Design, synthesis, and QSAR studies of novel lysine derives as amino-peptidase N/CD13 inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.04.012

A series of novel l-lysine derivatives were designed, synthesized, and assayed for their inhibitory activities on amino-peptidase N (APN)/CD13 and matrix metalloproteinase-2 (MMP-2). The preliminary biological test showed that most of the compounds displayed a high inhibitory activity against MMP-2 and a low activity against APN except compound B6 which exhibited good potency (IC₅₀ = 13.2 μM) similar with APN inhibitor Bestatin (IC₅₀=15.5 μM), and could be used as lead compound in the future.

Full text of DOI:10.1016/j.bmc.2008.04.012

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5473–5481
Design, synthesis, and QSAR studies of novel lysine
derives as amino-peptidase N/CD13 inhibitors
Qiang Wang,a Maoying Chen,a Huawei Zhu,a Jie Zhang,a Hao Fang,a
Binghe Wangb and Wenfang Xua,*
aDepartment of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, PR China
bDepartment of Chemistry, Georgia State University, Atlanta, GA 30303, USA
Received 30 January 2008; revised 6 April 2008; accepted 8 April 2008
Available online 11 April 2008
Abstract—A series of novel L-lysine derivatives were designed, synthesized, and assayed for their inhibitory activities on amino-pepti-
dase N (APN)/CD13 and matrix metalloproteinase-2 (MMP-2). The preliminary biological test showed that most of the compounds
displayed a high inhibitory activity against MMP-2 and a low activity against APN except compound B6 which exhibited good potency
(IC50 = 13.2 lM) similar with APN inhibitor Bestatin (IC50=15.5 lM), and could be used as lead compound in the future.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
group (ZBG); and Part C: heterocyclic rings or hydro-
phobic groups (Fig. 2).
As a zinc-dependent endopeptidase, amino-peptidase N
(APN) is also known as CD13. Scientific research has
been found that APN is over-expressed on tumor cells
and play an important role in extracellular matrix degra-
dation and invasion of tumor cells. Therefore, APN has
been a target for anticancer agents design. Since 1976, a
great amount of natural inhibitors have been found to
bind APN with high affinity (Fig. 1), including Bestatin
(1),1 probestin (2),2 amastatin (3),3 actinonin (4),4
phebestin ( 5),5 lapstatin (6),6 AHPA-Val (7),7 leuhistin
(8),8 curcumin (9)9, and pasmmaplin A (10).10
According to the literature, ZBG (part 2) could be car-
boxylic acid and hydroxymate. Then, the next step we
need to do should be not only the determination of
the hydrophobic fragment or heterocycle as part A
and part C, but also the choosing of a linker to connect
the three parts. In our preliminary studies, L-lysine was
selected as a linker due to its structure containing two
amino groups and one carboxylic acid which can be
used as ZBG. Therefore, a series of L-lysine derivatives
could be designed with different a-amino substitution
and N6-amino substitution. In this study, we reported
the preparation and in vitro APN inhibition studies of
N6-Cbz substituted L-lysine derivatives.
Our group has synthesized a series of 3-galloylamido-
N0-substituted 2,6-piperidinedione-N-acetamide pepti-
domimetics and found that the two target compounds
displayed a similar inhibitory activity compared with
Bestatin.11 In our on-going work, the strategy of
computer-aided molecular design has been used for
developing new peptidomimetics as APN inhibitors
and the docking studies have been done with natural
APN inhibitors listed in Figure 1. The results suggested
that the APN inhibitors can be divided into three parts.
Part A: hydrophobic fragment; Part B: zinc binding
2. Chemistry
The synthesis of target compounds is shown in Scheme
1. The N6-amino of L-lysine was selectively protected
by Cbz (carbobenzoxy) and then esterificated with HCl
in methanol to generate methyl 2-amino-6-(benzyloxy-
carbonylamino)hexanoate hydrochloride. Then, 10 dif-
ferent aromatic acids were converted to acyl chloride
and reacted with 2-amino group of L-lysine. Finally,
the ester functional group was hydrolyzed with NaOH/
H2O in methanol or treated with NH2OK in methanol
to get hydroxymic acid.
Keywords: Lysine derivatives; Synthesis; APN/CD13; Inhibitor;
QSAR.
*
Corresponding author. Tel./fax: +86 531 88382264; e-mail:
xuwenf@sdu.edu.cn
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.012

5474
Q. Wang et al. / Bioorg. Med. Chem. 16 (2008) 5473–5481
COOH
COOH
O
O
NH2
O
H
NH2
O
N
O
H2N
N
N
N
O
N
COOH
H
H
H
COOH
OH
OH
O
H
OH
N
OH
Bestatin (1)
N
Probestin(2)
Amastatin(3)
O
OH
H
NH2
OH
NHOH
H
N
COOH
N
COOH
NH
NH2
O
N
O
NH2
O
NH HN
O
Actinonin(4)
HO
O
AHPA-Val(7)
CH2OH
Lapstatin(6)
O
O
OH
OH
Phebestin(5)
OCH3
H
N
Br
O
Br
O
OCH3
OH
H2N
HO
N
S
S
COOH
N
H
N
H
HO
N
O
O
N
OH
HO
Psammaplin A(10)
Leuhistin(8)
Figure 1. Natural inhibitors of APN.
Curcumin(9)
malignant tumor, the inhibitory studies were performed
on both APN and MMP-2 so as to identify the selectiv-
ity of the target compounds.
From the results listed in Table 1, it can be found that
compound B6 (IC50 = 13.2 lM) is the most potent
APN inhibitor in all the target compounds, which showed
an inhibition similarto that of Bestatin (IC50 = 15.5 lM).
The inhibitory activity of the B series (R2:-NHOH) were
generally better than the A series (R2:-OH). It may be
caused by the hydroxymate residue in B the series, which
is a more strong zinc binding group than the carboxylic
acid. For the substitution of a-amino in L-lysine (R1), a
six-membered aromatic ring seems to be better than a
five-membered aromatic ring.
Figure 2. Docking the inhibitors showed in Figure 1 to APN.
However, most of the target compounds exhibited a
better inhibitory activity on MMP-2 than APN except
B6. This phenomenon was also found in our previous
studies on 3-galloylamido-N0-substituted 2,6-piperidine-
dione-N-acetamide peptidomimetics.11 The possible
reason may be the difference of the three-dimensional
structure of active sites of APN and MMP-2. By taking
3. Results and discussion
All the target compounds were tested for their inhibitory
ability against APN. Considering that matrix metallo-
proteinases-2 (MMP-2) also belong to the zinc-depen-
dent metalloproteinase and are associated with a
O
H2
N
H2
O
OH
NH2
HN
4
CO2
Cu
N
NH2
b
CO2
Cu
a
Cbz
Cbz
O
4
4
n
O2C
NH
N
H2N
O2C
H2N
Cbz
N
4
H2
H2
O
O
O
OH
c
d
H
N
4
H
Cbz
N
4
HCl
H2N
O
O
H2N
f
O
O
H
O
N
Cbz
R1
N
H
4
O
O
e
O
R1
Cl
R1
OH
g
h
O
NHOH
O
OH
O
O
H
H
N
Cbz
N
Cbz
R1
N
H
R1
N
4
4
H
O
O
Scheme 1. Reagents and conditions: (a) CuCO3Cu(OH)2, 1.2 N HCl, H2O, 80–90 ꢁC; (b) NaHCO3, CbzCl, 25 ꢁC; (c) EDTA SS, 25 ꢁC; (d) MeOH,
HCl, 25 ꢁC; (e) oxalyl chloride, CH2Cl2, ice-salt bath; (f) Et3N, CH2Cl2, 0 ꢁC; (g) 2 N NaOH, 1 N HCl, 25 ꢁC; (h) NH2OK, MeOH, 25 ꢁC.

Q. Wang et al. / Bioorg. Med. Chem. 16 (2008) 5473–5481
5475
Table 1. The structure and inhibitory activities of compounds against APN and MMP-2
H
N
H
N
R1
O
O
O
O
R2
Compound
R1
R2
IC50 (lM)
QSAR of model C
pIC50
MMP-2
APN
Res.
Act.
3.54
Pre.
A1
A2
A3
A4
–OH
–OH
–OH
–OH
8.76
2.93
8.49
3.14
291.6
197.4
3.58
ꢀ0.04
ꢀ0.01
ꢀ0.01
0.03
N
N
3.70
2.95
3.78
3.71
2.96
3.74
N
O
1120.7
166.8
N
S
A5
A6
–OH
–OH
12.7
17.8
1247.2
127.2
2.90
3.90
2.89
3.90
0.01
0.00
Cl
Cl
A7
A8
–OH
–OH
50.5
7.48
215.1
110.8
3.67
3.96
3.67
3.95
0.00
0.01
O2N
A9
–OH
–OH
19.2
13.1
25.5
202.2
188.0
91.2
3.69
3.73
4.04
3.69
3.73
3.99
0.00
0.00
0.05
O2N
Br
A10
B1
–NHOH
–NHOH
N
N
B2
14.3
97.0
4.01
4.02
ꢀ0.01
N
O
B3
B4
B5
B6
–NHOH
–NHOH
–NHOH
–NHOH
8.08
8.0
158.7
94.3
3.80
4.03
3.80
4.88
3.77
4.05
3.83
4.88
0.03
ꢀ0.02
ꢀ0.03
0.00
N
S
12.6
170.0
159.1
13.2
Cl
Cl
B7
B8
–NHOH
–NHOH
3.54
11.7
569.6
95.8
3.24
4.02
O2N
4.03
ꢀ0.01
(continued on next page)

5476
Q. Wang et al. / Bioorg. Med. Chem. 16 (2008) 5473–5481
Table 1 (continued)
Compound
R1
R2
IC50 (lM)
MMP-2
QSAR of model C
pIC50
APN
107.9
Res.
Act.
3.97
Pre.
3.99
B9
–NHOH
–NHOH
32.0
ꢀ0.02
O2N
B10
9.94
21.96
111.9
15.5
3.95
3.94
0.01
Br
Bestatin
an insight into the active site of APN(PDB code:
1HS6) and MMP-2 (PDB code: 1HOV, 1CK7,
1QIB), we found that the active site of APN was more
deeper than the active site of MMP-2, which leads to
the difficulty to access the active site of APN in most
compounds.
The QSAR studies were carried out with CoMFA12 of
Sybyl. The IC50 values were converted into pIC50
according to the formula: pIC50 = ꢀ lgIC50.
Considering that compounds A7 and B7 are different
from others, four models of QSAR have been studied
so as to obtain reasonable statistics. This is because
there is an olefin bond between the aromatic ring and
the amide in the R1 position of A7 and B7, which leads
to the significant structural difference in comparison to
other compounds. Therefore, results of the CoMFA
model will also be different irrespective to the training
set including them.
4. SAR studies
4.1. Dataset and molecular modeling
We chose B6 as a template molecule and tried to find
the pharmacophoric conformation. First, all the con-
formations of B6 searched by SYSTEM SEARCH in
Sybyl7.0 were docked to the active site of APN. From
the results, we found that there were two possibilities:
(1) Conformation I: R1 stretched into pocket A and
Cbz stretched into pocket C; (2) Conformation II:
Cbz stretched into pocket A and R1 stretched into
pocket C. The best docked conformation was picked
out for each of them (Fig. 3). Second, we take an en-
ergy analysis for the two conformations (Table 2). In
addition, considering the conformation of Bestatin
binding to APN in the X-ray crystal, the benzene ring
of Bestatin stretched into pocket A was more close to
its ZBG; we chose conformation I as the pharmaco-
phoric conformation of B6, which has a lower energy
and its R1 is more near to R2 (Fig. 4). Third, the tem-
plate molecule B6 was taken and the rest of the mole-
cules were aligned to it by the DATABASE
ALIGNMENT method in Sybyl (Fig. 5).
The steric and electrostatic CoMFA fields were calcu-
lated at each lattice intersection of a regularly spaced
˚
grid of 2.0 A in all the three dimensions within a defined
region. A sp3 carbon atom with +1.00 charge was used
as a probe atom. The steric and electrostatic fields were
truncated at +30.00 kcal molꢀ1, and the electrostatic
fields 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
r2 that resulted in optimum number of components and
lowest standard error of prediction were considered for
further analysis.
Figure 3. The docking results of B6.

Q. Wang et al. / Bioorg. Med. Chem. 16 (2008) 5473–5481
Table 2. The energy analysis of conformation I and conformation II
5477
(Fig. 6(a)). The green region surrounded the R2 group
which indicated that the bulky group of the B series
(R2:-NHOH) is better than that of the A series (R2 :-
OH), while the yellow area was mainly around R1,
which accorded with the nature of part A in docking
studies. This is because part A is a small pocket and
the bulky group substitution would be unfavorable for
APN inhibition. For example, compounds A7 and B7
showed a low APN inhibitory activity, which contain
the larger substitution of styrene in R1 position. In
Figure 6(b), the blue area was mainly surrounded by
the aromatic ring of R1 position and suggested that elec-
tronegativity groups, such as Cl, Br, and NO2, may en-
hance its activity. Based on our result, 2,4-dichloro
substitution exhibited better inhibitory activities on
APN, and compound B6 is the most potent and selective
compound in these L-lysine derivatives.
Energy analysis (kcal molꢀ1
)
Conformation I Conformation II
Bond stretching energy
Angle bend energy
Torsional energy
0.803
3.952
0.848
4.283
37.009
0.115
6.319
40.673
0.180
5.524
Out of plane bending energy
1–4 Van der Waals energy
Van der Waals energy
14.831
24.827
Total energy
63.029
76.334
4.2. Results and discussion
The LOO cross-validated q2 of the CoMFA model C is
0.737. The non cross-validated r2 for the model
established by the study is 0.997. The value of the
variance ratio F (n1 = 10, n2 = 8) is 289.708 and the stan-
dard error of the estimate (SEE) is 0.033. The contribu-
tion of electrostatic and steric is 50.4% and 49.6%,
respectively Finally, model C had a good predictability
as shown in, Figure 7.
5. Conclusions
In summary, we developed a series of novel L-lysine
derivatives as potential APN inhibitors. Among them,
compound B6 not only exhibited a similar inhibitory
activity compared with natural APN inhibitor Bestatin,
but also showed a significant selectivity for APN over
MMP-2. Therefore, B6 could be a lead compound
for us to search new L-lysine derivatives as APN
inhibitors.
According to the results in Table 3, the CoMFA model
C is the best one and the coefficient contour plots of
CoMFA model C are shown in Figure 7. It suggested
that the influence of steric field on APN inhibitory
activity is mainly located in the R1 and R2 positions
6. Experimental
6.1. APN inhibition assay
IC50 values against APN were determined as previously
described and by using L-Leu-p-nitroanilide as a sub-
strate and Microsomal amino-peptidase from Porcine
Kidney Microsomes (Sigma) as the enzyme in 50 mM
PBS, pH 7.2, at 37 ꢁC. The hydrolysis of the substrate
was monitored by following the change in the absor-
bance measured at 405 nm with the UV–vis spectropho-
tometer Pharmacia LKB, Biochrom 4060. All the
solutions of the inhibitors were prepared in the assay
buffer, and the pH was adjusted to 7.5 by the addition
of 0.1 M HCl or 0.1 M NaOH. All the inhibitors were
preincubated with APN for 30 min at room tempera-
ture. The assay mixture, which contained the inhibitor
solution (concentration dependent on the inhibitor),
the enzyme solution (4 lg/mL final concentration), and
the assay buffer, was adjusted to 200 lL.
Figure 4. Pharmacophoric conformation of B6 (Bestatin in the X-ray
crystal is showed in green).
6.2. MMP inhibition assay
Gelatinase A (MMP-2) and TNBS were purchased from
Sigma, and the substance was synthesized as described
by Vijaykumar et al. The gelatinase, substance, and
inhibitor were dissolved in sodium borate (pH 8.5,
50 mmol/L) and incubated for 30 min at 37 ꢁC, and then
0.03%TNBS was added and incubated for another
20 min, the resulting solution was detected under
450 nm wavelength to gain absorption.
Figure 5. Superposition of 20 inhibitors for CoMFA construction.

5478
Q. Wang et al. / Bioorg. Med. Chem. 16 (2008) 5473–5481
Figure 6. CoMFA coefficient contour map of model C (B6 is in the background). (a)Green (G) is the more bulky region, yellow (Y) is the less bulky
region; (b) red (R) color represents the negative charge region, blue (B) is the positive charge region.
stirred for 12 h, filtered and washed with water, repeated
this step again to get a white microcrystalline powder
(yield 88%, mp 230–236 ꢁC).
6.3.2. 2-Amino-6-(benzyloxycarbonylamino)hexanoate
hydrochloride ( 2). This compound was prepared as
described by Jordis14 with compound 1.
6.3.3. N6-[(benzyloxy)carbonyl]-N2-isonicotinoyl- L-lysine
hexanoate (3). To a (100 ml) two-necked flask equipped
with a stirring oar was dropwise added 2.0 g of isonicoti-
nic acid dissolved with 40 ml methylene chloride followed
by a solution of oxalyl chloride(1.1 equiv) in methylene
Figure 7. The predictability of the CoMFA model C.
chloride (10 ml) over a 10-min period. The temperature
was kept below ꢀ5 ꢁC by ice-salt bath. The reaction mix-
6.3. Chemistry: general procedures
ture was stirred for 5 h and then condensed by rotary
evaporation to remove methylene chloride and excess
oxalyl chloride.
6.3.1. N6-[(Benzyloxy)carbonyl]-L -lysine (1). The title
compound was prepared as described by Shen
Zongxuan.13
Compound 2 (3.0g) and TEA (2.0 equiv) were dissolved
in methylene chloride (50 ml) followed by a solution of
chloride (1.1 equiv) obtained above in methylene chlo-
ride (30 ml) dropwise overa10-min period, and the solu-
tion was detected by TLC (AcOEt/DAB-6, 1:2 (V/V)).
The mixture was condensed by rotary evaporation to re-
move the solvent. AcOEt (100 ml) was added to the
product, and then it was washed followed by 1 N HCl,
NaHCO3 SS, brine to neutral, and dried by Na2SO4.
It was filteered and condensed by rotary evaporation
to obtain crude compound 3, and then purified by col-
umn chromatography(AcOEt/DAB-6, 1:4 (V/V)) to ob-
tain a colorless oil (3.1g), yield 86%.
The starting materials, cupric subcarbonate (4.3 g,
18.1 mmol) and 1.2 N HCl (24 ml) were dissolved in
water (36 ml). To this mixture, L-lysine (4.3 g,
29.4 mmol) was added and heated to 80–90 ꢁC. The
resulting pale blue mixture was gently refluxed for 1 h.
The warm reaction mixture was filtered using vacuum
and washed with hot water (3· 10 ml). The combined
aqueous filtrates were cooled to room temperature.
NaHCO3 (3.37 g, 40.1 mmol) was added to the mixture
followed by a solution of benzylchloroformate (5.4 g,
31.8 mmol) in methylbenzol (30 ml) dropwise over
3 min period. The reaction mixture was stirred for
24 h, filtered, and washed with water (3· 30 ml). The
solid filter cake was dried in a vacuum dryer. The blue
solid cake was dissolved in EDTA SS (100 ml) and
The other N6-[(benzyloxy)carbonyl]-N2-L-lysine hexano-
ates were synthesized following the general procedure as
described above.
Table 3. Main parameters of four CoMFA models
Model
Opt. Com.
q2
R2
F
SEE
A (all)
B (–A7)
2
2
0.352
0.316
0.737
0.728
0.738
0.753
0.997
0.995
23.916
0.229
0.229
0.033
0.042
24.346
289.708
189.744
C (–B7)
D (–A7, –B7)
10
9

Q. Wang et al. / Bioorg. Med. Chem. 16 (2008) 5473–5481
5479
N6-[(Benzyloxy)carbonyl]- N2 -(pyrazin-2-ylcarbonyl)-L-
lysine hexanoate: (3.1 g, yield 86%).
J = 7.8 Hz, 1H, CONH), 7.851 (d, = 0.3 Hz, 1H,
ArH), 7.272–7.381 (m, 5H, ArH), 7.236–7.254 (m, 1H,
CONH), 7.175 (d, = 3.60 Hz, 1H, ArH); 6.633 (dd,
J = 1.80, 1.50 Hz, 1H, ArH); 4.990 (s, 2H, CH2), 4.293
(q, J = 13.72 Hz, 1H, CH), 2.977 (q, J = 12.45 Hz, 2H,
CH2), 1.726–1.773 (m, 2H, CH2), 1.236–1.392 (m, 4H,
CH2).
N6-[(Benzyloxy)carbonyl]-N2-furoyl-L-lysine hexanoate:
(2.9 g, yield 81%).
N6-[(Benzyloxy)carbonyl]-N2-(pyridin-3-ylcarbonyl)-L-
lysine hexanoate: (3.0 g, yield 85%).
6.3.7. N6-[(Benzyloxy)carbonyl]-N2-(pyridin-3-ylcarbon-
yl)-L-lysine (A4). White solid, yield 93%, mp 175.5–
N6-[(Benzyloxy)carbonyl]-N2-(2-thienylcarbonyl)-L-
lysine hexanoate: (3.2 g, yield 89%).
1
176.5 ꢁC. ESI-MS 386.7 (M+H); H NMR (DMSO-d6,
ppm): 12.621 (s, 1H, COOH), 9.031 (d, J = 2.1 Hz,
1H, ArH), 8.809 (d, J = 7.80 Hz, 1H, CONH), 8.713–
8.734 (m, 1H, ArH), 8.197–8.236 (m, 1H, ArH), 7.517
(dd, J = 4.80 Hz, 3.3 Hz, 1H, ArH), 7.291–7.379 (m,
5H, ArH), 7.250–7.269 (m, 1H, CONH), 4.985 (s, 2H,
CH2), 4.350 (q, J = 13.78 Hz, 1H, CH), 2.992 (q,
J = 12.37 Hz, 2H, CH2), 1.741–1.816 (m, 2H, CH2),
1.356–1.467 (m, 4H, CH2).
N6-[(Benzyloxy)carbonyl]- N2 -(2,4-dichlorobenzoyl)-L-
lysine hexanoate: (3.3 g, yield 78%).
N6-[(Benzyloxy)carbonyl]-N2 -cinnamoyl-L-lysine hexa-
noate: (2.7 g, yield 68%).
N6-[(Benzyloxy)carbonyl]- N2-(3-nitrobenzoyl)-L-lysine
hexanoate: (2.6 g, yield 65%).
6.3.8. N6-[(Benzyloxy)carbonyl]-N2-(2-thienylcarbonyl)-
L-lysine (A5). White solid, yield 81.5%, mp 58–60 ꢁC.
ESI-MS 391.5 (M+H); 1H NMR (DMSO-d6, ppm):
12.637 (s, 1H, COOH), 8.595 (d, J = 7.80 Hz, 1H,
CONH), 7.892 (d, J = 3.0 Hz, 1H, ArH), 7.769 (d,
J = 5.10 Hz, 1H, ArH), 7.293–7.381 (m, 5H, ArH),
7.250–7.269 (m, 1H, CONH), 7.158 (dd, J = 3.60,
1.20 Hz, 1H, ArH), 4.991 (s, 2H, CH2), 4.296 (q,
J = 13.56 Hz, 1H, CH), 2.987 (q, J = 12.39 Hz, 2H,
CH2), 1.716–1.821 (m, 2H, CH2), 1.337–1.406 (m, 4H,
CH2).
N6-[(Benzyloxy)carbonyl]- N2-(4-nitrobenzoyl)-L-lysine
hexanoate: (2.9 g, yield 79%).
N6-[(Benzyloxy)carbonyl]-N2-(4-bromobenzoyl)-L-lysine
hexanoate: (3.0 g, yield 78%).
6.3.4. N6-[(Benzyloxy)carbonyl]-N2-isonicotinoyl-L-lysine
(A1). Compound 3 1.5 g was dissolved in metha-
nol(20 ml), to the solution, 2 N NaOH (4 ml) was added.
The solution was detected by TLC. The mixture was
condensed by rotary evaporation to remove methanol,
and then acidified to pH 4–6 by 1 N HCl, and then ex-
tracted by AcOEt (3· 150 ml). The mixture was dried
by anhydrous Na2SO4, filtered and condensed by rotary
evaporation to obtain the crude target compound. It
was then recrystallized with AcOEt to give a white solid,
6.3.9. N6-[(Benzyloxy)carbonyl]-N2-(2,4-dichlorobenzoyl)-L-
lysine (A6). White solid, yield 86%, mp 130–131 ꢁC. ESI-
MS 453.4 (M+H); H NMR (DMSO-d6, ppm): 12.671
1
(s, 1H, COOH), 8.756 (d, J = 7.80 Hz, 1H, CONH),
7.681 (d, J = 1.80 Hz, 1H, ArH), 7.480–7.514 (m, 1H,
ArH), 7.299–7.435 (m, 6H, ArH), 7.241–7.299 (m, 1H,
CONH), 4.992 (s, 2H, CH2), 4.304 (q, = 13.27 Hz,
1H, CH), 2.988 (q, J = 12.45 Hz, 2H, CH2), 1.641–
1.771 (m, 2H, CH2), 1.211–1.286 (m, 4H, CH2).
1
yield 90.1%, mp 173–174 ꢁC.ESI-MS 386.7 (M+H); H
NMR (DMSO-d6, ppm): 12.699 (s, 1H, COOH),
8.904(d, J = 7.5 Hz, 1H, CONH), 8.736 (d, = 6.0 Hz,
2H, ArH), 7.788 (dd, = 1.50, 4.50 Hz, 2H, ArH),
7.293–7.379 (m, 5H, ArH), 7.250–7.269 (m, 1H,
CONH), 4.988 (s, 2H, CH2), 4.337 (q, = 13.80 Hz,
1H, CH), 2.992 (q, = 12.48 Hz, 2H, CH2), 1.796–1.822
(m, 2H, CH2), 1.296–1.411 (m, 4H, CH2).
6.3.10. N6-[(Benzyloxy)carbonyl]-N2-cinnamoyl-L-lysine
(A7). White solid, yield 82.7%, mp 56–57 ꢁC. ESI-MS
411.6 (M+H); H NMR (DMSO-d6, ppm): 12.628 (s,
1
1H, COOH); 8.352 (d, = 7.8 Hz, 1H, CONH); 7.463–
7.586 (m, 2H, Ar–H); 7.302–7.447 (m, 9H, ArH,
@CH); 7.248–7.286 (m, 1H, NH); 6.748 (d,
J = 15.9 Hz, 1H, @CH); 4.996 (s, 2H, CH2); 4.290 (q,
J = 13.58 Hz, 1H, CH); 2.977 (q, J = 12.29 Hz, 2H,
CH2); 1.618–1.765(m, 2H, CH2); 1.329–1.420 (m, 4H,
CH2).
The other compounds of A series were synthesized fol-
lowing the general procedure as described above.
6.3.5. N6-[(Benzyloxy)carbonyl]-N2-(pyrazin-2- ylcarbon-
yl)-L-lysine (A2). White solid, yield 88.9%, mp 134–
1
135 ꢁC. ESI-MS 387.6 (M+H); H NMR (DMSO-d6,
ppm): 12.852 (s, 1H, COOH), 9.188 (d, J = 1.5 Hz,
1H, ArH), 8.906 (s, 1H, ArH), 8.874 (d, J = 7.4 Hz,
1H, CONH); 8.761 (dd, = 1.50 Hz, 2.40Hz, 1H, ArH),
7.288–7.372 (m, 5H, ArH), 7.242–7.261 (m, 1H,
CONH), 4.974 (s, 2H, CH2), 4.434 (q, J = 13.60 Hz,
1H, CH), 2.967 (q, J = 12.37 Hz, 2H, CH2), 1.848–
1.873 (m, 1H, CH2), 1.311–1.415 (m, 4H, CH2).
6.3.11. N6-[(Benzyloxy)carbonyl]-N2-(3-nitrobenzoyl)-L-
lysine (A8). Yellow oil, yield 77.6%.ESI-MS 430.6
(M+H); 1H NMR (DMSO-d6, ppm): 12.596 (s, 1H,
COOH), 9.020 (d, = 7.50 Hz, 1H, CONH), 8.742 (s,
1H, ArH); 8.413 (d, J = 8.10 Hz, 1H, ArH); 8.333 (d,
= 7.80 Hz, 1H, ArH); 7.795 (t, J = 8.10 Hz, 1H, ArH);
7.324–7.374 (m, 5H, ArH); 7.265–7.288 (m, 1H,
CONH), 4.982 (s, 2H, CH2), 4.380 (q, J = 13.97 Hz,
1H, CH), 2.988 (q, J = 12.58 Hz, 2H, CH2), 1.812–
1.990 (m, 2H, CH2), 1.234–1.420 (m, 4H, CH2).
6.3.6. N6-[(Benzyloxy)carbonyl]-N2-2-furoyl-L-lysine (A3).
Yellow oil, yield 83.3%.ESI-MS 375.5 (M+H); 1H NMR
(DMSO-d6, ppm): 12.637 (s, 1H, COOH), 8.421 (d,

5480
Q. Wang et al. / Bioorg. Med. Chem. 16 (2008) 5473–5481
6.3.12. N6-[(Benzyloxy)carbonyl]-N2-(4-nitrobenzoyl)-L-
lysine (A9). Yellow solid, yield 82.7%, mp 47–49 ꢁC.
ESI-MS 430.6 (M+H); 1H NMR (DMSO-d6, ppm):
12.687 (s, 1H, COOH), 8.950 (d, J = 7.50 Hz, 1H,
CONH), 8.329 (d, J = 8.70 Hz, 2H, ArH); 8.116 (d,
J = 8.70 Hz, 2H, ArH); 7.291–7.379 (m, 5H, ArH);
7.246–7.266 (m, 1H, CONH), 4.982 (s, 2H, CH2),
4.365 (q, J = 13.85 Hz, 1H, CH), 3.001 (q,
J = 12.47 Hz, 2H, CH2), 1.809–1.991 (m, 2H, CH2),
1.152–1.233 (m, 4H, CH2).
6.3.17. N6-[(Benzyloxy)carbonyl]-N2-(pyridin-3-ylcarbon-
yl)-N1-hydroxy-L-lysinamide (B4). White solid, yield
82.4%, mp 142–143 ꢁC. ESI-MS 401.6 (M+H); 1H
NMR (DMSO-d6, ppm): 10.740 (s, 1H, CONH),
9.286 (s, 1H, OH); 9.033 (s, 1H, ArH), 8.706 (d,
J = 7.20 Hz, 2H, ArH), 8.220 (d, J = 7.80 Hz, 1H,
CONH); 7.496 (dd, J = 4.80, 5.10 Hz, 1H, ArH),
7.295–7.383 (m, 5H, ArH), 7.240–7.275 (m, 1H,
CONH),
4.989
(s,
2H,
CH2),
4.334
(q,
J = 13.56 Hz, 1H, CH), 2.973 (q, J = 12.24 Hz, 2H,
CH2), 1.703–1.751 (m, 2H, CH2), 1.198–1.360 (m,
4H, CH2).
6.3.13. N6-[(Benzyloxy)carbonyl]-N2-(4-bromobenzoyl)-
L-lysine (A10). White solid, yield 83%, mp 140–141 ꢁC.
ESI-MS 465.4 (M+H); 1H NMR (DMSO-d6, ppm):
12.603 (s, 1H, COOH), 8.671 (d, J = 7.80 Hz, 1H,
CONH), 7.832 (d, J = 8.10 Hz, 2H, ArH); 7.689 (d,
J = 8.10 Hz, 2H, ArH); 7.292–7.377 (m, 5H, ArH);
7.238–7.276 (m, 1H, CONH), 4.983 (s, 2H, CH2),
4.319 (q, J = 13.80 Hz, 1H, CH), 2.973 (q,
J = 12.41 Hz, 2H, CH2), 1.733–1.779 (m, 2H, CH2),
1.352–1.427 (m, 4H, CH2).
6.3.18. N6-[(Benzyloxy)carbonyl]-N2-(2-thienylcarbonyl)-
N1-hydroxy-L-lysinamide (B5). White solid, yield 73%,
mp 65–67 ꢁC. ESI-MS 406.6 (M+H); 1H NMR
(DMSO-d6, ppm): 10.714 (s, 1H, CONH), 8.852 (s,
1H, OH); 8.506 (d, J = 8.10 Hz, 1H, CONH), 7.921 (d,
J = 2.70 Hz, 1H, ArH), 7.753 (dd, J = 0.9, 4.80 Hz,
1H, ArH), 7.274–7.383 (m, 5H, ArH), 7.236–7.254 (m,
1H, CONH), 7.139 (dd, J = 3.90, 4.80 Hz, 1H, ArH),
4.988 (s, 2H, CH2), 4.268 (q, J = 13.72 Hz, 1H, CH),
2.960 (q, J = 12.78 Hz, 2H, CH2), 1.643–1.730 (m, 2H,
CH2), 1.226–1.433 (m, 4H, CH2).
6.3.14. N6-[(Benzyloxy)carbonyl]-N2-isonicotinoyl-N1-hydro-
xy-L-lysinamide (B1). This compound was prepared as
described by Hwan15 with compound 3, the other
compounds of A series were synthesized following the
general procedure of B1.
6.3.19. N6-[(Benzyloxy)carbonyl]-N2-(2,4-dichlorobenzoyl)-
N1-hydroxy-L-lysinamide (B6). White solid, yield
77.8%, mp 172–173 ꢁC. ESI-MS 468.5 (M+H); 1H
NMR (DMSO-d6, ppm): 10.707 (s, 1H, CONH),
8.910 (s, 1H, OH); 8.760 (d, J = 8.10 Hz, 1H,
CONH), 7.658 (d, J = 1.80 Hz, 1H, ArH), 7.445–
7.493 (m, 2H, ArH), 7.301–7.367 (m, 5H, ArH),
7.237–7.273 (m, 1H, CONH), 4.992 (s, 2H, CH2),
4.266 (q, J = 13.52 Hz, 1H, CH), 2.975 (q, J =
12.42 Hz, 2H, CH2), 1.610–1.657 (m, 2H, CH2),
1.288–1.398 (m, 4H, CH2).
White solid, yield 81%, mp 175–177 ꢁC. ESI-MS 401.6
(M+H); 1H NMR (DMSO-d6, ppm): 10.743 (s, 1H,
CONH), 8.873 (d, J = 1.50 Hz, OH); 8.792 (d,
J = 8.10 Hz, 1H, CONH), 8.712 (dd, J = 1.50, 4.50 Hz,
2H, ArH), 7.786 (dd, J = 1.50 Hz, 4.50 Hz, 2H, ArH),
7.294–7.380 (m, 5H, ArH), 7.235–7.274 (m, 1H,
CONH), 4.984 (s, 2H, CH2), 4.312 (q, J = 13.64 Hz,
1H, CH), 2.973 (q, J = 12.35 Hz, 2H, CH2),
1.673–1.744 (m, 2H, CH2), 1.266–1.416 (m, 4H,
CH2).
6.3.20. N6-[(Benzyloxy)carbonyl]-N2-cinnamoyl-N1-hydro-
xy-L-lysinamide (B7). White solid, yield 74.8%, mp
168–169 ꢁC. ESI-MS 426.5 (M+H); 1H NMR
(DMSO-d6, ppm): 10.723 (s, 1H, CONH); 8.868 (d,
J = 1.50 Hz, 1H, OH); 8.279 (d, J = 8.40 Hz, 1H,
NH); 7.539–7.566 (m, 2H, Ar–H); 7.288–7.450 (m,
9H, Ar–H, @CH); 7.232–7.269 (m, 1H, NH); 6.768
(d, J = 15.9 Hz, 1H, @CH); 4.992 (s, 2H, CH2);
4.260 (q, J = 13.63 Hz, 1H, CH); 2.967 (q, J =
12.46 Hz, 2H, CH2); 1.537–1.607 (m, 2H, CH2);
1.359–1.427 (m, 2H, CH2); 1.240–1.315 (m, 2H,
CH2).
6.3.15.
N6-[(Benzyloxy)carbonyl]-N2-(pyrazin-2-ylcar-
bonyl)-N1-hydroxy-L-lysinamide(B2). White solid, yield
1
73.3%, mp 55–57 ꢁC. ESI-MS 402.6 (M+H); H NMR
(DMSO-d6, ppm): 10.801 (s, 1H, CONH), 9.184 (d,
J = 1.50 Hz, 1H, OH), 8.962 (s, 1H, ArH); 8.899 (d,
J = 2.40 Hz, 1H, ArH), 8.755 (d, J = 1.50, 2.40 Hz,
1H, ArH); 8.576 (d, J = 8.40 Hz, 1H, CONH), 7.278–
7.377 (m, 5H, ArH), 7.226–7.264 (m, 1H, CONH),
4.975 (s, 2H, CH2), 4.395 (q, J = 13.72 Hz, 1H, CH),
2.959 (q, J = 12.49 Hz, 2H, CH2), 1.689–1.762 (m, 1H,
CH2), 1.234–1.435 (m, 4H, CH2).
6.3.21. N6-[(Benzyloxy)carbonyl]-N2-(3-nitrobenzoyl)-N1-
hydroxy-L-lysinamide (B8). White solid, yield 60%, mp
6.3.16. N6-[(Benzyloxy)carbonyl]-N2-2-furoyl-N1-hydro-
xy-L-lysinamide (B3). White solid, yield 80%, mp 55–
57 ꢁC. ESI-MS 390.6 (M+H); 1H NMR (DMSO-d6,
ppm): 10.700 (s, 1H, CONH), 8.858 (s, 1H, OH); 8.206
(d, J = 8.40 Hz, 1H, CONH), 7.837 (d, J = 1.2 Hz, 1H,
ArH), 7.282–7.384 (m, 5H, ArH), 7.233–7.269 (m, 1H,
CONH), 7.201 (d, J = 3.30 Hz, 1H, ArH); 6.617(dd,
J = 1.50, 3.30 Hz, 1H, ArH); 4.989 (s, 2H, CH2), 4.270 (q,
J = 13.63 Hz, 1H, CH), 2.963 (q, = 12.37 Hz, 2H,
CH2), 1.631–1.678 (m, 2H, CH2), 1.151–1.423 (m, 4H,
CH2).
1
177–179 ꢁC. ESI-MS 445.7 (M+H); H NMR (DMSO-
d6, ppm): 10.765 (s, 1H, CONH), 8.932 (d,
J = 7.50 Hz, 1H, OH); 8.856 (d, J = 7.50 Hz, 1H,
CONH), 8.752 (s, 1H, ArH); 8.392 (d, J = 8.10 Hz,
1H, ArH); 8.326 (d, J = 7.50 Hz, 1H, ArH); 7.771 (t,
J = 8.10 Hz, 1H, ArH); 7.290–7.375 (m, 5H, ArH);
7.236–7.269 (m, 1H, CONH), 4.980 (s, 2H, CH2),
4.346 (q, J = 13.67 Hz, 1H, CH), 2.975 (q,
J = 12.49 Hz, 2H, CH2), 1.693–1.765 (m, 2H, CH2),
1.274–1.424 (m, 4H, CH2).

Q. Wang et al. / Bioorg. Med. Chem. 16 (2008) 5473–5481
5481
6.3.22. N6-[(Benzyloxy)carbonyl]-N2-(4-nitrobenzoyl)-N1-
hydroxy-L-lysinamide (B9). White solid, yield 53.3%,
mp 184–186 ꢁC. ESI-MS 445.7 (M+H); 1H NMR
(DMSO-d6, ppm): 10.081 (s, 1H, CONH), 9.083 (s,
1H, OH), 8.869 (d, J = 7.50 Hz, 1H, CONH), 8.325 (d,
J = 8.70 Hz, 2H, ArH); 8.114 (d, J = 8.70 Hz, 2H,
ArH); 7.263–7.371 (m, 5H, ArH); 7.227–7.236 (m, 1H,
CONH), 4.988 (s, 2H, CH2), 4.321 (q, J = 13.85 Hz,
1H, CH), 2.985 (q, J = 12.47 Hz, 2H, CH2), 1.657–
1.880 (m, 2H, CH2), 1.079–1.381 (m, 4H, CH2).
Shandong Nature Science Foundation of China (Grant
No. Y2004C02).
References and notes
1. Umezawa, H.; Aoyagi, T.; Suda, H.; Hamada, M.
Antibiotics 1976, 29, 97–99.
2. Aoyagi, T.; Yoshida, S.; Nakamura, Y.; Shigihara, Y.
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3. Rich, D. H.; Moon, B. J.; Harbeson, S. J. Med. Chem.
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6.3.23. N6-[(Benzyloxy)carbonyl]-N2-(4-bromobenzoyl)-
N1-hydroxy-L-lysinamide (B10). White solid, yield 70%,
mp 157–159 ꢁC. ESI-MS 480.4 (M+H); 1H NMR
(DMSO-d6, ppm): 10.701 (s, 1H, CONH), 8.840 (d,
J = 1.20 Hz, 1H, OH); 8.553 (d, J = 8.10 Hz, 1H,
CONH), 7.833 (d, J = 8.70 Hz, 2H, ArH); 7.667 (d,
J = 8.40 Hz, 2H, ArH); 7.296–7.382 (m, 5H, ArH);
7.232–7.269 (m, 1H, CONH), 4.982 (s, 2H, CH2),
4.291 (q, J = 13.72 Hz, 1H, CH), 2.969 (q, J =
12.34 Hz, 2H, CH2), 1.678–1.707 (m, 2H, CH2), 1.235–
1.412 (m, 4H, CH2).
4. Umezawa, H.; Aoyagi, T.; Tanaka, T.; Suda, H. Antibi-
otics 1985, 38, 1629–1630.
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This work was supported by the National Natural
Foundation Research Grant (Grant Nos. 30772654
and 36072541) and the National High Technology Re-
search and Development Program of China (863 pro-
ject; Grant No. 2007AA02Z314) and Doctoral
Foundation of Ministry of Education of the People’s
Republic of China (Grant No. 20060422029) and the