/06 (H1N1), A/Wisconsin/67/
05 (H3N2), A/OK/3052/09 (H1N1pdm09), and B/Malaysia/5206/
04 were grown in embryonated chicken eggs. IC50 determinations
were carried out using the fluorogenic substrate 20-(4-methylum-
belliferyl)-a-D
-N-acetylneuraminic acid32 adapted to a 96-well
plate. The reactions included neuraminidase in the form of purified
intact virions, preincubated for 30 min at room temperature with
two-fold dilutions of inhibitor in 50 mM sodium acetate, pH 6.0,
6.2.4. Diethyl 2-[(3-bromo-propionyl)-(4-methoxycarbonyl-2-
methyl-phenyl)-amino]malonate (7)
To a solution of 3-bromopropionic acid (2.44 g, 16.0 mmol) in
toluene (25 mL), PCl3 (3.49 g, 25.4 mmol) was added and the mix-
ture was heated at 110 °C for 2 h. The mixture was cooled to room
temperature, and a solution of 6 (2.35 g, 7.26 mmol) in toluene
(10 mL) was added dropwise. Heating was continued at 100 °C
for an additional 20 h. The reaction mixture was concentrated
and diluted with EtOAc (100 mL), washed with sat.NaHCO3 solu-
tion (2 ꢂ 75 mL), water (2 ꢂ 75 mL) and brine (2 ꢂ 75 mL). The or-
ganic layer was dried over anhydrous Na2SO4, evaporated and the
crude product was purified by flash silica chromatography (25%
EtOAc in hexanes; Rf 0.5) to afford 7 as a colorless solid (2.86 g,
85.0%). mp 104–106 °C; 1H NMR (300 MHz, CDCl3) d; 1.12 (t,
J = 6 Hz, 3H), 1.31 (t, J = 7.2 Hz, 3H), 2.43 (s, 3H), 2.49–2.70 (m,
2H), 3.49–3.63 (m, 2H), 3.93 (s, 3H), 4.00–4.18 (m, 2H), 4.30 (q,
J = 7.2 Hz, 2H), 5.05 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.90 (dd,
J = 8.4 Hz, 1.5 Hz, 1H), 8.00 (s, 1H); 13C NMR (75 MHz, CDCl3) d
13.9, 14.1, 18.1, 26.1, 37.3, 52.5, 62.4, 65.2, 128.7, 129.7, 131.2,
132.9, 137.7, 142.9, 165.2, 165.6, 166.3, 170.1; MS (ES) m/z 458,
460 (M+1).
100
100
l
l
M CaCl2, 320
lM MgCl2 and 60 mM NaCl, before adding
M of the substrate (Sigma). The reaction mixtures were incu-
bated for 15 min at 37 °C, after which the reaction was stopped
with five volumes of 0.2 M glycine–NaOH, pH 11. The IC50s were
determined by Prism software using the log (inhibitor) versus nor-
malized response option and cross-checked by plotting the enzyme
fractional activity against the inhibitor concentration, and the IC50
was determined from the linear region of the dose–response curve.
6.2. General experimental procedures
NMR data were recorded on Bruker ARX 300 or 400 MHz spec-
trometer. Mass spectra were obtained using electrospray ioniza-
tion on a MicroMass Platform LCZ LC/MS with HP1100HPLC/
autoinjector and diode array detector. Flash column chromatogra-
phy was carried out using silica gel (40
performed on silica gel (25 m platesWhatman), and spots were
visualized with UV light (UV-254 nm). Melting points were
lm, Silicycle, Inc.). TLC was
l

4586
Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4582–4589
6.2.5. 5,5-Bis(ethoxycarbonyl)-1-(4-methoxycarbonyl-2-
methylphenyl)pyrrolidin-2-one (8)
6.2.8.2. 4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-1-yl)-3-(iso-
propoxymethyl)benzoic acid (10a). Colorless solid: mp 146–
To a suspension of NaH (0.300 g, 12.6 mmol) in anhydrous DMF
(35 mL) was added a solution of 7 (5.26 g, 11.5 mmol) in anhy-
drous DMF (20 mL) dropwise for a period of 15 min at 0 °C. The
mixture was stirred at room temperature for 4 h. The reaction mix-
ture was diluted with EtOAc (200 mL) and washed with water
(4 ꢂ 150 mL) and brine (2 ꢂ 150 mL). The organic layer was dried
over anhydrous Na2SO4,evaporated and the crude product was
purified by flash silica chromatography (25% EtOAc in hexanes, Rf
0.4) to afford pure 8 as a colorless solid (4.0 g, 92%); mp 68–
70 °C; 1H NMR (400 MHz, CDCl3) d 7.93 (s, 1H), 7.85 (d, J = 8.3 Hz,
1H), 7.52 (d, J = 8.3 Hz, 1H), 4.34 (m, 2H), 3.93 (m, 2H), 3.90 (s,
3H), 2.98 (m, 1H), 2.65 (m, 2H), 2.52 (m, 1H), 2.17 (s, 3H), 1.30
(t, J = 7.1 Hz, 6H), 0.88 (t, J = 7.1 Hz, 6H); 13C NMR (100 MHz, CDCl3)
d 13.8, 14.4, 18.5, 29.3, 29.7, 52.6, 62.7, 63.1, 74.6, 128.2, 128.6,
130.3, 132.2, 138.4, 140.6, 166.8, 167.0, 1170.2, 174.6; MS (ES)
m/z 378(M+1).
148 °C; yield 20%; TLC Rf 0.3, (EtOAc/MeOH, 4:1, v/v). 1H
NMR(400 MHz, CD3COCD3) d 1.01(d, J = 6.1 Hz, 6H), 2.12(m, 2H),
2.39(m, 2H), 3.09(d, J = 11.8 Hz, 1H), 3.14(d, J = 11.8 Hz, 1H),
3.40(d, J = 11.7 Hz, 1H), 3.46(d, J = 11.7 Hz, 1H), 3.55(m, 1H),
4.18(d, J = 12.9 Hz, 1H), 4.54(d, J = 12.9 Hz, 1H), 7.25(d, J = 8.3 Hz,
1H), 7.78(dd, J = 8.3, 2 Hz, 1H), 8.04(d, J = 2 Hz, 1H); 13C NMR
(100 MHz, CD3COCD3) d 21.3, 21.5, 25.1, 30.4, 63.0, 63.8, 65.8,
72.2, 72.4, 129.1, 129.4, 130.6, 131.0, 138.5, 139.9, 168.3, 178.1;
Purity by HPLC 100%; MS (ES) m/z 338 (M+1).
6.2.8.3. 4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-1-yl)-3-(2-
butoxymethyl)benzoic acid (10b).
Colorless solid: mp 156–
158 °C; yield 29%; TLC Rf 0.3, (AcOEt/MeOH, 4:1, v/v). 1H
NMR(400 MHz, CD3COCD3) d 0.76–0.81(m, 3H), 1.01–1.08(m, 3H),
1.35–1.52(m, 2H), 2.18(m, 2H), 2.38(m, 2H), 3.19–3.28(m, 2H),
3.41(m, 1H), 3.53–3.61(m, 2H), 4.23(d, J = 12.7 Hz, 0.5H), 4.33(d,
J = 12.7 Hz, 0.5H), 4.64(d, J = 12.6 Hz, 0.5H), 4.70(d, J = 12.6 Hz,
0.5H), 7.38(d, J = 8.2 Hz, 1H), 7.81(m, 1H), 8.14(m, 1H); 13C NMR
(100 MHz, CD3COCD3) d 10.4, 10.6, 19.8, 20.0, 31.3, 64.3, 64.4,
65.1, 65.2, 67.1, 67.3, 72.2, 72.3, 77.9, 78.2, 130.0, 130.1, 131.1,
131.2, 131.4, 131.8, 140.6, 140.8, 140.9, 141.2, 167.8, 167.9,
176.8, 176.9; Purity by HPLC 96%; MS (ES) m/z 352 (M+1).
6.2.6. Diethyl 1-(2-(bromomethyl)-4-
(methoxycarbonyl)phenyl)-5-oxopyrrolidine-2,2-dicarboxylate
(9)
To
a solution of 8 (1.6 g, 4.2 mmol) and AIBN (70 mg,
0.40 mmol) in CCl4 (36 mL), NBS (0.80 g, 4.6 mmol) was added.
The mixture was refluxed for 3 h. The solid was filtered and the fil-
trate was concentrated to dryness. The obtained residue was chro-
matographed on a flash silica gel column (hexane/EtOAc, 2:1 v/v)
to yield 9 (1.0 g, 52%) as a white solid. mp 58–60 °C; TLC Rf 0.4,
(hexane/EtOAc, 1:1 v/v). 1H NMR (400 MHz, CDCl3) d 0.86 (t,
J = 7.1 Hz, 3H), 0.95 (t, J = 7.1 Hz, 3H), 2.53 (m, 1H), 2.67 (m, 2H),
2.96 (m, 1H), 3.93 (s, 3H), 4.01 (m, 2H), 4.34 (m, 2H), 4.35 (d,
J = 11.4 Hz, 1H), 4.40 (d, J = 11.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H),
7.97 (dd, J = 1.9, 8.4 Hz, 1H), 8.21 (d, J = 1.9 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 13.7, 14.4, 29.3, 29.4, 29.8, 52.8, 62.8, 63.4,
74.6, 128.4, 130.5, 130.7, 132.9, 137.1, 140.5, 166.3, 166.4, 170.1,
175.2; MS (ES) m/z 456, 458(M+1).
6.2.8.4. 4-(2,2-Bis(hydroxymethyl)-3-((3-pentyloxy)methyl)-5-
oxopyrrolidin-1-yl)benzoic acid (10c).
Colorless solid: mp
177–179 °C; yield 20%; TLC Rf 0.3, (EtOAc/MeOH, 4:1, v/v). 1H
NMR(400 MHz, CD3COCD3) d 0.67(d, J = 7.5 Hz, 6H), 1.33(m, 2H),
2.08(m, 2H), 2.29(m, 2H), 3.09–3.17(m, 3H), 3.44(d, J = 11.5 Hz,
1H), 3.48(d, J = 11.5 Hz, 1H), 4.18(d, J = 12.9 Hz, 1H), 4.54(d,
J = 12.9 Hz, 1H), 7.28(d, J = 8.2 Hz, 1H), 7.71(dd, J = 8.2, 1.9 Hz,
1H), 8.07(d, J = 1.9 Hz, 1H); 13C NMR (100 MHz, CD3COCD3) d
10.2, 10.4, 14.8, 23.7, 26.1, 26.7, 26.9, 31.3, 64.3, 65.1, 67.6, 72.2,
83.4, 130.0, 130.4, 131.1, 1231.5, 140.5, 141.2, 167.8, 176.8; Purity
by HPLC 98%; MS (ES) m/z 366 (M+1).
6.2.7. Note
6.2.8.5.
(phenoxymethyl)benzoic acid (10d).
198–200 °C; yield 20%; TLC Rf 0.5, (EtOAc/MeOH, 4:1, v/v). 1H
NMR(400 MHz, CD3OD) 2.33(m, 2H), 2.63(m, 2H), 3.36(d,
4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-1-yl)-3-
In the following conversion of 9 to 10a–e, overall yields were
highest when intermediates were not isolated and purified.
Colorless solid: mp
d
6.2.8. 4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-1-yl)-3-
(alkoxymethyl)benzoic acid (10a–e)
J = 11.5 Hz, 1H), 3.42(d, J = 11.5 Hz, 1H), 3.64(d, J = 11.7 Hz, 1H),
3.70(d, J = 11.7 Hz, 1H), 5.04(d, J = 13.6 Hz, 1H), 5.40(d,
J = 13.6 Hz, 1H), 6.95(m, 1H), 7.04(d, J = 7.8 Hz, 2H), 7.28(m, 2H),
7.55(d, J = 8.2 Hz, 1H), 8.04(dd, J = 8.2, 1.9 Hz, 1H), 8.31(d,
J = 1.9 Hz, 1H); 13C NMR (100 MHz, CD3OD) d 26.6, 31.2, 64.5,
65.1, 67.2, 73.6, 116.3, 122.5, 130.8, 131.0, 132.4, 139.6, 139.9,
160.5, 169.6, 179.6; Purity by HPLC 97%; MS (ES) m/z 372 (M+1).
6.2.8.1. General procedure.
After the alcohol was treated
with 4 mL of butyllithium (1.6 M) at ꢀ10 °C, compound 9 (1 mmol)
was added at room temperature. The resulting mixture was stirred
for 15 h at room temperature. The reaction mixture was diluted
with ethyl acetate (50 mL), and washed with saturated NaHCO3
(3 ꢂ 20 mL), water (2 ꢂ 20 mL) and brine (2 ꢂ 20 mL). The organic
layer was dried (Na2SO4) and concentrated. The residue was chro-
matographed on a flash silica gel column to afford an oil. The oil
was dissolved in dry MeOH (3 mL) and THF (3 mL) and cooled to
0 °C under an argon atmosphere. Then NaBH4 (189 mg, 5.00 mmol)
was added in portions over 20 min. The mixture was allowed to
warm to room temperature, stirred for 1–2 h, and quenched by
adding cold water (5 mL), This mixture was then extracted with
EtOAc (3 ꢂ 10 mL), and the combined organic extracts were
washed with water (1 ꢂ 5 mL) and brine (1 ꢂ 5 mL), dried (Na2SO4)
and evaporated to dryness. The residue was chromatographed on a
flash silica gel column to afford a semisolid. The semisolid in MeOH
(5 mL) was treated with a 1 M solution of NaOH (5 mL) and the
solution was stirred at room temperature for 1–2 h. The mixture
was neutralized by using 1 N HCl. The solvent was evaporated
and the residue was purified by flash chromatography to obtain
the benzoic acids 10a–e. The three steps overall yields were calcu-
lated from bromo compounds 9 to the benzoic acids 10a–e.
6.2.8.6. 3-((Benzyloxy)methyl)-4-(2,2-bis (hydroxymethyl)-5-
oxopyrrolidin-1-yl)benzoic acid (10e).
Colorless solid: mp
198–200 °C; yield 20%; TLC Rf 0.5, (AcOEt/MeOH, 4:1, v/v). 1H
NMR(400 MHz, CD3COCD3) d 2.05(m, 1H), 2.15(m, 1H), 2.27(m,
1H), 2.38(m, 1H), 3.33–3.44(m, 2H), 4.13(w, 1H), 4.44–4.58(m,
5H), 7.14–7.31(m, 6H), 7.86(dd, J = 8.2, 1.9 Hz, 1H), 8.14(d,
J = 1.9 Hz, 1H); 13C NMR (100 MHz, CD3COCD3) d 22.6, 31.8, 62.7,
63.7, 69.7, 73.7, 128.8, 129.1, 129.6, 130.5, 130.6, 131.5, 138.7,
139.7, 142.0, 167.6, 175.5; Purity by HPLC 98%; MS (ES) m/z 386
(M+1).
6.2.8.7. General procedure for the palladium-catalyzed cross-
coupling reaction (11a–c).
To a solution of vinyl bromide a–c
(6.7 mmol) in dioxane (30 mL) at 25 °C under argon were succes-
sively added Et3N (3.70 mL, 26.9 mmol), Pd (PPh3)4 (779 mg,
0.670 mmol), and pinacolborane (2.90 mL, 20.2 mmol) dropwise.
The solution was heated to 80 °C for 1 h. After cooling, water

Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4582–4589
4587
(5 mL) was added dropwise followed by methyl 4-amino-3-iodo-
benzoate (1.5 g, 5.4 mmol) dissolved in dioxane (15 mL) and
Ba(OH)2ꢁ8H2O (6.40 g, 20.2 mmol). The solution was heated to
90 °C for 8 h. After cooling, the mixture was filtered through Celite,
brine was added (40 mL), and the solution was extracted with ethyl
acetate (3 ꢂ 50 mL). After drying over magnesium sulfate and evap-
oration of the solvents under vacuum, the residue was purified by
flash chromatography to obtain the benzoates
flash silica gel column to afford an oil. A solution of the oil in dry
DMF (4 mL) was added dropwise to a stirred suspension of NaH
(16 mg, 0.68 mmol) in dry DMF (4 mL) over 10 min under Ar. The
mixture was stirred at room temperature for 1 h. The NaH was
quenched with water (0.1 mL), and the mixture was diluted with
ethyl acetate (20 mL). This was washed with NaHCO3 (2 ꢂ 10 mL)
and brine (4 ꢂ 10 mL), dried (Na2SO4), filtered, and concentrated.
The obtained residue was chromatographed on a flash silica gel
column to obtain the benzoic acids 12a–b. The four step overall
yields are given below.
6.2.8.9. Methyl
4-amino-3-(2-propylpent-1-ynyl)benzoate
(11a). Colorless oil, yield 76%, TLC Rf 0.4 (hexane/EtOAc 1/1
v/v). 1H NMR(400 MHz, CDCl3) d 0.81(t, J = 7.4 Hz, 3H), 0.97(t,
J = 7.4 Hz, 3H), 1.40(m, 2H), 1.54(m 2H), 2.02(m, 2H), 2.17(m,
2H), 3.85(s, 3H), 4.10(w, 2H), 6.00(s, 1H), 6.64(d, J = 8.4 Hz,
1H),7.68(d, J = 2.0 Hz, 1H), 7.74(dd, J = 8.4, 2.0 Hz, 1H); 13C
NMR(100 MHz, CDCl3) d 14.4, 14.4, 21.6, 21.6, 33.1, 38.7, 52.0,
114.0, 119.5, 120.2, 123.6, 130.0, 132.5, 147.3, 149.1, 167.9; MS
(ES) m/z 262 (M+1).
6.2.9.2. Diethyl 1-(4-(methoxycarbonyl)-2-(2-propylpent-1-enyl)
phenyl)-5-oxopyrrolidine-2,2-dicarboxylate (12a).
Colorless
solid: mp 88–90 °C, yield 17%, TLC Rf 0.59 (hexane/EtOAc 1/1 v/v).
1H NMR(400 MHz, CDCl3) d 0.86(m, 3H), 0.93(t, J = 7.3 Hz, 3H),
0.94(t, J = 7.3 Hz, 3H), 1.30(m, 3H), 1.53(m 4H), 2.09(m, 4H),
2.42(m, 1H), 2.58(m, 2H), 2.98(m, 1H), 3.90(m, 1H), 3.91(s, 3H),
3.93(m, 1H), 5.90(s, 1H), 4.32(m, 2H), 7.58(d, J = 8.3 Hz, 1H),
7.90(dd, J = 8.3, 2.0 Hz, 1H), 7.94(d, J = 2.0 Hz, 1H); 13C
NMR(100 MHz, CDCl3) d 14.3, 14.7, 21.4, 22.0, 29.3, 29.4, 33.5,
39.0, 52.6, 62.9, 74.5, 120.3, 128.6, 129.1, 130.0, 131.8, 139.1,
140.3, 147.2, 167.1, 174.6, MS (ES) m/z 474 (M+1).
6.2.8.10. Methyl
(11b). Colorless oil, yield 73%, TLC Rf 0.4 (hexane/EtOAc 1/1
v/v). 1H NMR(400 MHz, CDCl3)
0.97(t, J = 7.6, 3H), 1.14(t,
4-amino-3-(2-ethylbut-1-ynyl)benzoate
d
J = 7.4 Hz, 3H), 2.08(q, J = 7.4 Hz, 2H), 2.23(q, J = 7.6 Hz, 2H),
3.85(s, 3H),4.07(w, 2H), 5.96(s, 1H), 6.64(d, J = 8.4 Hz, 1H), 7.69(d,
J = 2.0 Hz, 1H), 7.75(dd, J = 8.4, 2.0 Hz, 1H); 13C NMR(100 MHz,
CDCl3) d 12.9, 13.0, 24.1, 28.6, 51.5, 113.6, 117.8, 119.2, 123.2,
129.7, 132.0, 148.6, 150.2, 167.4; MS (ES) m/z 234 (M+1).
6.2.9.3. Diethyl 1-(2-(2-ethylbut-1-enyl)-4-(methoxycarbonyl)
phenyl)-5-oxopyrrolidine-2,2-dicarboxylate (12b).
less solid: mp 85–87 °C, yield 20%, TLC Rf 0.53 (hexane/EtOAc 1/1
v/v). 1H NMR(300 MHz, CDCl3)
0.91(m, 3H), 1.04(m, 6H),
Color-
d
1.26(m, 3H), 2.14(m, 4H), 2.58(m, 2H), 3.89(s, 3H), 4.44–3.8(m,
4H), 5.90(s, 1H), 7.59(d, J = 8.3 Hz, 1H), 7.88(dd, J = 8.3, 2.0 Hz,
1H), 7.92(d, J = 2.0 Hz, 1H); 13C NMR(75 MHz, CDCl3) d 12.6, 12.8,
14.2, 24.2, 28.7, 28.8, 29.0, 52.2, 62.4, 74.1, 118.3, 128.2, 128.9,
129.6, 131.2, 138.7, 139.9, 149.6, 166.7, 174.3; MS (ES) m/z 446
(M+1).
6.2.8.11. Methyl 4-amino-3-(2-methylprop-1-ynyl) benzoate
(11c).
Colorless oil, yield 70%, TLC Rf 0.4 (hexane/EtOAc 1/1
v/v). 1H NMR(400 MHz, CDCl3) d 1.70(d, J = 0.4 Hz, 3H), 1.91(d,
J = 0.8 Hz, 3H), 3.84(s, 3H), 4.12(w, 2H), 6.00(s, 1H), 6.64(d,
J = 8.4 Hz, 1H), 7.69(d, J = 1.7 Hz, 1H), 7.75(dd, J = 8.4, 1.7 Hz, 1H);
13C NMR(100 MHz, CDCl3) d 19.8, 19.8, 26.3, 52.0, 114.1, 119.5,
120.2, 123.5, 132.5, 139.3, 149.1, 167.8; MS (ES) m/z 206 (M+1).
6.2.9.4. Diethyl 1-(4-(methoxycarbonyl)-2-(2-methylprop-1-
enyl) phenyl)-5-oxopyrrolidine-2,2-dicarboxylate (12c).
To
6.2.8.12. Note.
In the following conversion of 11a–b to 12a–
a solution of 11c (2.60 g, 12.6 mmol) and diethyl ketomalonate
(6.6 g, 38 mmol) in toluene (130 mL), p-toluene sulfonic acid
(242 mg, 1.30 mmol) and molecular sieves (13 g) were added.
The reaction mixture was refluxed overnight. The cooled reaction
mixture was washed with saturated NaHCO3 (3 ꢂ 80 mL) and brine
(2 ꢂ 80 mL). The organic layer was dried (Na2SO4), filtered, and
evaporated to dryness. The residue was chromatographed on a
flash silica gel column to afford an oil. To a solution of the oil in
1,2-dichloroethane (30 mL), NaBH4 (630 mg, 33.2 mmol) was
added followed by the slow addition of glacial acetic acid
(2.5 mL). After the mixture was stirred overnight at rt, the reaction
was quenched by adding cold water (2 mL) and diluted with ethyl
acetate (90 mL). The mixture was washed with saturated NaHCO3
(2 ꢂ 30 mL) and brine (2 ꢂ 30 mL), and the organic layer was dried
(Na2SO4), filtered, and evaporated to dryness. The residue was
chromatographed on a flash silica gel column to afford an oil. To
a solution of the oil in toluene (20 mL), phosphorus trichloride
(3.60 mL, 28.7 mmol) was added. The mixture was refluxed over-
night. After quenching with water (50 mL), the reaction mixture
was diluted with ethyl acetate (100 mL). The combined aqueous
layers were extracted with ethyl acetate (2 ꢂ 50 mL). The organic
layers were combined, washed with saturated NaHCO3
(2 ꢂ 60 mL) and brine (2 ꢂ 60 mL), dried (Na2SO4), filtered and
evaporated to dryness. The residue was chromatographed on a
flash silica gel column to afford an oil. A solution of the oil in dry
DMF (20 mL) was added dropwise to a stirred suspension of NaH
(128 mg, 3.2 mmol) in dry DMF (20 mL) over 10 min under Ar.
The mixture was stirred at room temperature for 2 h. The NaH
was quenched with water (0.5 mL), and the mixture was diluted
with ethyl acetate (100 mL). The mixture was washed with
b, and for 11c–12c, overall yields were highest when intermediates
were not isolated and purified.
6.2.9. Diethyl 1-(-2-alkenyl-4-methoxycarbonylphenyl)-5-
oxopyrrolidine-2,2-dicarboxylate (12a–b)
6.2.9.1. General procedure.
To a solution of methyl 3-alke-
nyl-4-aminobenzoate (11a–b) (3.83 mmol) and diethylketomalo-
nate (1.33 g, 7.66 mmol) in toluene (40 mL), p-toluene sulfonic
acid (73 mg, 0.38 mmol) and molecular sieves (4 g) were added.
The reaction mixture was refluxed 15 h, then transferred to a
separatory funnel and washed with saturated NaHCO3
(3 ꢂ 40 mL) and brine (2 ꢂ 40 mL). The organic layer was dried
(Na2SO4), filtered, and evaporated to dryness. The residue was
chromatographed on a flash silica gel column to afford an oil. To
a
solution of the oil in 1,2-dichloroethane (8 mL), NaBH4
(144 mg, 3.80 mmol) was added followed by slow addition of gla-
cial acetic acid (0.3 mL). After the mixture was stirred 15 h at rt, the
reaction was quenched by adding cold water (1 mL). The mixture
was washed with saturated NaHCO3 (2 ꢂ 10 mL) and brine
(2 ꢂ 10 mL), and the organic layer was dried (Na2SO4), filtered,
and evaporated to dryness. The residue was chromatographed on
a flash silica gel column to afford an oil. To a solution of the oil
in toluene (5 mL), phosphorus trichloride (0.40 mL, 4.6 mmol)
was added. The mixture was refluxed 15 h. The cooled reaction
mixture was washed with water (2 ꢂ 5 mL). The combined aque-
ous layers were extracted with ethyl acetate (2 ꢂ 10 mL). The or-
ganic layers ware combined, and washed with saturated NaHCO3
(2 ꢂ 10 mL) and brine (2 ꢂ 10 mL), dried (Na2SO4), filtered, and
evaporated to dryness. The residue was chromatographed on a

4588
Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4582–4589
NaHCO3 (2 ꢂ 50 mL) and brine (4 ꢂ 50 mL), dried (Na2SO4), fil-
2.66–2.50(m, 2H), 3.22(d, J = 11.2 Hz, 1H), 3.43(d, J = 11.2 Hz, 1H),
3.62(d, J = 11.8 Hz, 1H), 3.73(d, J = 11.8 Hz, 1H), 6.24(d, J = 8.0 Hz,
1H), 7.46(d, J = 8.0 Hz, 1H), 7.94(d, J = 8.0 Hz, 1H), 7.96(s, 1H); 13C
NMR(75 MHz, CD3OD) d 11.6, 11.8, 23.7, 25.3, 28.2, 29.3, 29.8,
63.0, 64.0, 71.3, 78.0, 120.0, 128.3, 129.4, 131.8, 138.8, 138.9,
148.5, 177.1; MS (ES) m/z 348 (M+1).
tered, and evaporated to dryness. The residue was chromato-
graphed on
a flash silica gel column to afford a colorless
semisolid. The semisolid was dissolved in THF (6 mL). Palladium
on carbon (30 mg, 5%) was added. The mixture was stirred under
a hydrogen atmosphere (approximately one atmosphere from an
attached balloon reservoir) for 4 h. The mixture was filtered and
concentrated to yield 12c (0.6 g, 11% for five steps, TLC Rf 0.50
(hexane/EtOAc 1/1 v/v) as a colorless solid; mp 73–75 °C. 1H
NMR(300 MHz, CDCl3) d 0.96–0.90(m, 9H), 1.29(t, J = 5.3 Hz, 3H),
1.98(m, 1H), 2.20(dd, J = 5.9, 11.9 Hz, 1H), 2.36(dd, J = 5.0,
11.9 Hz, 1H), 2.44(m, 1H), 2.65(m, 2H), 3.05(m, 1H), 3.84(m, 1H),
3.90(s, 3H), 4.01(m, 1H), 4.29–4.35(m, 2H), 7.55(d, J = 8.2 Hz, 1H),
7.87(dd, J = 8.2, 2.0 Hz, 1H), 7.94(d, J = 2.0 Hz, 1H); 13C
NMR(75 MHz, CDCl3) d 13.2, 13.9, 22.3, 22.9, 27.6, 28.8, 29.0,
39.8, 62.2, 62.6, 74.2, 127. 6, 128.8, 129.7, 130.6, 140.3, 141.0,
165.9, 166.6, 169, 7, 174.7; MS (ES) m/z 420 (M+1).
6.2.9.7. 4-(2,2-Bis (hydroxymethyl)-5-oxopyrrolidin-1-yl)-3-
isobutylbenzoic
acid (13c).
Compound 12c (419 mg,
1.00 mmol) was dissolved in dry MeOH (3 mL) and THF (3 mL)
and cooled to 0 °C under argon. NaBH4 (190 mg, 5.00 mmol) was
added. The mixture was warmed to room temperature, stirred
for 2 h, and quenched by adding cold water (0.5 mL). The mixture
was evaporated to dryness leaving a solid which was dissolved in
dichloromethane (20 mL). The organic layer was washed brine
(2 ꢂ 10 mL), dried (Na2SO4), filtered, and evaporated to dryness.
The residue was dissolved in MeOH (2 mL) and 1 M NaOH (2 mL)
was added. After the mixture was stirred for 2 h at room tempera-
ture, the pH was adjusted to 2 with 1 M HCl, and the mixture was
evaporated to dryness, leaving a solid which was dissolved in
methanol (3 mL). The organic layer was concentrated. The obtained
residue was chromatographed on silca gel (EtOAc/MeOH/
AcOH = 40:2:1, Rf = 0.57) to yield acid 13c (99 mg, 31% in two
steps) as a colorless solid. Mp 168–170 °C: 1H NMR(300 MHz,
CD3OD) d 1.10(d, J = 2.5 Hz, 3H), 1.12(d, J = 2.5 Hz, 3H), 2.31(m,
1H), 2.46–2.50(m, 2H), 2.60–2.88(m, 4H), 3.52(d, J = 11.2 Hz, 1H),
3.62(d, J = 11.2 Hz, 1H), 2.69–2.42(m, 3H), 2.85(m, 1H), 3.35(d,
J = 11.1 Hz, 1H), 3.46(d, J = 11.1 Hz, 1H), 3.80(d, J = 11.7 Hz, 1H),
3.83(d, J = 11.7 Hz, 1H), 7.62(d, J = 8.2 Hz, 1H), 8.06(dd, J = 8.2,
2.0 Hz, 1H), 8.20(d, J = 2.0 Hz, 1H); 13C NMR(100 MHz, CD3OD) d
21.2, 21.9, 25.3, 27.4, 30.0, 39.1, 63.2, 63.6, 71.4, 127.3, 129.6,
130.2, 130.5, 139.6, 141.8, 168.2, 177.9; Purity by HPLC 97%; MS
(ES) m/z 322 (M+1).
6.2.9.5. 4-(2,2-Bis (hydroxymethyl)-5-oxopyrrolidin-1-yl)-3-(2-
propylpentyl)benzoic
acid
(13a).
Compound
12a
(0.30 mmol) was dissolved in THF (3 mL). Palladium on carbon
(14 mg, 5%) was added. The mixture was stirred under a hydrogen
atmosphere (approximately one atmosphere from an attached bal-
loon reservoir) for 5 h. The mixture was filtered and evaporated to
dryness. The residue was dissolved in dry MeOH (1 mL) and THF
(1 mL) and cooled to 0 °C under argon. NaBH4 (54.0 mg,
1.46 mmol) was added. The mixture was warmed to room temper-
ature, stirred for 5 h, and quenched by adding cold water (0.1 mL).
The mixture was evaporated to dryness leaving a solid which was
dissolved in dichloromethane (10 mL). The organic layer was
washed with brine (2 ꢂ 5 mL), dried (Na2SO4), filtered, and evapo-
rated to dryness. The residue was dissolved in MeOH (2 mL) and
1 M NaOH (2 mL) was added. After the mixture was stirred for
2 h at room temperature, the pH was adjusted to 2 with 1 M HCl,
and the mixture was evaporated to dryness, leaving a solid which
was dissolved in methanol (3 mL). The organic layer was concen-
trated. The obtained residue was chromatographed on a flash silica
gel column to obtain the benzoic acids 13a. Colorless solid: mp
203–204 °C, yield 42% for three steps, TLC Rf 0.38 (AcOEt/MeOH/
AcOH 9:1:0.5, v/v/v). 1H NMR(400 MHz, CD3OD) d 0.78(m, 6H),
1.33–1.11(m, 8H), 1.76(m, 1H), 2.20(m, 2H), 2.44–2.32(m, 2H),
2.54(m, 1H), 2.73(m, 1H), 3.24(d, J = 11.2 Hz, 1H), 3.34(d,
J = 11.2 Hz, 1H), 3.50(d, J = 11.6 Hz, 1H),3.54(d, J = 11.6 Hz, 1H),
7.33(d, J = 8.1 Hz, 1H), 7.78(d, J = 8.1 Hz, 1H), 7.90(s, 1H); 13C
NMR(100 MHz, CD3OD) d 15.2, 21.3, 21.4, 27.2, 32.0, 37.0, 37.3,
37.7, 38.3, 65.0, 65.4, 73.3, 129.0, 131.5, 132.3, 132.6, 141.5,
143.8, 170.2, 179.8; Purity by HPLC 98%; MS (ES) m/z 378 (M+1).
6.2.9.8. 4-(2,2-Bis (hydroxymethyl)-5-oxopyrrolidin-1-yl)-3-(2-
ethylbutyl)benzoic acid (14b).
Compound 13b (20.0 mg,
0.058 mmol) was dissolved in THF (0.5 mL). Palladium on carbon
(2 mg, 5%) was added. The mixture was stirred under a hydrogen
atmosphere (approximately one atmosphere from an attached bal-
loon reservoir) for 5 h. The mixture was filtered and concentrated
to yield 14b (20 mg, 99%) as a colorless solid. Mp 168–170 °C; 1H
NMR(300 MHz, CD3OD) d 0.91(t, J = 7.3 Hz, 6H), 1.36(m, 4H),
1.70(m, 1H), 2.30(m, 2H), 2.69–2.42(m, 3H), 2.85(m, 1H), 3.35(d,
J = 11.1 Hz, 1H), 3.46(d, J = 11.1 Hz, 1H), 3.61(d, J = 11.7 Hz, 1H),
3.66(d, J = 11.7 Hz, 1H), 7.43(d, J = 8.2 Hz, 1H), 7.89(d, J = 8.2 Hz,
1H), 8.01(s, 1H); 13C NMR(75 MHz, CD3OD) d 10.0, 10.1, 25.0,
25.2, 25.4, 30.2, 34.4, 40.2, 63.3, 63.6, 71.5, 127.2, 129.7, 130.6,
130.8, 139.7, 142.0, 168.4, 178.0. MS (ES) m/z 350 (M+1).
6.2.9.6. 4-(2,2-Bis (hydroxymethyl)-5-oxopyrrolidin-1-yl)-3-(2-
ethylbut-1-enyl)benzoic
acid (13b).
Compound 12b
Acknowledgment
(168 mg, 0.380 mmol) was dissolved in dry MeOH (1.2 mL) and
THF (1.2 mL) and cooled to 0 °C under argon. NaBH4 (71.0 mg,
1.89 mmol) was added. The mixture was warmed to room temper-
ature, stirred for 5 h, and quenched by adding cold water (0.1 mL).
The mixture was evaporated to dryness leaving a solid which was
dissolved in dichloromethane (10 mL). The organic layer was
washed with brine (2 ꢂ 5 mL), dried (Na2SO4), filtered, and evapo-
rated to dryness. The residue was dissolved in MeOH (3 mL) and
1 M NaOH (3 mL) was added. After the mixture was stirred for
1 h at room temperature, the pH was adjusted to 2 with 1 M HCl,
and the mixture was evaporated to dryness, leaving a solid which
was dissolved in methanol (2 mL). The organic layer was concen-
trated. The obtained residue was chromatographed on silca gel
(EtOAc/MeOH/AcOH = 40:2:1, Rf = 0.48) to yield acid 13b (47 mg,
36% in two steps) as a colorless solid: mp 189–191 °C; 1H
NMR(300 MHz, CD3OD) d 1.15–1.09(m, 6H), 2.45–2.15(m, 6H),
This work was supported by NIAID Grant AI 062950 to W.J.B.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.001.
References and notes
1. World Health Organization WHO factsheet 211: influenza; 2009 http://
www.who.int/mediacentre/factsheets/fs211/en/.
2. Webby, R. J.; Webster, R. G. Science 2003, 302, 1519.
3. Kilbourne, E. D. Emerg. Infect. Dis. 2006, 12, 9.
4. Fraser, C.; Donnelly, C. A.; Cauchemez, S.; Hanage, W. P.; Van Kerkhove, M. D.;
Ollingsworth, T. D.; Griffin, J.; Baggaley, R. F.; Jenkins, H. E.; Lyons, E. J.; Jombart,
T.; Hinsley, W. R.; Grassly, N. C.; Balloux, F.; Ghani, A. C.; Ferguson, N. M.;

Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4582–4589
4589
Rambaut, A.; Pybus, O. G.; Lopez-Gatell, H.; Apluche-Aranda, C. M.; Chapela, I.
B.; Zavala, E. P.; Guevara, D. M. E.; Checchi, F.; Garcia, E.; Hugonnet, S.; Roth, C.
Science 2009, 324, 1557.
19. Ward, R. C. Structure 1997, 5, 1139.
20. Singh, S.; Jedrzejas, M. J.; Air, G. M.; Luo, M.; Laver, G. W.; Brouillette, W. J. J.
Med. Chem. 1995, 38, 3217.
5. Nicholson, K. G. Vaccines 2009, 8(4), 373.
6. Sambhara, S.; Stephenson, I. Vaccines 2009, 8(4), 375.
21. Jadrezas, M. J.; Singh, S.; Brouillette, W. J.; Laver, W. G.; Air, G. M.; Luo, M.
Biochemistry 1995, 34, 3144.
7. Trollfors, B. Acta Paediatr. 2006, 95(7), 774.
8. Carrat, F.; Flahault, A. Vaccine 2007, 25, 6852.
9. Palese, P. Nat. Med. 2004, 10, S82.
22. Atigadda, V. R.; Brouillette, W. J.; Duarte, F.; Ali, S. M.; Babu, Y. S.; Bantia, S.;
Chand, P.; Chu, N.; Montgomery, J. A.; Walsh, D. A.; Sudbeck, E. A.; Finley, J.;
Luo, M.; Air, G. M.; Laver, G. W. J. Med. Chem. 1999, 42, 2332.
23. Brouillette, W. J.; Atigadda, V. R.; Luo, M.; Air, G. M.; Babu, S.; Bantia, S. Bioorg.
Med. Chem. Lett. 1999, 9, 1901.
24. Brouillette, W. J.; Bajpai, S. N.; Ali, S. M.; Velu, S. E.; Atigadda, V. R.; Lommer, B.
S.; Finley, J. B.; Luo, M.; Air, G. M. Bioorg. Med. Chem. 2003, 11, 2739.
25. Smith, P. W.; Sollis, S. L.; Howes, P. D.; Cherry, P. C.; Cobley, K. N.; Taylor, H.;
Whittington, A. R.; Scicinski, J.; Bethell, R. C.; Taylor, N.; Skarzynski, A. C.; Singh,
O.; Wonacott, A.; Varghese, J.; Coleman, P. Bioorg. Med. Chem. Lett. 1996, 6,
2931.
10. Colman, P. M. In Influenza Viruses: Influenza Virus Neuraminidase, Enzyme and
antigen; Krug, R. M., Ed.; Plenum Press: New York, 1989; pp 175–218.
11. Air, G. M.; Brouillette, W. J. Influenza virus antiviral targets. In Antiviral
Research; LaFemina, R., Ed.; ASM Press: Washington, DC, 2009; pp 187–207.
12. von Itzstein, M.; Wen-Yang, W.; Gaik, B. K.; Michael, S. P.; Jeffrey, C. D.; Betty, J.;
Tho, V. P.; Mark, L. S.; Hume, F. W.; Stuart, W. O.; Peter, M. C.; Joseph, N. V.;
Michael, R.; Jacqueline, M. W.; Richard, C. B.; Vanessa, J. H.; Janet, M.; Cameron,
J. M.; Charles, R. P. Nature 1993, 363, 418–423.
13. Hayden, F. G.; Treanor, J. J.; Betts, R. F.; Lobo, M.; Esinhart, J. D.; Hussey, E. K. J.
Am. Med. Assoc. 1996, 275, 295.
26. Sollis, S. L.; Smith, P. W.; Howes, P. D.; Cherry, P. C.; Bethell, R. C. Bioorg. Med.
Chem. Lett. 1996, 6, 1805.
14. Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.; Swaminathan, S.;
Bischofberger, N.; Chen, M. S.; Mendel, D. B.; Tai, C. Y.; Laver, W. G.; Stevens, R.
C. J. Am. Chem. Soc. 1997, 119, 681.
15. Weixing, L.; Escarpe, P. A.; Eisenberg, E. J.; Cundy, K. C.; Sweet, C.; Jakeman, K.;
Merson, J.; Lew, W.; Williams, M.; Zhang, L.; Kim, C.; Bischofberger, N.; Chen,
M. S.; Mendel, D. B. Antimicrob. Agents Chemother. 1998, 42, 647.
16. Babu, Y. S.; Chand, P.; Bantia, S.; Kotian, P.; Dehghani, A.; El-Kattan, Y.; Lin, T.;
Hutchison, T. L.; Elliott, A. J.; Parker, C. D.; Ananth, S. L.; Horn, L. L.; Laver, G. W.;
Montgomery, J. A. J. Med. Chem. 2000, 43, 3482.
17. Wang,G. T.;Chen,Y.;Wang,S.;Gentles,R.;Sowin, T.;Kati,W.;Muchmore, S.;Giranda,
V.; Stewart, K.; Sham, H.; Kempf, D.; Laver, W. G. J. Med. Chem. 2001, 44, 1192.
18. Stoll, V.; Stewart, K. D.; Maring, C. J.; Muchmore, S.; Giranda, V.; Gu, Y.-G. Y.;
Wang, G.; Chen, Y.; Sun, M.; Zhao, C.; Kennedy, A. L.; Madigan, D. L.; Xu, Y.;
Saldivar, A.; Kati, W.; Laver, G.; Sowin, T.; Sham, H. L.; Greer, J.; Kempf, D.
Biochemistry 2003, 42, 718.
27. Yang, L.; Qi, C.-M.; Zhang, G.-X.; Zou, N.-Z. J. Het. Chem. 2003, 40, 1107.
28. Bantia, S.; Parker, D.; Ananth, L.; Horn, L.; Andries, K.; Chand, P.; Kotian, P.;
Dehghani, A.; El-kattan, Y.; Lin, T.; Hutchison, T.; Montgomery, J.; Kellog, D.;
Babu, Y. Antimicrob. Agents Chemother. 2001, 45, 1162.
29. Russell, R. J.; Haire, L. F.; Stevens, D. J.; Collins, P. J.; Lin, Y. P.; Blackburn, G. M.;
Hay, A. J.; Gamblin, S. J.; Skehel, J. J. Nature 2006, 443, 45.
30. Smith, P. W.; Sollis, S. L.; Howes, P. D.; Cherry, P. C.; Cobley, K. N.; Taylor, H.;
Whittington, A. R.; Scicinski, J.; Bethell, R. C.; Taylor, N.; Skarzynski, A. C.; Singh,
O.; Wonacott, A.; Varghese, J.; Coleman, P. Bioorg. Med. Chem. Lett. 1996, 6,
2931.
31. Sollis, S. L.; Smith, P. W.; Howes, P. D.; Cherry, P. C.; Bethell, R. C. Bioorg. Med.
Chem. Lett. 1805, 1996, 6.
32. Potier, M.; Mameli, L.; Belisle, M.; Dallaire, L.; Melancon, S. B. Anal. Biochem.
1979, 94, 287.