Design, synthesis, inhibitory activity, and SAR studies of pyrrolidine derivatives as neuraminidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.01.020

A series of pyrrolidine derivatives were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influenza A virus (H3N2). All compounds were synthesized in good yields starting from commercially 4-hydroxy-l-proline using a suitable synthetic strategy. These compounds showed potent inhibitory activity against influenza A neuraminidase. Within this series, five compounds, 6e, 9c, 9e, 9f, and 10e, have good potency (IC₅₀ = 1.56-2.71 μM) which are compared to that the NA inhibitor Oseltamivir (IC₅₀ = 1.06 μM), and could be used as lead compoundS in the future.

Full text of DOI:10.1016/j.bmc.2007.01.020

Bioorganic & Medicinal Chemistry 15 (2007) 2749–2758
Design, synthesis, inhibitory activity, and SAR studies of
pyrrolidine derivatives as neuraminidase inhibitors
Jie Zhang,a Qiang Wang,a Hao Fang,a Wenfang Xu,a,* Ailin Liub and Guanhua Dub
aDepartment of Medicinal Chemistry, School of Pharmacy, Shandong University, 44,
Wenhuaxi Road, Ji’nan, Shandong 250012, PR China
bInstitute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
Received 5 December 2006; revised 13 January 2007; accepted 17 January 2007
Available online 19 January 2007
Abstract—A series of pyrrolidine derivatives were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influ-
enza A virus (H3N2). All compounds were synthesized in good yields starting from commercially 4-hydroxy-L-proline using a suit-
able synthetic strategy. These compounds showed potent inhibitory activity against influenza A neuraminidase. Within this series,
five compounds, 6e, 9c, 9e, 9f, and 10e, have good potency (IC50 = 1.56–2.71 lM) which are compared to that the NA inhibitor
Oseltamivir (IC50 = 1.06 lM), and could be used as lead compoundS in the future.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
mucus of respiratory tract and reducing the propensity
of the virus particles to aggregate. Despite the homolog-
ical identity of NA in different strains being only about
30%, the catalytic site of neuraminidase in all influenza
A and B viruses is completely conserved.5 Therefore,
NA has been regarded as an attractive target for antivi-
ral drug development. Now, two NA inhibitors, Zanam-
ivir and Tamiflu, have been confirmed as effective and
safe for the treatment of influenza and approved by
FDA.6
Influenza is an acute viral infection of the upper respira-
tory tract that can affect millions of people every year.1
Vaccines against influenza virus are ineffective due to the
rapid emergence of mutant viral antigens. The M2 pro-
tein ion channel blockers, for example amantadine and
rimantadine, are only effective on type A influenza with
undesirable side effects and rapidly generated resistant
mutants.2 Because effective and safe anti-influenza ther-
apeutics are lacking, developing effective anti-influenza
agents has become a high-priority and attractive area
in drug discovery.
In 1999, Luo et al., proposed that the enzymatic active
site contains four anti-parallel strands arranged in a
propeller fashion.7 Each monomeric subunit has an
active site cavity lined with 10 conserved residues
and four water molecules. The inhibitors bind to the
active site with the carboxylic acid binding to the triad
guanidine groups of the three arginine residues,
Arg118, Arg371, and Arg292, which are located as a
cluster on one side of the active site. Opposite to the
guanidine triad, there is a hydrophobic pocket formed
by side chains of Trp178, Ile 222, and part of Arg224.
In the design of inhibitor Oseltamivir,22 an amino
group was used to replace the 4-OH of SA and inter-
act with a negatively charged pocket formed by
Glu119 and Glu227. Wang et al.8 derived an ‘airplane’
model of the NA active site as illustrated in Figure 1
to summarize the basic structural requirements of a
potent NA inhibitor.
Hemagglutinin (HA) and neuraminidase (NA) are two
glycoproteins in viral surface and essential for viral rep-
lication, infectivity, and the infective cycle of influenza.
HA is known to mediate binding of viruses to target
cells via terminal sialic acid (SA) residue in glycoconju-
gates.3 In contrast to HA activity, NA catalyzes removal
of terminal SA linked to glycoproteins and glycolipids.
It has been suggested that NA is not only crucial in
the release of virion progeny away from infected cells,4
but also important in the movement of the virus through
Keywords: Neuraminidase inhibitor; SAR study; Pyrrolidine deriva-
tives; Peptidomimetics.
*
Corresponding author. Tel./fax: +86 531 88382264; e-mail:
xuwenf@sdu.edu.cn
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.020

2750
J. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 2749–2758
peptidomimetics have been designed by acylating pyr-
rolidine with diclofenac which is a known NSAIDs so
as to enhance the stability of the target compounds.12
Keep or converted to NH2
HO
Keep or converted to
COOCH3, CONHOH
COOH
CH3
N
O
Figure 1. ‘Airplane’ model of NA active site (Ref. 8).
amino acid or diclofenac
According to the studies on NA active site and SAR of
published NA inhibitors, inhibition of the NA is mainly
determined by the relative positions of the substituents
(carboxylate, glycerol, acetamido, and hydroxyl) of the
central ring. Currently, several pyrrolidine compounds
have been found to possess potent NA inhibitory activ-
ities.7,8 For example, A-3156758 (1) is highly active in
cell culture against a variety of strains of influenza A
and B viruses. This urges us to develop new NA inhibi-
tors based on pyrrolidine derivatives which contain dif-
ferent substituents (carboxylate, guanidino, acetamino,
and alkyl) to interact with the four binding pockets of
the NA active site.9
2. Chemistry
The synthesis of pyrrolidine derivatives possessing NA
inhibitory activities is shown in Scheme 1. The starting
materials, Boc-protected amino acids (3) and the 4-hy-
droxy-L-proline methyl ester hydrochloride (4), were
prepared according to the literature.10 The Boc-amino
acids (3) were activated with DCC and HOBt and then
coupled with compound 4 to yield 5a–10a. The methy-
lester 5a–10a was then hydrolyzed with NaOH/H2O or
treated with NH2OK to obtain relative carboxylic acids
5b–10b or hydroximic acids 5c–10c.13
O
HO
H
OH
CH3
4-Amino-pyrrolidine derivatives were prepared from
intermediate 5a–10a. First, the hydroxyl group of 5a–
10a was convert to mesyl group by methanesulfonate
and then reacted with sodium azide to generate configu-
ration inverted azide. Amino derivatives 5d–10d can be
synthesized by hydrogenation using Pd–CaCO3. The
methyl ester 5d–10d can be transformed to correspond-
ing carboxylic acids 5e–10e and hydroximic acids 5f–10f
as the methods mentioned before.
AcHN
N
H
COOH
N
OMe
O
Me
2. NAHYP
1
In our previous work, the 4-hydroxy-L-proline had been
used to prepare a series of pyrrolidine derivatives as ma-
trix metalloproteinase (MMP) and aminopeptidase-N
(APN) inhibitors.10,11 We first screened all the pyrroli-
dine derivatives in our compound library including not
only the target compounds and intermediates we synthe-
sized before, but also some commercially available com-
pounds. The pharmacological result showed that
NAHYP, one anti-inflammation compound (2, Oxacep-
rol), exhibited modest activity against influenza virus A
(H3N2) neuraminidase (IC50 = 48.73 lM) and could be
used as lead compound in future.
The Boc-protecting group can be easily removed with
3 N HCl in ethyl acetate to give hydrochloride salts of
pyrrolidine derivatives 5g–10g. Finally, the amino group
was acetylated with acetic anhydride to yield 5h–10h.
The synthesis of peptidomimetics that possess NA
inhibitory activity is shown in Scheme 2. This scheme
is similar to Scheme 1, the coupling of diclofenac with
L-hydroxyproline using the strategy of synthesis of
dipeptide.
In order to improve the affinity of lead compounds, we
optimized the structure of NAHYP with the following
chemical modification: (i) N-acyl group in pyrrolidine
ring was changed to other Boc-protected or free amino
acid residues; (ii) 2-carboxylic acid can be kept or con-
verted to other derivatives such as methyl ester or
hydroxymate; (iii) keep hydroxy in 4-position or
converted to free amino group. Despite the designed
dipeptides serving as target compounds, a series of
3. Results and discussion
All the target compounds (47 compounds) were tested
for their ability to inhibit neuraminidase. Preliminary re-
sult showed that 24 compounds displayed inhibitory
activities with IC50 value from 1.56 to 90.79 lM (Table
1). Compound 10e with NH2 group and methionine as

J. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 2749–2758
2751
OH
O
h
N
NHOH
OH
R
d
O
NHBoc
R1
H
5c~10c
N
COOCH3
HCl
OH
O
h
h
N
HO
O
R
3
O
R2
c
N
R
a,b
N
COOH
NH2
COOCH3
R
5g~10g
NHBoc
NHBoc
5a~10a
R
5b~10b
O
i
N
Boc
e,f,g
H
OH
NH2
4
R1
NH2
c
h
O
R
O
N
O
N
COOH
N
R2
R
NHBoc
5e~10e
COOCH3
R
NHAc
5h~10h
NHBoc
5d~10d
NH2
d
O
N
CONHOH
NHBoc
5f~10f
R
Scheme 1. Reagents and conditions: (a) DCC, HOBt, THF, 0 °C; (b) compound 3, NMM, THF; (c) MeOH, NaOH/H2O, 25 °C; (d) NH2OK,
MeOH, 65 °C; (e) MsCl, Et3N, DCM; (f) NaN3, DMF, 65 °C; (g) 5% Pd–CaCO3, H2, MeOH; (h) HCl/EtOAC, EtOAc, 25 °C; (i) Ac2O, Et3N, DCM.
H2
CONHOH
C
H2
C
N
NH
O
Cl
Cl
OH
NH
H2
C
d
c
O
Cl
COOCH3
Cl
OH
N
11c
a,b
NH
O
Cl
Cl
H2
C
H
N
COOH
COOH
OH
COOCH3
HCl
N
NH
NH
11a
O
Cl
Cl
Cl
HO
3
OH
e,f,g
11b
H2
C
N
O
Cl
H2
C
c
COOCH3
NH2
N
NH
11e
O
Cl
Cl
H2
C
NH2
d
CONHOH
11d
N
NH
O
Cl
Cl
NH2
11f
Scheme 2. Reagents and conditions: (a) DCC, HOBt, THF, 0 °C; (b) compound 3 NMM, THF; (c) MeOH, NaOH/H2O, 25 °C; (d) NH2OK, MeOH,
65 °C; (e) MsCl, Et3N, DCM; (f) NaN3, DMF, 65 °C; (g) 5% Pd–CaCO3, H2, MeOH.
hydrophobic side-chain showed the best inhibitory
activity (IC50 = 1.56 lM). Three compounds containing
isoleucine (10c, 10e, and 10f) exhibited good activities
(2.10–2.71 lM). Compounds, 11c and 11f, had lower
IC50 value (8.54 and 3.57 lM), and they possibly had
anti-inflammatory activity. Generally, the compound
with NH2 and –COOH group showed better activities
than that with OH and –CONHOH group.
In summary, our studies have discovered a new series of
pyrrolidine derivatives that have potent NA inhibitory
activity. The binding of compound 10e in the active site
of NA is shown in Figure 2, and we found that the
–CO2H or –CONHOH group of the target compounds
interacts with the positively charged site 1 of the NA ac-
tive site (Fig. 1), the exocyclic OH or NH2 group binds
to the negatively charged site 2, and the Boc group

2752
J. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 2749–2758
Table 1. The structure and in vitro inhibitory activities of compounds against NA
O
R1
C R3
N
R2
Compound
R1
R2
R3
IC50(lM)
pICa5ct
0
pICp5re
0
Res.
2
5b
CH3CO
Boc-Leu
Boc-Leu
Boc-Leu
Boc-Phe
Boc-Phe
Boc-Phe
Boc-Phe
Boc-Val
Boc-Val
Boc-Val
Boc-Ala
Boc-Ala
Boc-Ala
Boc-Ala
Boc-Ile
Boc-Ile
Boc-Ile
Boc-Met
DCAa
OH
OH
OH
OH
48.73
11.51
3.87
3.55
4.47
4.96
4.76
5.12
4.96
5.19
4.97
4.85
4.75
4.88
3.54
3.57
4.27
4.39
5.22
5.10
5.23
5.36
4.31
4.70
4.40
5.07
3.72
5.49
3.64
4.92
4.91
4.92
5.08
5.08
5.15
5.03
4.94
4.94
5.03
3.59
3.62
4.26
4.22
5.12
5.22
5.15
5.35
4.26
4.69
4.41
5.11
4.22
À0.091
À0.445
0.049
5c
OH
NHOH
NHOH
OH
5f
6b
NH2
OH
6.13
2.85
À0.162
0.033
6c
6e
OH
NHOH
OH
4.27
2.40
À0.12
0.037
NH2
NH2
OH
6f
7b
NHOH
OH
4.17
4.63
À0.060
À0.093
À0.188
À0.148
À0.048
À0.052
0.014
7c
7e
OH
NHOH
OH
6.09
4.29
NH2
OH
8a
8d
OMe
OMe
OH
90.79
83.69
15.82
12.62
2.15
NH2
NH2
NH2
OH
8e
8f
9c
NHOH
NHOH
OH
0.177
0.096
9e
NH2
NH2
NH2
OH
2.71
2.10
À0.122
0.080
0.010
9f
10e
NHOH
OH
OH
1.56
19.81
8.54
11b
11c
0.051
0.001
DCA
DCA
OH
NHOH
OH
11e
11f
NH2
NH2
NH2
16.23
3.57
À0.015
À0.040
À0.497
DCA
Leu
NHOH
OMe
5g
Oseltamivir
48.01
1.06
a DCA is 2-(2-(2,6-dichlorophenylamino)phenyl)acetic acid.
proline we used appeared to be an ideal starting material
because of its low cost and commercial abundance.
4. SAR studies
4.1. Dataset and molecular modeling
We used Sybyl7.0 program to carry out the SAR studies
of these pyrrolidine derivatives. The CoMFA studies
were carried out with the QSAR model of Sybyl. The
test set consisted of 5b and 7e, the other 22 compounds
composed of the training set. The IC50 values were con-
verted into pIC50 according to the formula:
pIC50 = log10IC50.
Based on the docking results, the template molecule 2
was taken and the rest of the molecules were aligned
to it using the pyrrolidine as scaffold by DATABASE
ALIGNMENT method in the Sybyl. The aligned mole-
cules are shown in Figure 3.
Figure 2. FlexX-docked result. Compound10e in the active site of
neuraminidase (PDB ID:1nnc). The yellow lines and numbers show the
potential hydrogen bonds and bond length.
interacts with the hydrophobic site 3 via one of the
methyl groups. The hydrophobic side chain occupied
the hydrophobic pocket of site 4. Compared to the other
research, we reported a more convenient and economi-
cal method for the synthesis of pyrrolidine NA inhibi-
tors. Compared to the other research, 4-hydroxy-L-
The steric and electrostatic CoMFA fields were calculat-
ed at each lattice intersection of a regularly spaced grid
˚
of 2.0 A in all three dimensions within defined region.
An sp3 carbon atom with +1.00 charge was used as a
probe atom. The steric and electrostatic fields were trun-
cated at +30.00 kcal molÀ1, and the electrostatic fields

J. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 2749–2758
2753
steric and electrostatic fields displayed good correlation
with the LOO cross-validated q2 of 0.852 at five opti-
mum components and SEE of 0.096. For CoMFA, a
five-component model was obtained with q2, r2, F, and
SEE of 0.852, 0.978, 141.328, and 0.096, respectively.
We also found in the CoMFA model that the contribu-
tions of steric and electrostatic fields are 0.541 and
0.459, respectively.
From Figure 4(b) we can find that the CoMFA model
can predict 7e well, but not very well 5b in the test set
and 11b in the training set. That may have been caused
by the sample size being much more lower when the
actual pIC50 < 4.7 than it >4.7.
5. Conclusions
Figure 3. Superposition of 22 inhibitors for CoMFA construction.
We have described the synthesis and properties of a
series of pyrrolidine derivatives as influenza NA inhib-
itors. Several compounds were shown to possess
potent influenza NA inhibitory activity, although in
all cases, measured activity was lower than that of
Oseltamivir. The most potent compound of the series
is compound 10e (IC50 = 1.56 lM), which, in addition
to good enzyme inhibitory activity, displays potent
anti-viral activity in vitro. We reported a more conve-
nient and economical method for the synthesis of pyr-
rolidine NA inhibitors. Compared to the other
research, 4-hydroxy-L-proline we used appeared to be
an ideal starting material because of its low cost and
commercial abundance. Accurate structural knowledge
of the inhibitors bound to the active site made it clear
that at least three regions of the active site of NA
needed to be occupied to establish a consistent bind-
ing orientation, S1, S2, and S3, (Fig. 1) for our
NAHYP-based pyrrolidine derivatives. Establishing a
consistent binding mode was critical to predictive
structure-based drug design and discovering potent
compounds in the nanomolar range that would poten-
tially be useful for antiviral therapy. The compounds
we have got all showed potent NA inhibitory activity,
and this finding could be used to design further influ-
enza NA inhibitors.
were ignored at the lattice points with maximal steric
interactions.
PLS method was used to linearly correlate the CoMFA
fields to the inhibitory activity values. The cross-valida-
tion analysis was performed using the leave one out
(LOO) method in which one compound is removed from
the dataset and its activity is predicted using the model
derived from the rest of the dataset. The cross-validated
q2 (0.852) that resulted in optimum number of compo-
nents (n = 5) and lowest standard error of prediction
were considered for further analysis. We have evaluated
different filter value r and at least selected r as
2.00 kcal molÀ1 to speed up the analysis and reduce
noise.
4.2. Results and discussion
Only from the docked results we can obtain the conclu-
sions: the order of increasing activity is R2: –NHOH>
–OH>–OMe, R3: –NH2>–OH, which we can also find
from the actual results. We have found two potential
hydrogen bonds (respectively to Glu277 and Glu227)
when the R3 is –NH2 (Fig. 2), which was not existing
when the R3 was –OH. The QSAR model obtained from
Figure 4. (a) The most active molecule F5 is shown in the background. Red (R) color represents the negative charge region, blue (B) is the positive
charge region, green (G) is the more bulky region, and yellow (Y) is the less bulky region. (b) The predictability of the CoMFA model.

2754
J. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 2749–2758
6. Experimental
separated by filtration and the filtrate would be used
in the next step.
6.1. Neuraminidase inhibition assay
Compound (3) was suspended in anhydrous THF, and
2.02 g N-methylmorpholine (NMM) (20 mmol) was
added. After half-an-hour, the filtrate was added. The
reaction mixture was stirred at room temperature over-
night. The dicyclohexylurea (DCU) was filtered off and
THF was evaporated in vacuo. The residues obtained
after evaporation were dissolved in 150 mL EtOAc
and the DCU was filtered off. The filtrate was washed
with 10% citric acid, brine, and saturated NaHCO3 solu-
tion, and then was dried with NaSO4. The solvent was
evaporated to give a residue that was chromatographed
on a silica column to give the title dipeptide 5a (yield:
The in vitro assay is based on the method reported by
Guahua Du.14,15 The strain A (Yuefang 72-243 A and
Jifang 90-15 A) influenza viruses, which were donated
by Chinese Centers for Disease Control, were used as
source of NA. The NA was obtained by the method de-
scribed by Laver.16 The compound 2’-(4-methylumbel-
liferyl)-a-D-acetylneuraminic acid (MUNANA) is the
substrate of NA. And cleavage of this substrate by
NA produces a fluorescent product, which can emit an
emission wavelength of 460 nm with an excitation wave-
length of 355 nm. The intensity of fluorescence can re-
flect the activity of NA sensitively.
1
83.2%) as an oil. ESI-MS m/z 359.6 (M+H); H NMR
(DMSO-d6, ppm): d 1.40 (s, 9H); 0.94 (d, J = 6.6 Hz,
3H); 0.97 (d, J = 6.5 Hz, 3H); 3.71 (s, 3H); 4.64
(t,J = 8.3 Hz, 1H); 1.50 (m, 2H); 1.74 (m, 1H); 2.33
(m, 1H); 1.99 (m, 1H); 3.06 (br, 1H); 3.69 dd, J = 3.7,
10.9 Hz, 1H); 3.97 (d, J = 10.9 Hz, 1H); 5.22 (d, J =
8.5 Hz, 1H); 4.40 (m, 1H); 4.55 (s, 1H); 6.80 (d,
J = 8.3 Hz, 1H).
In the enzyme reaction system, there were 30 lL of the
enzyme in 33 mmol/L MES buffer (pH 3.5), 10 lL of
4 mmol/L CaCl2, 20 lL of 20 lmol/L MUNANA,
and 30 lL water in a 96-well microplate. The terminal
volume was 100 lL. After 10 min at 37 °C, 150 lL of
14 mmol/L NaOH in 83% ethanol was added to
0.1 mL of the reaction mixture to terminate the reac-
tion. The intensity of fluorescence was quantitated in
Fluostar Galaxy (excitation, 360 nm; emission,
450 nm), and substrate blanks were subtracted from
the sample readings. The IC50 was calculated by plot-
ting percent inhibition versus the inhibitor concentra-
tion and determination of each point was performed
in duplicate.
6.2.3.1. Methyl-1-(2-(tert-butoxycarbonyl)-3-phenyl-
propanoyl)-4-hydroxypyrrolidine-2-carboxylate (6a). Yield:
1
81.7%, ESI-MS 393.3 (M+H), H NMR (DMSO-d6): d
1.30 (s, 9H); 3.62 (s, 3H); 1.89 (m, 1H); 2.06 (m, 1H);
2.69 (m, 1H); 2.78 (m, 1H); 4.03 (d, J = 7.1 Hz, 1H);
4.06 (d, J = 5.7 Hz, 1H); 4.35(m, 1H); 4.36 (m, 1H); 5.21
(t, J = 3.7 Hz, 1H); 6.92 (m, 1H); 7.18 (m, 2H); 7.28 (m,
2H); 6.92 (d, J = 7.6 Hz, 1H).
6.2. Chemistry: General procedures
6.2.3.2. Methyl-1-(2-(tert-butoxycarbonyl)-4-methyl-
pentanoyl)-4-hydroxypyrrolidine-2-carboxylate (7a). Yield:
80.5%, ESI-MS: 359.6 (M+H). H NMR (DMSO-d6): d
1.40 (s, 9H); 0.95 (d, J = 6.7 Hz, 3H); 0.97 (d,
J = 6.5 Hz, 3H); 3.72 (s, 3H); 4.65 (t, J = 8.3 Hz, 1H);
1.50 (m, 2H); 1.74 (m, 1H); 2.34 (m, 1H); 1.99 (m, 1H);
3.06 (br, 1H); 3.69 (dd, J = 3.7 Hz, 10.9 Hz, 1H); 3.97 (d,
J = 10.9 Hz, 1H); 5.23 (d, J = 8.5 Hz, 1H); 4.40 (m, 1H);
4.55 (s, 1H); 6.81 (d, J = 8.3 Hz, 1H).
All reactions except those in aqueous media were carried
out by standard techniques for the exclusion of mois-
ture. All reactions were monitored by thin-layer chro-
matography on 0.25 mm silica gel plates (60GF-254)
and visualized with UV light, or iodine vapor. 1H
NMR spectra were determined on a Brucker Avance
600 spectrometer using TMS as an internal standard.
ESI-MS were determined on an API 4000 spectrometer.
Melting points were determined on an electrothermal
melting point apparatus and are uncorrected. Anhy-
drous reactions were carried out in oven-dried glassware
under a nitrogen atmosphere.
1
6.2.3.3. Methyl-1-(2-(tert-butoxycarbonyl)-3-methylb-
utanoyl)-4-hydroxypyrrolidine-2-carboxylate (8a). Yield:
1
79.9%, ESI-MS 345.5 (M+H), H NMR (DMSO-d6): d
1.38 (s, 9H); 0.84 (d, J = 6.5 Hz, 3H); 0.89 (d,
J = 6.6 Hz, 3H); 1.91 (m, 2H); 2.09 (m, 1H); 3.60 (s,
3H); 3.65 (m, 2H); 4.34 (t, J = 7.8 Hz, 1H); 4.03 (m,
1H); 6.68 (d, J = 8.7 Hz, 1H).
6.2.1. (2S,4R)-Methyl-4-hydroxypyrrolidine-2-carboxyl-
ate hydrochloride (3). The title compound was prepared
as described by Jordis in (1S,4S)-2-thia-5-azabicyclo-
[2.2.1]heptane.17
6.2.3.4. Methyl-1-(2-(tert-butoxycarbonyl)propanoyl)-
4-hydroxypyrrolidine-2-carboxylate (9a). Yield: 75.6%,
ESI-MS 317.5 (M+H), 1H NMR (DMSO-d6): d 1.37
(s, 9H); 1.14 (d,J = 7.0 Hz, 3H); 1.91 (m, 1H); 2.10 (m,
1H); 3.59 (s, 3H); 4.03 (q,J = 7.1 Hz, 1H); 4.23
(t,J = 7.2 Hz, 1H); 4.34 (m, 2H); 5.19 (m, 1H); 6.89
(m, J = 7.7 Hz, 1H).
6.2.2. (2S,3S)-2-(tert-Butoxycarbonyl)-3-methylpentanoic
acid (N-Boc-L-isoleucine) (4). The title compound was
prepared as described by Haaina.18
6.2.3. (2S,4R)-Methyl-1-((3S)-2-(tert-butoxycarbonyl)-3-
methylpentanoyl)-4-hydroxypyrrolidine-2-carboxylate(5a).19
A solution of DCC (4.48 g, 21.6 mmol) in 30 mL anhyd
THF was added dropwise to a solution of 4.62 g N-Boc-
L-isoleucine (20 mmol) and 2.92 g HOBt (21.6 mmol) in
60 mL of anhyd THF at 0 °C for a period of about 20 h
to get the HOBt active ester. Then the urea which was
6.2.3.5. Methyl-1-(2-(tert-butoxycarbonyl)-4-(methyl-
thio)butanoyl)-4-hydroxypyrrolidine-2-carboxylate (10a).
Yield: 82.5%, ESI-MS 377.5 (M+H), 1H NMR (DMSO-
d6): d 1.38 (s, 9H); 1.91 (s, 3H); 3.58 (s, 3H); 1.78 (m,

J. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 2749–2758
2755
1H); 2.11 (m, 1H); 2.46 (m, 1H); 2.51 (m, 1H); 2.04 (m,
2H); 3.71 (m, 1H); 4.35 (m, 1H); 5.19 (m, 1H); 4.03 (q,
J = 7.1 Hz, 1H); 4.27 (t, J = 8.0 Hz, 1H); 6.96 (d,
J = 8.6 Hz, 1H).
(m, 1H); 4.37 (m, 1H); 7.08 (m, 1H); 7.19 (m, 2H);
7.29 (m, 2H); 6.99 (d, J = 8.3 Hz, 1H).
6.2.6.2. Methyl-4-amino-1-(2-(tert-butoxycarbonyl)-4-
methylpentanoyl)pyrrolidine-2-carboxylate (7d). Yield:
61.2%, ESI-MS 358.5 (M+H), H NMR (DMSO-d6):
1
6.2.3.6.
Methyl-1-(2-(2-(2,6-dichlorophenylamino)-
phenyl)acetyl)-4-hydroxypyrrolidine-2-carboxylate (11a).
Yield: 83.7%, mp = 99.8–101.2 °C, ESI-MS 423.5
(M+H), 1H NMR (DMSO-d6): d 3.609 (s, 3H); 1.99
(m, 1H); 2.09 (m, 1H); 3.34 (s, 2H); 3.78 (m, 2H);
4.38 (t, J = 7.8 Hz, 1H); 5.28 (m,1H); 6.29 (m,1H);
6.87 (t,J = 7.5, 7.3 Hz, H); 7.06 (dt, J = 6.9, 7.2,
2.3 Hz, 1H); 7.16 (q, J = 8.1 Hz, 1H); 7.25
(dd,J = 7.2, 0.69 Hz, 1H); 7.51 (d, J = 8.1 Hz, 1H);
7.54 (s, 1H).
d1.36 (s, 9H); 0.88 (d, J = 7.9 Hz, 3H); 0.90 (d,
J = 4.2 Hz, 3H); 3.59 (s, 3H); 1.41 (m, 2H); 1.52 (m,
1H); 1.55 (m, 1H); 2.34 (m, CH2, 1H); 4.26 (t, CH,
J = 8.1 Hz, 1H); 3.88 (dd, CH, J = 3.7 Hz, 10.6 Hz,
1H); 4.20 (m, CH, 1H); 3.43 (d, CH2, J = 7.0 Hz, 1H);
6.90 (d, CH2, J = 8.1 Hz, 1H); 6.90 (d, NH,
J = 8.1 Hz, 1H).
6.2.6.3. Methyl-4-amino-1-(2-(tert-butoxycarbonyl)-3-
methylbutanoyl)pyrrolidine-2-carboxylate (8d). Yield:
1
6.2.4. (2S,4R)-Methyl-1-((3S)-2-(tert-butoxycarbonyl)-3-
methylpentanoyl)-4-hydroxypyrrolidine-2-carboxylate (5a1).
To a solution of 5a (7.16 g, 20 mmol) in 100 mL CH2Cl2
at 0 °C was added Et3N (3.5 mL, 25 mmol) followed by
dropwise addition of methanesulfonyl chloride (1.7 mL,
22 mmol). The reaction mixture was stirred at 0 °C for
45 min and warmed to room temperature with stirring
for 4 h. The following mixture was diluted with 50 mL
CH2Cl2. The organic layer was washed with saturated
NaHCO3 solution, H2O, and brine in turn, dried with
Na2SO4, filtered, and evaporated to afford crude mesy-
late 5a1, which was of suitable purity to use directly in
the next step (yield: 82.3%).
59.8%, ESI-MS 344.6 (M+H), H NMR (DMSO-d6): d
1.36 (s, 9H); 0.86 (d, J = 6.5 Hz, 3H); 0.92 (d,
J = 6.6 Hz, 3H); 3.60 (s, 3H); 1.54 (m, 1H); 1.94 (m,
1H); 2.35 (m, 1H); 3.16 (m, 1H); 3.43 (m, H); 3.99 (m,
2H); 4.26 (t, J = 8.2 Hz, 1H); 6.75 (d, J = 8.4 Hz, 1H).
6.2.6.4. Methyl-4-amino-1-(2-(tert-butoxycarbonyl)-
propanoyl)pyrrolidine-2-carboxylate (9d). Yield: 61.3%,
ESI-MS 316.5 (M+H), 1H NMR (DMSO-d6): d 1.36
(s, 9H); 1.16 (d, J = 6.8 Hz, 3H); 3.60 (s, 3H); 3.80 (q,
J = 4.3 Hz, 1H); 4.21 (t, J = 7.5 Hz, 1H); 2.29 (m, 2H);
3.60 (m, 2H); 4.05 (m, 1H); 6.90 (d, J = 7.4 Hz, 1H).
6.2.6.5. Methyl-4-amino-1-(2-(tert-butoxycarbonyl)-4-
(methylthio)butanoyl)pyrrolidine-2-carboxylate (10d). Yield:
60.5%, ESI-MS 376.5 (M+H), H NMR (DMSO-d6): d
1.36 (s, 3H); 2.06 (s, 3H); 3.17 (s, 3H); 1.55 (m, 2H);
1.80 (m, 2H); 2.32 (m, 2H); 3.30 (m, 1H); 3.62 (m, 2H);
3.92 (m, 1H); 4.28 (t, J = 8.1 Hz, 1H); 7.09 (d, J =
7.5 Hz, 1H).
6.2.5. (2S,4S)-Methyl-4-azido-1-((3S)-2-(tert-butoxycar-
bonyl)-3-methylpentanoyl)pyrrolidine-2-carboxylate (5a2).20
To a solution of 5a1 (8.72 g, 20 mmol) in 30 mL anhyd
DMF was added ground sodium azide (1.39 g,
21.4 mmol), and the reaction mixture was heated at
55 °C for 10 h. The reaction mixture was cooled to room
temperature and then partitioned between water and
EtOAc. The organic layer was washed with brine, dried
with Na2SO4, filtered, and evaporated to get pale yellow
oil (yield: 63.5%).
1
6.2.6.6. Methyl-4-amino-1-(2-(2-(2,6-dichlorophenyla-
mino)phenyl)acetyl)pyrrolidine-2-carboxylate (11d). Yield:
63.7%, mp = 59.8–61.4 °C, ESI-MS: 422.5 (M+H), 1H
NMR (DMSO-d6): d 1.60 (m, 1H); 2.35 (m, 1H); 3.59 (s,
3H); 3.76 (s, 2H); 3.25 (m, 1H); 3.49 (m, 1H); 3.92 (m,
1H); 4.31 (t, J = 7.8 Hz, 1H); 6.29 (m, 1H); 6.86 (t,
J = 7.0 Hz, 1H); 7.05 (t, J = 7.3 Hz, 1H); 7.15 (q,
J = 8.1 Hz, 1H); 7.23 (d, J = 7.2 Hz, 1H); 7.50 (d,
J = 8.2 Hz, 2H); 7.73 (s, 1H).
6.2.6. (2S,4S)-Methyl-4-amino-1-((3S)-2-(tert-butoxycar-
bonyl)-3-methylpentanoyl)pyrrolidine-2-carboxylate (5d).21
In hydrogen atmosphere, 7.66 g compound 5a2 (20 mmol)
was dissolved in anhyd methanol, and 5% Pd/CaCO3
(0.77 g) was added. The resulting solution was stirred for
10 h under 760 mm Hg pressure, during which the hydro-
gen was bubbled intermittently. The resulting mixture was
filtered and the filtrate was evaporated to give pale yellow
oil, which was purified by flash chromatography on silica
gel to give 5d as pale yellow oil (yield: 60.8%). ESI-MS
6.2.7. 1-(2-(tert-Butoxycarbonyl)-4-methylpentanoyl)-4-
hydroxypyrrolidine-2-carboxylic acid (5b).22 Compound
5a (7.66 g, 20 mmol) was dissolved in 100 mL MeOH,
and 20 mL of 2 mol/L NaOH was added over 5min with
good stirring. This was allowed to stir for 24h at room
temperature. The reaction mixture was filtered and
adjusted to pH 5–6 with 80% acetic acid/water. The
residual solution was evaporated to remove MeOH
and then was acidified to pH 2–3 with 40% citric acid
solution and extracted with EtOAc. The extract was
washed with saturated NaCl, dried over Na2SO4. The
solvent was removed in vacuo to obtain 5b (yield
1
m/z 358.5 (M+H); H NMR (DMSO-d6, ppm): d 1.36 (s,
9H); 0.82 (t, J = 7.5 Hz, 3H); 0.88 (d,J = 6.6 Hz, 3H);
1.09 (m, 2H); 1.56 (m, 1H); 1.71 (m, 1H); 2.33 (m, 1H);
3.17 (m, 2H); 3.42 (m, 1H); 4.01 (m, 1H); 4.25 (t,
J = 8.1 Hz, 1H); 6.87 (d,J = 8.7 Hz, 1H).
6.2.6.1. Methyl-4-amino-1-(2-(tert-butoxycarbonyl)-3-
phenylpropanoyl)pyrrolidine-2-carboxylate (6d). Yield:
1
1
60.5%, ESI-MS 392.5 (M+H), H NMR (DMSO-d6): d
79.3%, mp = 79–80 °C). ESI-MS m/z 345.4 (M+H); H
1.28 (s, 9H); 3.61 (s, 3H); 1.59 (m, 1H); 2.40 (m, 1H);
2.75 (m, 1H); 2.91 (m, 1H); 3.76 (d, J = 5.8 Hz, 1H);
3.84 (d, J = 4.4 Hz, 1H); 4.30 (t, J = 7.8 Hz, 1H); 4.10
NMR (DMSO-d6, ppm): d 1.37 (s, 9H); 0.80 (t, 3H);
0.87 (d, J = 6.6 Hz, 3H); 1.04 (m, 2H);1.68 (m, 1H);
1.88 (m, 1H); 2.08 (m, 1H); 3.64 (m, 1H); 4.05 (m,

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J. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 2749–2758
2H); 5.17 (m, 1H); 4.23 (t, J = 8.1 Hz, 1H); 6.78 (d,
J = 9.0 Hz, 1H).
was stirred at 65 °C for 10 h, then 7.5 g silica gel was added
and evaporated to give pale yellow powder, which was
purified by column chromatography to provide 4.67 g of
compound 5c as a white solid, which appeared red in
FeCl3. Yield: 62.5%, mp = 94 –95 °C, ESI-MS m/z 360.6:
6.2.7.1. 1-(2-(tert-Butoxycarbonyl)-3-phenylpropanoyl)-
4-hydroxypyrrolidine-2-carboxylic acid (6b). Yield:
79.5%, mp = 96–97 °C, ESI-MS 379.6 (M+H), 1H
NMR (DMSO-d6): d 1.29 (s, 9H); 1.91 (m, 1H); 2.10
(m, 1H); 2.75 (m, 1H); 2.87 (m, 1H); 3.56 (d,
J = 3.6 Hz, 1H); 3.62 (d, J = 4.5 Hz, 1H); 4.29 (m,
1H); 4.34 (m, 1H); 5.16 (t, J = 3.7 Hz, 1H); 6.94 (m,
1H); 7.19 (m, 2H); 7.27 (m, 2H); 12.42 (s, 1H); 6.94 (d,
J = 8.5 Hz, 1H).
1
(M+H); H NMR (DMSO-d6, ppm): d 1.36 (s, 9H); 0.80
(t, J = 7.2 Hz, 3H); 0.83 (d, J = 6.4 Hz, 3H); 1.05 (m,
2H); 1.68 (m, 1H); 1.91 (m, 1H); 2.07 (m, 1H); 3.61 (m,
1H); 3.99 (m, 1H); 4.19 (m, 1H); 5.07 (m, 1H); 4.33 (t,
J = 7.3 Hz, 1H); 6.68 (d, J = 8.7 Hz, 1H).
6.2.8.1. tert-Butyl-1-(4-hydroxy-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-1-oxo-3-phenylpropan-2-ylcarbamate (6c).
Yield: 62.3%, mp = 104–105 °C, ESI-MS: 394.5 (M+H),
1H NMR (DMSO-d6): d 1.28 (s, 9H); 1.90 (m, 1H); 1.96
(m, 1H); 2.70 (m, 1H); 2.87 (m, 1H); 3.52 (d, J = 4.6 Hz,
1H); 3.63 (d, J = 4.5 Hz, 1H); 4.24 (t, J = 7.5 Hz, 1H);
4.36 (m, 1H); 5.11 (m, 1H); 6.93 (m, 1H); 7.21 (m,
2H); 7.28 (m, 2H); 8.80 (s, 1H); 10.53 (s, 1H); 6.93 (d,
J = 8.4 Hz, 1H).
6.2.7.2. 1-(2-(tert-Butoxycarbonyl)-4-methylpentanoyl)-
4-hydroxypyrrolidine-2-carboxylic acid (7b). Yield:
78.7%, mp = 167–168 °C, ESI-MS: 345.5 (M+H), 1H
NMR (DMSO-d6): d 1.42 (s, 9H); 0.95 (d, J = 6.6 Hz,
3H); 0.97 (d, J = 6.5 Hz, 3H); 4.77 (t, J = 8.3 Hz, 1H);
1.53 (m, 2H); 1.57 (m, 1H); 2.35 (m, 1H); 1.68 (m,
1H); 3.60 (dd, J = 3.5, 11.8 Hz, 1H); 4.21 (d,
J = 11.2 Hz, 1H); 5.21 (d, J = 8.1 Hz, 1H); 4.46 (m,
1H); 4.55 (s, 1H); 6.85 (d, J = 8.9 Hz, 1H).
6.2.8.2. tert-Butyl-1-(4-hydroxy-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-4-methyl-1-oxopentan-2-ylcarbamate (7c).
Yield: 62.1%, mp = 100–101 °C, ESI-MS: 360.6 (M+H),
1H NMR (DMSO-d6): d1.42 (s, 9H); 0.93 (d, J = 6.6 Hz,
3H); 0.96 (d, J = 6.4 Hz, 3H); 1.52 (m, 2H); 4.57 (t,
J = 8.3 Hz, 1H); 1.72 (m, 1H); 2.78 (m, 1H); 2.27 (m,
1H); 3.87 (dd, J = 3.6, 9.6 Hz, 1H); 4.41 (s, 1H); 3.74
(m, 1H); 3.98 (d, J = 11.0 Hz, 1H); 5.77 (d, J = 8.6 Hz,
1H); 6.79 (d, J = 8.2 Hz, 1H).
6.2.7.3. 1-(2-(tert-Butoxycarbonyl)-3-methylbutanoyl)-
4-hydroxypyrrolidine-2-carboxylic acid (8b). Yield:
77.9%, mp = 171.9–176.3 °C, ESI-MS: 331.5 (M+H),
1H NMR (DMSO-d6): d 1.37 (s, 9H); 0.83 (d,
J = 6.6 Hz, 3H); 0.89 (d, J = 6.7 Hz, 3H); 1.90 (m,
2H); 2.08 (m, 1H); 3.61 (m, 2H); 4.03 (m, 1H); 4.26 (t,
J = 8.1 Hz, 1H); 5.13 (m, 1H); 6.64 (d, J = 8.7 Hz,
1H); 12.35 (s, 1H).
6.2.8.3. tert-Butyl-1-(4-hydroxy-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-ylcarbamate (8c).
Yield: 60.7%, mp = 90–91 °C, ESI-MS 346.4: (M+H),
1H NMR (DMSO-d6): d 1.37 (s, 9H); 0.82 (d,
J = 6.5 Hz, 3H); 0.89 (d, J = 6.5 Hz, 3H); 1.86 (m,
2H); 1.91 (m, 1H); 3.60 (m, 2H); 3.99 (m, 1H); 4.20 (t,
J = 7.8 Hz, 1H); 5.07 (m, 1H); 6.60 (d, J = 8.6 Hz,
1H); 8.76 (s, 1H); 10.52 (s, 1H).
6.2.7.4. 1-(2-(tert-Butoxycarbonyl)propanoyl)-4-hydro-
xypyrrolidine-2-carboxylic acid (9b). Yield: 76.5%,
mp = 169.7–170.4 °C, ESI-MS: 303.6 (M+H), 1H
NMR (DMSO-d6): d 1.36 (s, 9H); 1.13 (d, 3H); 1.90
(m, 1H); 2.06 (m, 1H); 3.59 (q, J = 7.4 Hz, 1H); 4.26
(m, 2H); 4.34 (t, J = 2.2 Hz, 1H); 5.15 (m, 1H); 6.87
(d,J = 7.6 Hz, 1H).
6.2.7.5. 1-(2-(tert-Butoxycarbonyl)-4-(methylthio)bu-
tanoyl)-4-hydroxypyrrolidine-2-carboxylic acid (10b). Yield:
79.1%, mp = 132.6–133.9 °C, ESI-MS: 363.6 (M+H), H
NMR (DMSO-d6): d 1.37 (s, 9H); 2.04 (s, 3H); 1.58 (m,
2H); 1.77 (m, 2H); 2.04 (m, 2H); 3.24 (m, 1H); 3.63 (m,
2H); 4.29 (t, J = 7.5 Hz, 1H); 5.20 (m, 1H); 7.03 (d,
J = 6.6 Hz, 1H).
6.2.8.4. tert-Butyl-1-(4-hydroxy-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-1-oxopropan-2-ylcarbamate (9c). Yield:
59.8%, mp = 98–99 °C, ESI-MS 318.5: (M+H), 1H
NMR (DMSO-d6): d 1.36 (s, 9H); 1.14 (d, J = 7.0 Hz,
3H); 1.87 (m, 1H); 1.98 (m, 1H); 4.02 (q, J = 7.2 Hz,
1H); 4.20 (t, J = 7.5 Hz, 1H); 3.48 (m, 1H); 3.60 (m,
1H); 4.36 (m, 1H); 6.87 (d, J = 7.8 Hz, 1H); 10.35 (s,
1H); 10.51 (s, 1H).
1
6.2.7.6. 1-(2-(2-(2,6-Dichlorophenylamino)phenyl)ace-
tyl)-4-hydroxypyrrolidine-2-carboxylic acid (11b). Yield:
82.3%, mp = 116–117 °C, ESI-MS: 409.6 (M+H), 1H
NMR(DMSO-d6): d 1.94 (m, 1H); 2.12 (m, 1H); 3.30
(s, 2H); 3.72 (m, 2H); 4.30 (t, J = 7.8 Hz, 1H); 5.20 (m,
1H); 6.29 (m, 1H); 6.85 (t, J = 7.4 Hz, 1H); 7.05
(dt,J = 8.2, 7.6, 1.3 Hz, 1H); 7.15 (q, J = 8.1 Hz, 1H);
7.24 (dd, J = 6.9, 0.9;Hz, 1H); 7.50 (d, J = 7.9 Hz, 1H);
7.64 (s, 1H).
6.2.8.5. tert-Butyl-1-(4-hydroxy-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-4-(methylthio)-1-oxobutan-2-ylcarbamate
(10c). Yield: 60.7%, mp = 66 –67 °C, ESI-MS 378.6:
1
(M+H), H NMR (DMSO-d6): d 1.38 (s, 9H); 2.09 (s,
3H); 1.86 (m, 2H); 1.98 (m, 3H); 2.50 (m, 3H); 3.63
(m, 1H); 4.31 (m, 2H); 4.20 (t, J = 7.8 Hz, 1H); 5.14
(m, 1H); 6.98 (d, J = 8.1 Hz, 1H); 8.83 (s, 1H); 10.58
(s, 1H).
6.2.8.
tert-Butyl-1-(4-hydroxy-2-(hydroxycarbamoyl)-
6.2.8.6. 1-(2-(2-(2,6-Dichlorophenylamino)phenyl)ace-
tyl)-N,4-dihydroxypyrrolidine-2-carboxamide (11c). Yield:
63.7%, mp = 102.6–104.2 °C, ESI-MS: 424.5 (M+H), H
NMR (DMSO-d6): d 1.75 (m, 1H); 1.93 (m, 1H); 3.31 (s,
2H); 3.74 (m, 2H); 4.26 (t, J = 7.5 Hz, 1H); 5.15 (d,
pyrrolidin-1-yl)-3-methyl-1-oxopentan-2-ylcarbamate (5c).23
Compound 5a (7.66 g, 20 mmol) was dissolved in 100 mL
anhyd MeOH and 15 mL MeOH with NH2OK (prepared
by Fieser and Fieser. Vol. 1, p 478). The resulting solution
1

J. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 2749–2758
2757
J = 3.9 Hz, 1H); 6.28 (m, 1H); 6.84 (t, J = 7.3 Hz, 1H);
7.04 (dt, J = 7.8, 8.1, 1.3 Hz, 1H); 7.23 (dd, J = 7.3,
0.9 Hz, 1H); 7.50 (d, J = 8.0 Hz, 2H); 7.72 (s, 1H).
6.2.10.6. 4-Amino-1-(2-(2-(2,6-dichlorophenylamino)-
phenyl)acetyl)pyrrolidine-2-carboxylic acid (11e). Yield:
62.5%, mp = 196–197 °C, ESI-MS: 408.5 (M+H), 1H
NMR (DMSO-d6): d 2.11 (d, J = 14.1 Hz, 1H); 4.41
(d, J = 9.2 Hz, 1H); 3.53 (dd, J = 5.2, 13.4 Hz, 2H);
3.76 (s, 2H); 3.70 (m, 1H); 3.75 (m, 1H); 6.29 (d,
J = 7.9 Hz, 1H); 6.82 (t, J = 6.8 Hz, 1H); 7.03 (t,
J = 7.4 Hz, 1H); 7.15 (q, J = 8.1 Hz, 1H); 7.23 (d,
J = 6.4 Hz, 1H); 7.50 (d, J = 4.4 Hz, 2H); 8.18 (s, 1H).
6.2.9. (2S,4S)-4-Azido-1-((3S)-2-(tert-butoxycarbonyl)-
3-methylpentanoyl)pyrrolidine-2-carboxylic acid (5d1).
Compound 5d was converted to compound 5d1 as de-
scribed for compound 5b (yield 77.8%).
6.2.10. (2S,4S)-4-Amino-1-((3S)-2-(tert-butoxycarbonyl)-
3-methylpentanoyl)pyrrolidine-2-carboxylic acid (5e).
Compound 5d1 was converted to compound 5e as de-
scribed for compound 5d. Yield: 59.7%, mp = 160.7–
161.8 °C, ESI-MS m/z 344.6: (M+H); 1H NMR
(DMSO-d6, ppm): d 1.36 (s, 9H); 0.81 (t,J = 7.2 Hz,
3H); 0.86 (d, J = 6.5 Hz, 3H); 1.11 (m, 2H); 1.69 (m,
1H); 1.88 (m, 1H); 2.27 (m, 1H); 3.85 (m, 2H); 4.05
(m, 1H); 3.95 (t, J = 8.7 Hz, 1H); 5.76 (m, 1H); 6.93
(d, J = 8.7 Hz, 1H).
6.2.11. tert-Butyl-1-(4-amino-2-(hydroxycarbamoyl)pyrr-
olidin-1-yl)-3-methyl-1-oxopentan-2-ylcarbamate
(5f).
Compound 5d was converted to compound 5f as de-
scribed for compound 5c. Yield 63.4%, mp = 115.8–
116.9 °C, ESI-MS: m/z 359.6 (M+H); 1H NMR
(DMSO-d6,ppm): d 1.36 (s, 9H); 0.80 (t, J = 7.2 Hz,
3H); 0.86 (d, J = 6.6 Hz, 3H); 1.09 (m, 2H); 1.48 (m,
1H); 1.66 (m, 1H); 2.30 (m, 1H); 3.39 (m, 1H); 3.56
(m, 1H); 3.96 (m, 2H); 4.13 (t, J = 6.2 Hz, 1H); 6.87
(d, J = 8.2 Hz, 1H).
6.2.10.1. 4-Amino-1-(2-(tert-butoxycarbonyl)-3-phe-
nylpropanoyl)pyrrolidine-2-carboxylic acid (6e). Yield:
61.2%, mp = 175–176 °C, ESI-MS: 376.4 (M+H), 1H
NMR (DMSO-d6): d 1.29 (s, 9H); 1.97 (m, 1H); 2.23
(m, 1H); 2.74 (m, 1H); 2.91 (m, 1H); 3.51 (d,
J = 5.5 Hz, 1H); 3.85 (d, J = 5.1 Hz, 1H); 3.79 (m,
1H); 4.09 (m, 1H); 4.54 (t, J = 7.7 Hz, 1H); 7.04 (m,
1H); 7.19 (m, 2H); 7.26 (m, 2H); 8.90 (br, 2H); 7.04
(d, J = 8.5 Hz, 1H).
6.2.11.1. tert-Butyl-1-(4-amino-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-1-oxo-3-phenylpropan-2-ylcarbamate (6f).
Yield: 61.7%, mp = 119–120 °C, ESI-MS: 391.0 (M+H),
1H NMR (DMSO-d6): d 1.28 (s, 9H); 1.72 (m, 1H); 2.37
(m, 1H); 2.75 (m, 1H); 2.85 (m, 1H); 3.52 (d, J = 3.5 Hz,
1H); 3.85 (d, J = 5.6 Hz, 1H); 3.65 (m, 1H); 4.21 (m,
1H); 4.29 (t, J = 7.6 Hz, 1H); 7.09 (m, 1H); 7.19 (m,
2H); 7.25 (m, 2H); 7.09 (d, J = 8.2 Hz, 1H).
6.2.10.2. 4-Amino-1-(2-(tert-butoxycarbonyl)-4-meth-
ylpentanoyl)pyrrolidine-2-carboxylic acid (7e). Yield:
60.7%, mp = 181–182 °C, ESI-MS: 342.5 (MÀH), 1H
NMR (DMSO-d6): d 1.36 (s, 9H); 0.85 (d, J = 3.4 Hz,
3H); 0.89 (d, J = 6.3 Hz, 3H); 1.62 (m, 2H); 2.24 (m,
1H); 4.50 (t, J = 5.1 Hz, 1H); 1.89 (m, 2H); 3.66 (m,
1H); 4.07 (dd, J = 8.9, 18.3 Hz, 1H); 3.82 (m, 1H);
3.76 (d, J = 5.4 Hz, 1H); 6.95 (d, J = 12.3 Hz, 1H);
8.81 (br, 2H); 6.91 (d, J = 8.2 Hz, 1H).
6.2.11.2. tert-Butyl-1-(4-amino-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-4-methyl-1-oxopentan-2-ylcarbamate (7f).
Yield: 62.3%, mp = 126–127 °C, ESI-MS: 359.6 (M+H),
1H NMR (DMSO-d6): d 1.36 (s, 9H); 0.84 (d,
J = 7.9 Hz, 3H); 0.88 (d, J = 4.21 Hz, 3H); 1.57 (m,
2H); 1.65 (m, 1H); 1.68 (m, 1H); 2.31 (m, 1H); 4.15 (t,
J = 5.2 Hz, 1H); 3.85 (dd, J = 9.8, 9.9 Hz, 1H); 3.58
(m, 1H); 3.34 (d, J = 4.4 Hz, 1H); 3.58 (d, J = 5.3 Hz,
1H); 6.92 (d, J = 7.9 Hz, 1H).
6.2.10.3. 4-Amino-1-(2-(tert-butoxycarbonyl)-3-meth-
ylbutanoyl)pyrrolidine-2-carboxylic acid (8e). Yield:
59.9%, mp = 235–236 °C, ESI-MS: 330.5 (M+H), 1H
NMR (DMSO-d6): d 1.36 (s, 9H); 0.87 (d, J = 4.7 Hz,
3H); 0.91 (d, J = 6.6 Hz, 3H); 1.91 (m, 2H); 2.21 (m,
1H); 3.61 (m, 1H); 3.77 (m, 2H); 4.05 (m, 1H); 3.90 (t,
J = 8.3 Hz, 1H); 6.80 (d, J = 8.4 Hz, 1H); 8.74 (br, 2H).
6.2.11.3. tert-Butyl-1-(4-amino-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-ylcarbamate (8f).
Yield: 61.5%, mp = 108–109 °C, ESI-MS: 345.6 (M+H),
1H NMR (DMSO-d6): d1.36 (s, 9H); 0.85 (d, J = 6.5 Hz,
3H); 0.91 (d, J = 6.6 Hz, 3H); 1.89 (m, 2H); 2.23 (m,
1H); 3.23 (m, 1H); 3.37 (m, 1H); 3.92 (m, 2H); 4.07 (t,
J = 7.4 Hz, 1H); 6.73 (d, J = 8.2 Hz, 1H).
6.2.10.4. 4-Amino-1-(2-(tert-butoxycarbonyl)propano-
yl)pyrrolidine-2-carboxylic acid (9e). Yield: 59.6%,
mp = 248–249 °C, ESI-MS: 302.8 (M+H), 1H NMR
(DMSO-d6): d 1.36 (s, 9H); 1.15 (d, J = 6.7 Hz, 3H);
3.79 (q, J = 9.3 Hz, 1H); 4.17 (t, J = 7.8 Hz, 1H); 2.31
(m, 2H); 3.39 (m, 2H); 3.84 (m, 1H); 7.02 (d,
J = 7.0 Hz, 1H).
6.2.11.4. tert-Butyl-1-(4-amino-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-1-oxopropan-2-ylcarbamate (9f). Yield:
61.2%, mp = 144–146 °C, ESI-MS: 317.8 (M+H), 1H
NMR (DMSO-d6): d 1.36 (s, 9H); 1.15 (d, J = 6.7 Hz,
3H); 3.80 (q, J = 9.2 Hz, 1H); 4.27 (t, J = 8.0 Hz, 1H);
2.35 (m, 2H); 3.44 (m, 2H); 3.85 (m, 1H); 7.03 (d,
J = 7.0 Hz, 1H).
6.2.10.5. 4-Amino-1-(2-(tert-butoxycarbonyl)-4-(meth-
ylthio)butanoyl)pyrrolidine-2-carboxylic acid (10e). Yield:
61.7%, mp = 152.4–153.7 °C, ESI-MS: 362.6 (M+H),
1H NMR (DMSO-d6): d 1.36 (s, 9H); 2.05 (s, 3H); 1.84
(m, 2H); 1.94 (m, 1H); 2.29 (m, 1H); 2.40 (m, 2H); 3.55
(m, 1H); 3.75 (m, 2H); 4.16 (m, 1H); 4.26 (t, J = 6.1 Hz,
1H); 7.11 (d, J = 8.1 Hz, 1H).
6.2.11.5. tert-Butyl-1-(4-amino-2-(hydroxycarbamoyl)-
pyrrolidin-1-yl)-4-(methylthio)-1-oxobutan-2-ylcarbamate
(10f). Yield: 60.7%, mp = 108–109 °C, ESI-MS: 377.7
1
(M+H), H NMR (DMSO-d6): d 1.36 (s, 9H); 2.05 (s,
3H); 1.75 (m, 2H); 2.01 (m, 2H); 2.47 (m, 2H); 3.61
(m, 1H); 3.71 (m, 2H); 4.30 (m, 1H); 4.20 (t,
J = 4.1 Hz, 1H); 7.04 (d, J = 7.3 Hz, 1H).

2758
J. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 2749–2758
6.2.11.6. 4-Amino-1-(2-(2-(2,6-dichlorophenylamino)-
phenyl)acetyl)-N-hydroxypyrrolidine-2-carboxamide (11f).
Yield: 65.6%, mp = 159–160 °C, ESI-MS: 423.5
Acknowledgment
We thank Yumei Yuan, Jianzhi Gong, and Yu Liu for
their contributions to this work. This work was support-
ed by the National Nature Science Foundation of China
(Grant No. 36072541).
(M+H), 1H NMR (DMSO-d6):
d
2.03 (d,
J = 14.0 Hz, 1H); 4.39 (d, J = 9.1 Hz, 1H); 3.50 (dd,
J = 5.1, 13.2 Hz, 2H); 3.75 (s, 2H); 3.71 (m, 1H);
3.72 (m, 1H); 6.31 (d, J = 7.9 Hz, 1H); 6.83 (t,
J = 6.8 Hz, 1H); 7.05 (t, J = 7.2 Hz, 1H); 7.17 (q,
J = 8.0 Hz, 1H); 7.28 (d, J = 6.2 Hz, 1H); 7.50 (d,
J = 4.2 Hz, 2H); 8.20 (s, 1H).
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