Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors

Article abstract of DOI:10.3109/14756361003698147

A series of novel L-isoserine derivatives were synthesised and evaluated for their ability to inhibit aminopeptidase N (APN)/CD13. In our preliminary biological results, some of these compounds possessed a potent inhibitory activity against the APN. Within this series, compound 14b not only showed similar enzyme inhibition (IC₅₀ of 12.2μM) compared with the positive control bestatin (half maximal inhibitory concentration (IC ₅₀) of 7.3μM), but also had a potent antiproliferative activity against human cancer cell lines cells.

Full text of DOI:10.3109/14756361003698147

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(2): 302–310
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756361003698147
ReseaRch aRtIcle
Synthesis of a novel series of L-isoserine derivatives
as aminopeptidase N inhibitors
Kanghui Yang, Jinghong Fen, Hao Fang, Lei Zhang, Jianzhi Gong, and Wenfang Xu
Department of Medicinal Chemistry, School of Pharmacy, Shan Dong University, 44 West Culture Road,
Ji′nan, P R China
abstract
A series of novel L-isoserine derivatives were synthesised and evaluated for their ability to inhibit aminopeptidase N
(APN)/CD13. In our preliminary biological results, some of these compounds possessed a potent inhibitory activity
against the APN. Within this series, compound 14b not only showed similar enzyme inhibition (IC50 of 12.2μM)
compared with the positive control bestatin (half maximal inhibitory concentration (IC50) of 7.3μM), but also had a
potent antiproliferative activity against human cancer cell lines cells.
Keywords: Aminopeptidase N inhibitor, L-isoserine, synthesis
Introduction
binding modes, our group has developed various APN
inhibitors, including L-lysine derivates [12] and AHP
A(α-Amino-β-Hydroxyl-phenylbutanoic acid) derivates,
[13] which both showed potent APN inhibitory activities.
In our recent screening studies, L-isoserine showed the
ability to inhibit APN at an half maximal inhibitory con-
centration (IC50 ) value of 563 μM, which could serve as
a new lead compound for further chemical modification
and optimisation.
It has been reported that L-isoserine-L-leucine dipep-
tide shows moderate aminopeptidase B inhibitory activ-
ity (IC50 of 140 μM) [14]. is evidence indicates that the
incorporation of an amino acid to L-isoserine may con-
tribute to its inhibitory activity against APN. erefore in
our current work, different amino acids have been cou-
pled with L-isoserine by forming dipeptides or tripep-
tides. is article will describe the synthesis, preliminary
biological evaluation and structure-activity relationship
(SAR) study of such a novel series of L-isoserine deriva-
tives as potent APN inhibitors.
Aminopeptidase N (APN EC 3.4.11.2), also known as
CD13, is a zinc-dependent membrane metalloprotease
which belongs to the M1 family of ectoenzymes [1]. It usu-
ally forms a noncovalently bound homodimer on the cell
membrane of monocytes, myeloid, epithelial cells of the
intestine and kidney, fibroblasts and tumour cells [2–3].
isenzymeisabletocleavetheN-terminalresiduesfrom
polypeptide chains, as well as play an important role in
modulating bioactive peptides or protein responses such
as modification, activation, and degradation. APN is also
involved in various physiological processes, such as cell
proliferation, secretion, invasion and angiogenesis [4–5].
For example, APN is over-expressed on the tumour cell
surface for regulating ECM degradation and the invasion
of tumour cells [6]. erefore, inhibition of APN/CD13
could prevent the spread and metastasis of cancer cells
since several APN inhibitors have been well studied clini-
cally over the last two decades. Among the APN inhibitors,
bestatin is a representative molecule for the treatment of
adult acute nonlymphocytic leukemia [7].
Materials and methods
So far, X-ray crystal structures for APN for both the
holo- and apo-forms [8] have been resolved along with
a number of different ligands, such as bestatin [9–10],
arginine and lysine [11]. According to the literature for
Chemistry
e starting material L-Isoserine (7), is a white solid,
(mp 199-201°C, [α]25D = −32.5° (c 1, H2O)), was purchased
Address for Correspondence: Wenfang Xu, Department of Medicinal Chemistry, School of Pharmacy, Shan Dong University, 44 West Culture
Road, Ji′nan, P R China. Fax: (86)53188382264. Tel: (86)53188382264. E-mail: xuwenf@yahoo.cn
(Received 19 October 2009; revised 26 January 2010; accepted 10 February 2010)
302

Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors 303
from JiaDing District, Shanghai, China. Unless otherwise
residue was recrystallised with MeOH and ether to give
specified, all reagents and buffer salts were purchased
from commercial vendors and used without further
purification. e solvents were distilled prior to use and
flash chromatography was performed using silica gel (60
Å, 200 300 mesh). Melting points were determined on
an electrothermal melting point apparatus in an uncor-
rected form. Proton nuclear magnetic resonance (1H
NMR) spectra were determined on a Brucker Avance
600 spectrometer using TMS as an internal standard in
DMSO-d6 solutions. Chemical shifts were reported in
delta (δ) units, parts per million (ppm) downfield from
trimethylsilane. High-resolution mass spectral (HRMS)
data are reported as m/e (relative intensity). e opti-
cal rotation was determined on a GYROMAT-HP Digital
Automatic Polarimeter. All reported yields correspond to
purified products.
6a (0.56 g) as a white crystals, yield: 85.3%, mp 190–193°C,
1
[α]25D = +14.2° (c 1, MeOH). H-NMR: (DMSO-d6) δ 8.403
(d, J= 7.8 Hz, 1H, -CONH-), 8.054 (s, 3H, NH3), 7.285–7.202
(m, 5H, Ar-H), 4.482–4.472 (m, 1H, CH), 4.351–4.318 (m,
1H, CH), 3.573 (s, 3H, -OCH3), 3.11–3.092 (m, 1H, CH2),
2.791–2.751 (m, 1H, CH2), 1.527–1.495 (m, 1H, CH),
1.486–1.351 (m, 2H, CH2), 0.861 (d, J= 6.6 Hz, 3H, CH3),
0.831 (d, J= 6.6 Hz, 3H, CH3); ESI-MS m/z: 293.1 [M+H]+.
Compounds 6b, 6c and 6d were prepared following
the general procedure as described above.
L-phenylalanyl-L-leucine methyl ester hydrochloride (6b)
Whitecrystals,yield:79.4%,mp141–143°C;[α]25D = +17.5°
(c 1, MeOH); 1H-NMR: (DMSO-d6) δ 8.578 (d, J = 7.8
Hz, 1H, -CONH-), 8.108 (s, 3H, NH3), 7.28–7.182
(m, 5H, Ar-H), 4.622–4.587 (m, 1H, CH), 4.325–4.198
(m, 1H, CH), 3.628 (s, 3H, -OCH3), 2.793–2.605 (m, 1H,
CH2), 2.787–2.52 (m, 1H, CH2), 1.652–1.604 (m, 1H,
CH), 1.541–1.479 (m, 2H, CH2), 0.893 (d, J = 6.6 Hz, 3H,
CH3), 0.842 (d, J = 6.6 Hz, 3H, CH3); ESI-MS m/z: 293.2
[M+H]+.
General procedure for the synthesis of 2a, 2b, 4a and 4b
e title compound L-leucine methyl ester hydrochloride
(2a) and L-phenylalanine methyl ester hydrochloride
(2b) were prepared from L-leucine and L-phenylalanine
according to the methods described in the literature [15].
Using the same materials, the title compound L-leucine
benzyl ester hydrochloride (4a) and L-phenylalanine
benzyl ester hydrochloride (4b) were prepared as
described by Kim et al. [16].
L-leucyl-L-phenylalanine benzyl ester hydrochloride (6c)
A white solid, yield: 83.9%, mp 160–162°C; [α]25D = +6.3°
(c 1, H2O); 1H-NMR: (DMSO-d6) δ 9.203 (d, J= 7.8 Hz, 1H,
-CONH-), 8.345 (s, 3H, NH3), 7.363–7.226 (m, 10H, Ar-H),
5.09–5.04 (m, 2H, Ar-CH2-O-), 4.612–4.576 (m, 1H, CH),
3.805–3.781 (m, 1H, CH), 3.102–3.032 (m, 2H, Ar-CH2-),
1.671–1.627 (m, 1H, CH), 1.522–1.499 (m, 2H, CH2), 0.85
(d, J = 6.6 Hz, 3H, CH3), 0.829 (d, J = 6.6 Hz, 3H, CH3);
ESI-MS m/z: 369.3 [M+H]+.
General procedure for the synthesis of 6a, 6b, 6c and 6d
To a 200 mL solution of compound 3a (0.92 g, 4 mmol),
2b (0.88 g, 4.4 mmol), and hydroxybenzotriazole (HOBt)
(0.65 g, 4.8 mmol), 4-Dimethylaminopyridine (DMAP)
(0.09 g, 0.8 mmol) in dry DCM, was added TEA (1.33 g,
13.2 mmol). e reaction mixture was gently cooled to
0°C in an ice bath. To the reaction mixture was added
dropwise a solution of 1-ethyl-3-(3-dimethylaminopro-
pyl) carbodiimide (EDCI) (1.53 g, 8 mmol) in DCM for
1 h. After removal of the ice bath, the reaction mixture
was stirred at room temperature for 12h and then filtered
to remove the precipitate. e filtrate was washed with a
1 mol/L citric acid solution, saturated NaHCO3 and brine,
dried over Na2SO4, and evaporated in vacuo. e residue
was purified by flash column chromatography (EtOAc/
PE, 1:3(V/V)) to give the desired compound 5a as a
L-phenylalanyl-L-leucine benzyl ester hydrochloride (6d)
A white solid, yield: 81.8%, mp 161–163°C; [α]25D = +5.6°
(c 1, H2O); 1H-NMR: (DMSO-d6) δ 9.187 (d, J = 7.8,
1H, -CONH-), 8.32 (s, 3H, NH3), 7.389–7.24 (m, 10H,
Ar-H), 5.168–5.105 (m, 2H, Ar-CH2-O-), 4.398–4.36 (m,
1H, CH), 4.133–4.111 (m, 1H, CH), 3.162–3.13 (m, 1H,
Ar-CH2-), 2.953–2.917 (m, 1H, Ar-CH2-), 1.72–1.674 (m,
1H, CH), 1.678–1.526 (m, 2H, CH2), 0.896 (d, J = 6.6 Hz,
3H, CH3), 0.848 (d, J = 6.6 Hz, 3H, CH3); ESI-MS m/z:
369.2 [M+H]+.
1
white solid (1.48 g), yield: 94.2%, mp 70–73°C. H-NMR:
Synthesis of (S)-3-(tert-butoxycarbonylamino)-2-
hydroxypropanoic acid (8)
(DMSO-d6) δ 7.919 (d, J= 7.8 Hz, 1H, -CONH-), 7.287–
7.214 (m, 5H, Ar-H), 5.807 (d, J= 5.4 Hz, 1H, -CONH-),
4.474–4.459 (m, 1H, CH), 4.346–4.311 (m, 1H, CH), 3.568
(s, 3H, -OCH3), 3.102–3.089 (m, 1H, CH2), 2.787–2.75 (m,
1H, CH2), 1.526–1.501 (m, 1H, CH), 1.435 (s, 9H, CH3),
1.475–1.328 (m, 2H, CH2), 0.863 (d, J= 6.6 Hz, 3H, CH3),
0.828 (d, J= 6.6 Hz, 3H, CH3); ESI-MS m/z: 393.2 [M+H]+.
To a solution of compound 5a (0.78 g, 2 mmol) in dry
EtOAc at 0°C was added dropwise a solution of EtOAc
(10 mL) saturated by dry HCl gas. e reaction solu-
tion was stirred at 0°C for 2h, and then the temperature
was raised to room temperature and the reaction pro-
ceeded for 5 h before being concentrated in vacuo. e
Compound 8 was synthesised from L-Isoserine follow-
ing the general procedure as described above (prepara-
tion of 3a). a colourless solid, yield: 93.5%, mp 85–88°C;
1
[α]25D = +6.7° (c 1, MeOH); H-NMR: (DMSO-d6) δ 7.934
(d, J= 7.8 Hz, 1H, -CONH-), 6.54 (s, 1H, OH), 4.674–4.651
(m, 1H, CH), 3.457 (dd, J= 13.8 Hz, J= 8.4 Hz, 1H, CH2),
3.241 (dd, J= 13.8 Hz, J= 8.4 Hz, 1H, CH2), 1.435 (s, 9H,
CH3); ESI-MS m/z: 206.1 [M+H]+.
General procedure for the synthesis of 11a–11d and 13a–13d
Compound 9a was synthesised from N protected
L-Isoserine (8) and L-phenylalanine methyl ester
© 2012 Informa UK, Ltd.

304 K. Yang et al.
hydrochloride (2b) following the general procedure as
described above (preparation of 5a), yield: 84.5%, mp
4.372–4.335 (m, 1H, CH), 4.287–4.256 (m, 1H, CH), 3.088
(dd, J= 12.6 Hz, J= 8.4 Hz, 1H, CH), 2.778 (dd, J= 12.6 Hz,
J= 8.4 Hz, 1H, CH), 1.7–1.594 (m, 2H, CH2), 1.567–1.522
(m, 1H, CH), 0.882 (d, J= 6.6 Hz, 3H, CH3), 0.839 (d, J= 6.6
Hz, 3H, CH3); HRMS (FAB) calcd for C16H24N2O4 309.1809;
found: 309.1807 (M+H)+.
1
95–97°C; [α]25D =-9.72° (c 1, MeOH); H-NMR (DMSO-d6)
δ 7.921 (d, J=7.8 Hz, 1H, -CONH-), 7.287–7.362 (m, 2H,
Ar-H), 7.177–7.166 (m, 3H, NH3), 6.526 (s, 1H, OH), 5.801
(d, J=5.4 Hz, 1H, -CONH-), 4.604–4.567 (m, 1H, CH),
3.889–3.869 (m, 1H, CH), 3.627 (s, 3H, -OCH3), 3.173–3.135
(m, 1H, Ar-CH2- ), 3.075 (dd, J=13.8 Hz, J=8.4 Hz, 1H,
CH2), 3.032 (dd, J=13.8 Hz, J=8.4 Hz, 1H, CH2), 2.817–2.77
(m, 1H, Ar-CH2-),1.435 (s, 9m, CH3); HRMS (FAB) calcd
for C18H26N2O6 389.1683; found: 389.167 [M+Na]+.
L-Isoseryl-L-leucyl-L-phenylalanine methyl ester
hydrochloride (13a)
A white solid, yield: 64.3%, mp 65–68°C; [α]25D = -33.2°
(c 1, H2O); 1H-NMR (DMSO-d6) δ 8.588 (d, J= 7.8 Hz,
1H, -CONH-), 8.12 (s, 3H, NH3), 7.843 (d, J= 7.8 Hz, 1H,
-CONH-), 7.298–7.2 (m, 5H, Ar-H), 6.485 (s, 1H, OH),
4.486–4.499 (m, 1H, CH), 4.368–4.329 (m, 1H, CH), 4.12–
4.199 (m, 1H, CH), 3.579 (s, 3H, -OCH3), 3.102–3.093 (m,
1H, Ar-CH2-), 3.055 (dd, J= 13.80 Hz, J= 8.4 Hz, 1H, CH2),
2.977 (dd, J= 13.8 Hz, J= 8.4 Hz, 1H, CH2), 2.798–2.752 (m,
1H, Ar-CH2-), 1.528–1.494 (m, 1H, CH), 1.483–1.359 (m,
2H, CH2), 0.861 (d, J= 6.6 Hz, 3H, CH3), 0.831 (d, J= 6.6
Hz, 3H, CH3); HRMS (FAB) calcd for C19H29N3O5 380.2180;
found: 380.2174 (M+H)+.
Compound 11a was obtained through the deprotec-
tion of compound 9a in the saturated HCl/EtOAc solu-
tion as the preparation of 6a, yield: 77.7%, mp 138–140°C;
1
[α]25D = -19.8° (c 1, H2O). H-NMR (DMSO-d6) δ 8.25 (d,
J= 7.8 Hz, 1H, -CONH-), 8.094 (s, 3H, NH3), 7.294–7.27
(m, 2H, Ar-H), 7.23–7.197 (m, 3H, Ar-H), 6.541 (s, 1H,
OH), 4.6–4.563 (m, 1H, CH), 4.203–4.173 (m, 1H, CH),
3.107 (s, 3H, -OCH3), 3.101 (dd, J= 13.8 Hz, J= 8.4 Hz, 1H,
CH2), 3.05 (dd, J= 13.8 Hz, J= 8.4 Hz, 1H, CH2), 2.927 (dd,
J= 13.2 Hz, J= 9 Hz, 1H, Ar-CH2-), 2.569 (dd, J= 13.2 Hz,
J= 9 Hz, 1H, Ar-CH2-); HRMS (FAB) calcd for C13H18N2O4
267.1339; found: 267.1335 [M+H]+.
L-Isoseryl-L-phenylalanyl-L-leucine methyl ester
Compounds 9c–9d were prepared from N protected
L-Isoserine and amino acid methyl/benzyl ester hydro-
chlorides (2a,4a and 4b) and compounds 10a–10d were
prepared from N protected L-Isoserine and dipeptide
methyl/benzyl ester hydrochlorides(6a, 6b, 6c and 6d).
Compounds11c–11dand13a–13dwereobtainedthrough
the deprotection of compounds 9c–9d and 10a–10d.
hydrochloride (13b)
A white solid, yield: 50.9%, mp 77–80°C; [α]25D = -26.7°
(c 1, H2O); 1H-NMR (DMSO-d6) δ 8.631(d, J= 7.8 Hz,
1H, -CONH-), 8.1 (s, 3H, NH3), 7.862 (d, J= 7.8 Hz, 1H,
-CONH-), 7.298–7.195 (m, 5H, Ar-H), 6.512 (s, 1H, OH),
4.633–4.596 (m, 1H, CH), 4.33–4.269 (m, 1H, CH), 4.166–
4.145 (m, 1H, CH), 3.639 (s, 3H, -OCH3), 3.077 (dd, J= 13.8
Hz, J= 8.4 Hz, 1H, CH2), 2.918 (dd, J= 13.8 Hz, J= 8.4 Hz,
1H, CH2), 2.895–2.55 (m, 1H, Ar-CH2-), 2.568–2.521 (m,
1H, Ar-CH2-), 1.656–1.597 (m, 2H, CH2), 1.544–1.497 (m,
1H, CH), 0.894 (d, J= 6.6 Hz, 3H, CH3), 0.847 (d, J= 6.6 Hz,
3H, CH3); HRMS (FAB) calcd for C19H29N3O5 380.2180;
found: 380.2179 (M+H)+.
L-Isoseryl-L-leucine methyl ester hydrochloride (11b)
A white solid, yield: 63.1%, mp 120–123°C; [α]25D = -36.1°
(c 1, H2O); 1H-NMR (DMSO-d6) δ 8.054 (d, J= 7.8 Hz, 1H,
-CONH-), 7.903 (s, 3H, NH3), 6.427 (s, 1H, OH), 4.607–
4.593 (m, 1H, CH), 4.186–4.152 (m, 1H, CH), 3.218 (s, 3H,
-OCH3), 3.106 (dd, J= 13.8 Hz, J= 8.4 Hz, 1H, CH2), 3.023
(dd, J= 13.8 Hz, J= 8.4 Hz, 1H, CH2), 1.523–1.704 (m, 3H,
CH2CH), 0.926–0.947 (m, 6H, CH3); HRMS (FAB) calcd for
C10H20N2O4 233.1496; found: 233.1493 [M+H]+.
L-Isoseryl-L-leucyl-L-phenylalanine benzyl ester
hydrochloride (13c)
A white solid, yield: 52.2%, mp 135–137°C; [α]25D = -30.5°
(c 1, H2O); 1H-NMR (DMSO-d6) δ 8.631(d, J = 7.8 Hz,
1H, -CONH-), 8.134 (s, 3H, NH3), 7.827 (d, J = 7.8 Hz,
1H, -CONH-), 7.368–7.311 (m, 3H, Ar-H), 7.27–7.202
(m, 7H, Ar-H), 6.493 (s, 1H, -OH), 5.088–5.033 (m, 2H,
Ar-CH2-O-), 4.547–4.51 (m, 1H, CH), 4.381–4.341 (m,
1H, CH), 4.216–4.201 (m, 1H, CH), 3.084–3.074 (m, 1H,
Ar-CH2-), 3.067 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH2),
2.993 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH2), 2.782–2.63 (m,
1H, Ar-CH2-),1.512-1.35 (m, 3H, CH2CH), 0.844 (d, J = 6.6
Hz, 3H, CH3), 0.797 (d, J = 6.6 Hz, 3H, CH3); HRMS (FAB)
calcd for C25H33N3O5 456.2493; found: 456.2490 (M+H)+.
L-Isoseryl-L- phenylalanine benzyl ester hydrochloride (11c)
A white solid, yield: 76.6%, mp 177–179°C; [α]25D = −23.3°
1
(c 1, H2O); H-NMR (DMSO-d6) δ 8.285 (d, J = 7.8 Hz,
1H, -CONH-), 8.032 (s, 3H, NH3), 7.391–7.303 (m, 5H,
Ar-H), 7.274–7.177 (m, 5H, Ar-H), 6.511 (s, 1H, OH),
5.14–5.097 (m, 2H, Ar-CH2-O-), 4.644–4.607 (m, 1H,
CH), 4.195–4.164 (m, 1H, CH), 3.126 (dd, J = 13.8 Hz,
J = 8.4 Hz, 1H, CH2), 3.028 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H,
CH2), 2.95–2.93 (m, 1H, Ar-CH2-), 2.601–2.566 (m, 1H,
Ar-CH2-); HRMS (FAB) calcd for C19H22N2O4 343.1652;
found: 343.1650 (M+H)+.
L-Isoseryl-L-phenylalanyl-L-leucine benzyl ester
L-Isoseryl-L-leucine benzyl ester hydrochloride (11d)
hydrochloride (13d)
A white solid, yield: 84.6%, mp 98–103°C; [α]25D = -25.7°
(c 1, H2O); 1H-NMR (DMSO-d6) δ 8.387 (d, J= 7.8 Hz,
1H, -CONH-), 8.189 (s, 3H, NH3), 7.405–7.324 (m, 5H,
Ar-H), 6.48 (s, 1H, OH), 5.187–5.13 (m, 2H, Ar-CH2-O-),
A white solid, yield: 58.1%, mp 140–142°C; [α]25D = -28.6°
(c 1, H2O); 1H-NMR (DMSO-d6) δ 8.644 (d, J= 7.8 Hz,
1H, -CONH-), 8.037 (s, 3H, NH3), 7.852 (d, J= 7.8 Hz, 1H,
-CONH-), 7.377–7.319 (m, 3H, Ar-H), 7.242–7.171 (m, 5H,
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors 305
Ar-H), 6.494 (s, 1H, OH), 5.166–5.121 (m, 2H, Ar-CH2-O-),
(d, J= 7.8 Hz, 1H, -CONH-), 7.265–7.176 (m, 5H, Ar-H),
6.524 (s, 1H, OH), 4.637–4.6 (m, 1H, CH), 4.274–4.236 (m,
1H, CH), 4.159–4.095 (m, 1H, CH), 3.09 (dd, J= 13.8 Hz,
J= 8.4 Hz, 1H, CH2), 2.905 (dd, J= 13.8 Hz, J= 8.4 Hz, 1H,
CH2), 2.896–2.882 (m, 1H, Ar-CH2), 2.539–2.505 (m, 1H,
Ar-CH2), 1.72–1.526 (m, 3H, CH2CH), 0.907 (d, J= 6.6 Hz,
3H, CH3), 0.853 (d, J= 6.6 Hz, 3H, CH3); HRMS (FAB) calcd
for C18H27N3O5 366.2023; found: 366.2027 (M+H)+.
4.64–4.603 (m, 1H, CH), 4.385–4.348 (m, 1H, CH), 4.142–
4.126 (m, 1H, CH), 3.028 (dd, J= 13.8 Hz, J= 8.4 Hz, 1H,
CH2), 2.912–2.892 (m, 1H, Ar-CH2-), 2.851 (dd, J= 13.8 Hz,
J= 8.4 Hz, 1H, CH2), 2.54–2.504 (m, 1H, Ar-CH2-),1.657–
1.529 (m, 3H, CH2CH), 0.906 (d, J= 6.6 Hz, 3H, CH3), 0.846
(d, J= 6.6 Hz, 3H, CH3); HRMS (FAB) calcd for C25H33N3O5
456.2493; found: 456.2496 (M+H)+.
General procedure for the synthesis of 12a–12b and 14a–14b
Compound 9c (1.77g, 4 mmol) was hydrogenated in abso-
lute MeOH (30mL) with a catalytic quantity of 10% Pd/C
(0.17g). e mixture was stirred for 10h under normal
pressure then filtered. e filtrate was evaporated to give
a white solid. e solid was dissolved in dry EtOAc (5mL)
at 0°C and added dropwise to a solution of EtOAc (10mL)
saturated by dry HCl gas. e reaction solution was
stirred at 0°C for 12h to provide a white solid. e solu-
tion was removed by filtration, and the remaining solid
was recrystallised with MeOH and Et2O to give 12a white
crystals (0.91g), yield: 78.9%, mp 187–189°C; [α]25D =-13.4°
In vitro APN inhibition assay
e IC50 values against APN were determined using
L-Leu-p-nitroanilide as the substrate and microsomal
aminopeptidase from porcine kidney microsomes
(Sigma) in 50 mM PBS, pH 7.2, at 37°C. e hydrolysis of
the substrate was monitored by following the change in
the absorbance measured at 405 nm with a UV-Vis spec-
trophotometer Pharmacia LKB, Biochrom 4060. All solu-
tions of inhibitors were prepared in the assay buffer, and
the pH was neutralised to 7.5 by the addition of either
0.1 M HCl or 0.1 M NaOH.
e samples and positive controls were serial diluted
to various concentrations: 1280 μg/mL, 320 μg/mL,
80 μg/mL, and 20 μg/mL, 5 μg/mL and 1.25 μg/mL. All
compounds were pre-incubated with APN on a 96-well
plate for 30 min. e assay mixture, including the com-
pound solution, the enzyme solution (5 μg/mL final
concentration) and the assay buffer, was adjusted to give
a total volume of 200 μL. Fifteen minutes later, the absor-
bance (OD405) of the wells was recorded on a microplate
reader. e percentage inhibition was calculated accord-
ing to the absorbance of the assay wells relative to those
of the control wells. Finally, the IC50 values were deter-
mined using a linear regression analysis of the concen-
tration/inhibition data.
1
(c 1, H2O); H-NMR (DMSO-d6) δ 12.964 (s, 1H, COOH),
8.081 (s, 3H, NH3), 8.07 (d, J=8.4 Hz, 1H, -CONH-), 7.275
(t, J=7.8 Hz, 2H, Ar-H), 7.223–7201 (m, 3H, Ar-H), 6.527
(s, 1H, OH), 4.543–4.507 (m, 1H, CH), 4.183–4.169 (m,
1H, CH), 3.113 (dd, J=13.8 Hz, J=8.4 Hz, 1H, CH2), 3.031
(dd, J=13.8 Hz, J=8.4 Hz, 1H, CH2), 2.957–2.938 (m, 1H,
Ar-CH2-), 2.62–2.561 (m, 1H, Ar-CH2-); HRMS (FAB) calcd
for C12H16N2O4 253.1183; found: 253.1180 (M+H)+.
L-Isoseryl-L-leucine hydrochloride (12b)
A white solid, yield: 88.3%, mp 213–215°C; [α]25D = -44.5°
1
(c 1, H2O); H-NMR (DMSO-d6) δ 12.713 (s, 1H, COOH),
8.102 (s, 3H, NH3), 8.088 (d, J= 8.4 Hz, 1H, -CONH-), 6.441
(s, 1H, OH), 4.236–4.229 (m, 1H, CH), 4.274–4.251 (m,
1H, CH), 3.086 (dd, J= 12.6 Hz, J= 7.2 Hz, 1H, CH2), 2.784
(dd, J= 12.6 Hz, J= 7.2 Hz, 1H, CH2), 1.681–1.585 (m, 2H,
CH2), 1.556–1.513 (m, 1H, CH), 0.893 (d, J= 6.6 Hz, 3H,
CH3), 0.854 (d, J= 6.6 Hz, 3H, CH3); HRMS (FAB) calcd for
C9H18N2O4 219.1339; found: 219.1340 (M+H)+.
In vitro MMP-2 inhibition assay
Gelatinase A (MMP-2) and picrylsulphonic acid (TNBS)
were purchased from Sigma. Succinylated gelatin was
synthesised as described by Baragi et al. [17]. e com-
pound samples were assayed for inhibitory activity
against MMP-2 in 96-well microplates using succinylated
gelatin as the substrate. e compounds and gelatinase
were dissolved in sodium borate buffer (pH 8.5, 50mM),
and incubated at 37°C for 30 min. e substrate was added
and incubated at 37°C for another 60 min. en 0.03%
picrylsulphonic acid solution was added and incubated
at room temperature for an additional 20 min. e result-
ing solutions were detected under 450 nm wavelength to
gain absorbance (OD450). MMP-2 inhibition activity was
measured by the following formula [17]: Inhibition ration
(%) = [OD450(100%)− OD450(compound)]/[OD450(100%)−
OD450(blank)]×100%. e IC50 values were obtained
from the above inhibitory rates using the OriginPro 7.5
software.
L-Isoseryl-L-leucyl-L-phenylalanine hydrochloride (14a)
A white solid, yield: 56.9%, mp 90–92°C; [α]25D = -34.7° (c 1,
1
H2O); H-NMR (DMSO-d6) δ 12.69 (s, 1H, COOH), 8.468
(d, J= 7.8 Hz, 1H, -CONH-), 8.119 (s, 3H, NH3), 7.836 (d,
J= 7.8 Hz, 1H, -CONH-), 7.245–7.168 (m, 5H, Ar-H), 6.52
(s, 1H, OH), 4.652–4.573 (m, 1H, CH), 4.263–4.225 (m,
1H, CH), 4.176–4.084 (m, 1H, CH), 3.986 (dd, J= 13.8 Hz,
J= 8.4 Hz, 1H, CH2), 2.887 (dd, J= 13.8 Hz, J= 8.4 Hz, 1H,
CH2), 2.812–2.798 (m, 1H, Ar-CH2), 2.51–2.439 (m, 1H,
Ar-CH2), 1.502–1.414 (m, 3H, CH2CH), 0.862 (d, J= 6.6 Hz,
3H, CH3), 0.813 (d, J= 6.6 Hz, 3H, CH3); HRMS (FAB) calcd
for C18H27N3O5 366.2023; found: 366.2026 (M+H)+.
L-Isoseryl-L-phenylalanyl-L-leucine hydrochloride (14b)
A white solid, yield: 53.6%, mp 87–89°C; [α]25D = -35.1°
(c 1, H2O); H-NMR (DMSO-d6) δ 12.647 (s, 1H, COOH),
8.475 (d, J= 7.8 Hz, 1H, -CONH-), 8.109 (s, 3H, NH3), 7.847
In vitro HL-60 and SKOV3 cells viability assay
e HL-60 and SKOV3 Cells were grown in RPMI1640
medium with 10% fetal bovine serum at 37°C in a
1
© 2012 Informa UK, Ltd.

306 K. Yang et al.
5% CO2 humidified incubator. Cell proliferation was
determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-
2.5-diphenyl-2H-tetrazolium bromide) assay. Briefly,
10,000 HL-60 cells (or 5,000 SKOV3 cells) per well
were plated in a 96-well plate, cultured for 4 h in com-
plete growth medium, then treated with 400 μg/mL,
200 μg/mL, 100 μg/mL, 50 μg/mL and 25 μg/mL of com-
pounds for 48 h, respectively. An aliquot of 0.5% MTT
(10 μL/well) was then added to each well. After further
incubation for 4 h, the formazan formed from the MTT
was extracted by adding DMSO and mixing for 10 min.
e optical absorbance was read with an micoplate
reader at 550 nm, and the IC50 values were calculated
according to a regression analysis of the concentration/
inhibition data.
Results and discussion
Chemistry
L-leucine (1a) and L-phenylalanine (1b) were used as
the starting materials to yield their methyl esters 2a and
2b, and then protected by (Boc)2O to obtain 3a and 3b.
e carboxylic acids of 3a–3b were protected using ben-
zyl chloride, and then the Boc group was deprotected
with 3 N HCl in ethyl acetate to give 4a and 4b. N-Boc-
protected amino acids (3a and 3b) were coupled with
Scheme1. Reagents and conditions: a. CH3OH, HCl; b. (Boc)2O, CH3OH, NaOH; c. i) K2CO3, KI, TBAI, benzyl chloride, CH3COCH3; ii) HCl/
anhydrous EtOAc; d. EDCI, HOBt/anhydrous, DCM, 2a~2b,4a~4b, 0°C to room temperature; e. HCl/anhydrous EtOAc.
Scheme2. Reagents and conditions: a. (Boc)2O, CH3OH, NaOH; b. EDCI, HOBt/anhydrous, DCM, 2a~2b,4a~4b,6a~6d, 0°C to room
temperature; c. HCl/anhydrous EtOAc; d. i) 15% Pd/C, CH3OH. ii) HCl/anhydrous EtOAc.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors 307
Table 1. e structures and IC50 values of target compounds against APN and MMP-2.
Compound
R1
R2
APN/IC50a (μM)
MMP/IC50a (μM)
11a
-CH3
301.9 1.3
>1000
11b
11c
CH3
-Bn
229.2 0.6
99.5 2.1
681.7 2.8
328.5 1.9
11d
12a
-Bn
-H
195.1 0.7
249.1 0.9
>1000
710.3 2.3
12b
-H
199.2 3.1
364.2 4.0
Compound
R3
R4
R5
APN /IC50 (μM)
MMP/IC50 (μM)
13a
-CH3
139.6 0.5
65.1 0.7
69.1 1.4
32.2 2.3
20.9 0.8
12.2 1.6
125.6 2.4
13b
13c
13d
14a
14b
-Bn
-CH3
-Bn
-H
225.4 1.1
236..6 3.3
333.7 2.7
115.0 0.9
203.4 2.9
-H
Bestatin
7.3 0.2
167 2.8
a. Each value represents the mean of three experiments, standard deviation is given.
© 2012 Informa UK, Ltd.

308 K. Yang et al.
intermediates (2a, 2b, 4a and 4b) by a classical EDCI/
HOBt method, and then converted into 6a–6d by depro-
tecting the Boc group (Scheme 1). Using the same proce-
dure, Boc-protected L-isoserine 8 was coupled with the
intermediate (2a, 2b, 4a and 4b) and 6a–6d to yield the
dipeptide 9a–9d and tripeptide 10a–10d containing pro-
tecting group. Finally, target compounds 11a–11d and
12a–12d were obtained by easily cleaving Boc, while the
target compounds 13a–13b and 14a–14b were obtained
from 9c–9d and 10c–10d by first reacting in a hydrogen
atmosphere with 15% Pd/C and then deprotecting Boc
(Scheme 2).
MTT assay due to APN being over-expressed. In addi-
tion, the human ovarian carcinoma cell line (SKOV3)
was chosen as an additional model for MTT assays.
According to the results (Table 2), all the test com-
pounds showed cytostatic activity except for compound
13d. It should also be pointed out that compound 14b
not only displays the most potent inhibition on APN,
but also has a higher antiproliferative activity than
bestatin.
In order to determine the interaction between our tar-
get compounds with APN, the most potent compound,
14b, was constructed with a Sybyl/Sketch module and
optimised using the Tripos force field. e docking study
performed with the Sybyl/FlexX module was based on
the active site of the APN co-crystal structure with besta-
tin (PDB code: 2DQM). e docking results showed that
the carbonyl group and hydroxyl group of compound
14b could chelate with the zinc ion in APN (Figures 1
and 2). e terminal amino group of 14b could form a
hydrogen bond with Glu264 and the carboxylic group of
14b is able to interact with Arg825. In addition, the pheny-
lalanine residue of 14b can insert an S1′ pocket which is
surrounded by Gly261, Ala262 and Glu298, while the leucine
residue in 14b interacts with the S2′ pocket containing
In vitro APN and MMP assays
All the target compounds were tested for their inhibi-
tory potential against APN and MMP-2 and the results
are shown in Table 1. e matrix metalloproteinase-2
(MMP-2) also belongs to the zinc-dependent metallo-
proteinase family and is associated with tumour invasion
and metastasis. ese inhibitory studies were performed
on both APN and MMP-2 so as to identify the selectivity
of the target compounds.
From the results of the enzyme assay, compound
14b (IC50 of 12.2 μM) is the most potent APN inhibitor
of all the target compounds, and showed an inhibi-
tory activity similar to that of bestatin (IC50 of 7.3 μM).
In addition, the dipeptides derivatives (11a, 11b, 11c,
11d, 12a and 12b) showed less inhibition than their
tripeptide analogues (13a, 13b, 13c, 13d, 14a and 14b).
is phenomenon suggests that the third amino acid
residue helps to increase the interaction with the APN
binding site. One the other hand, the tripeptide com-
pounds (13c, 13d and 14b) with a scaffold of isoser-L-
Phe-L-Leu exhibited a better inhibitory activity than
that of the compounds (13a, 13b and 14a) with an isos-
er-L-Leu-L-Phe scaffold. As for the compounds (13c,
13d and 14b) with a substituted R5, the hydrogen seems
to be the best substitution for this position. A possible
reason may be that the free terminal carboxylate group
could enhance the hydrogen bond interaction with the
active sites of APN.
Met263 and Tyr376
.
Table2. In vitro antiproliferative inhibiting potency of
compounds 11c, 13c, 13d and 14b.
Compound
11c
13c
HL-60 IC50a (mM)
0.33 0.06
0.31 0.05
>1000
SKOV3 IC50a (mM)
0.28 0.03
0.07 0.02
>1000
13d
14b
Bestatin
0.17 0.03
0.87 0.07
0.38 0.07
1.98 0.09
Each value represents the mean values with S.E values for 5
independent experiments.
In our previous studies, most of the compounds tested
showed better inhibition on MMP-2 than APN [18].
However, most of these L-isoserine dipeptides and trip-
eptides derivatives exhibited a better inhibitory activity
on APN than MMP-2. is result may be due to the dif-
ferences between the 3D structure of the active sites of
APN and MMP-2. According to the binding site of APN
(PDB code: 1HS6) and MMP-2 (PDB code: 1HOV, 1CK7,
1QIB), the active site of APN was much deeper than the
active site of MMP-2 [17]. e scaffold of our L-isoserine
derivatives are linear structures, and therefore there is
easier access to the APN active site.
Antiproliferative activity assays on HL-60 and
SKOV3 cells
e human promyelocytic leukaemia cell line (HL-60)
was used to evaluate antiproliferative activity with the
Figure1. e docking result for 14b with the active site of APN
(PDB: 2DQM).
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors 309
Figure2. e docking results for 14b with APN showed by LIGPLOT.
conclusion
Declaration of interest
In summary, we have described the synthesis and per-
formed a SAR study for a novel series of L-isoserine
derivatives as inhibitors of APN. Most of these com-
pounds possessed potent activity toward APN. Among
them, compound 14b exhibited a potent inhibitory
activity and a significant selectivity for APN over MMP-2.
Furthermore, this compound also showed good cell-
based inhibitory activity, and could be used as a poten-
tial lead compound for new anti-cancer agents in the
future.
is work was supported by National Natural Foundation
Research Grant (Grant No. 9071304; No 30772654) and
National High Technology Research and Development
Program of China (863 proJect; Grant No 2007AA02Z314)
and Doctoral Foundation of Ministry of Education of the
People’s Republic of China (Grant No 20060422029).
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