Synthesis and bioactivity of a Goralatide analog with antileukemic activity

Article abstract of DOI:10.1016/j.bmc.2015.04.061

Natural tetrapeptide Goralatide (AcSDKP) is a selective inhibitor of primitive haematopoietic cell proliferation. It is not stable in vivo and decomposes within 4.5 min when applied to live cells. In this work we developed an analog of Goralatide that exhibits cytotoxicity towards human myeloid HL-60, HEL, Nalm-6 leukemia cells, endothelial HUVEC, glioblastoma U251 and transformed kidney 293T cells. The Goralatide analog showed significant stability in organic solution with no tendency to degrade oxidatively.

Full text of DOI:10.1016/j.bmc.2015.04.061

Bioorganic & Medicinal Chemistry 23 (2015) 5056–5060
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and bioactivity of a Goralatide analog with antileukemic
activity
Zhiliang Li a, Iryna O. Lebedyeva a,b, Vita M. Golubovskaya c, William G. Cance c, Khalid A. Alamry d,
Hassan M. Faidallah d, C. Dennis Hall a, , Alan R. Katritzky a,
⇑
a Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, United States
b Department of Chemistry and Physics, Georgia Regents University, 1120 15th Street SCI W3005, Augusta, GA 30912, United States
c Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263, United States
d Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural tetrapeptide Goralatide (AcSDKP) is a selective inhibitor of primitive haematopoietic cell prolif-
eration. It is not stable in vivo and decomposes within 4.5 min when applied to live cells. In this work we
developed an analog of Goralatide that exhibits cytotoxicity towards human myeloid HL-60, HEL, Nalm-6
leukemia cells, endothelial HUVEC, glioblastoma U251 and transformed kidney 293T cells. The Goralatide
analog showed significant stability in organic solution with no tendency to degrade oxidatively.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 2 February 2015
Revised 13 April 2015
Accepted 21 April 2015
Available online 14 May 2015
Keywords:
Anticancer
Antileukemic
Goralatide
Peptide
Peptidomimetic
1. Introduction
are stable in the blood18–22 and inhibit primitive hematopoietic cell
proliferation.17 Gaudron et al. synthesized a series of AcSDKP
Goralatide (AcSDKP) (1a, Fig. 1) first isolated from fetal calf
bone marrow,1 shows selective protection of human hematopoietic
progenitor cells during chemotherapy.2 In vivo, Goralatide pre-
vents murine hematopoietic stem cells and progenitors from
entering into S-phase following administration of lethal doses of
anti-cancer drugs3 or irradiation.4,5 High levels of AcSDKP 1a are
markers of thymosin b4 gene overexpression found in various leu-
kemic cell samples.6 Goralatide is a natural and specific substrate
of the N-terminus active site of human angiotensin-converting
enzyme.7–10 New properties have recently been added to the
Goralatide bioactivity profile that enhance the myelopoietic
response to granulocyte-macrophage colony-stimulating factor
(GM-CSF),11 protect stem cells from hyperthermic damage,12 and
block doxorubicin-induced toxicity in vivo.2 Goralatide also pre-
vents skin and hair aging,13 stimulates angiogenesis14 and shows
anti-inflammatory activity.15,16
peptidomimetic analogs such as AcSer
W
(CH2-NH)Asp-LysPro,
(CH2-N)Pro, the
Ac-Ser-Asp (CH2NH)-LysPro, Ac-Ser-Asp-Lys
W
W
17,18
C-terminus modified tetrapeptide Ac-Ser-Asp-Lys-Pro-NH2
(1b, Fig. 1) and the chirally distinct mimetic AcSDDKP.17 These
peptidomimetics reduce in vitro the proportion of murine col-
ony-forming units granulocyte/macrophage in S-phase and inhibit
entry into the cycle of high proliferative colony-forming cells.17
They also show resistance to an angiotensin-converting enzyme
which prevents rapid elimination of peptidomimetics from
plasma.19 Thierry et al. synthesized a number of AcSDKP analogs
(NAcSD, NAcSEKP, NAcADKP, NAcSDbKP) and molecular fragments
(SDK, SD, DKP, DK, SDKP) to identify the minimal sequence that is
responsible for the biological activity of Goralatide. Synthetic ana-
logs and di-, or tri-peptide sequences were then studied for cellular
interactions between T-cell and erythrocytes in Rosette
Formation.20 The same group reported syntheses of several R sub-
stituted AcSDKP analogs, where R = Boc, Fmoc, Ant, Suc, coumarin
(1d–h, Fig. 1).
Despite a wide range of possible bioapplications, Goralatide 1a
(Fig. 1) has an extremely short half-life of 4.5 min in plasma.17,18
Consequently AcSDKP-derived analogs have been designed that
All of these analogs retained the antiproliferative activity and
toxicity of Goralatide. The change of
-Lys for -Arg in the original molecule shows that polar groups
in the molecule are critical for the expression of biological
L-Ser for homo-L-Ser (1c) and
⇑
L
L
Corresponding author. Tel.: +1 (352) 392 0554; fax: +1 (352) 392 9199.
E-mail address: charlesdennishall@gmail.com (C. Dennis Hall).
Deceased author.
http://dx.doi.org/10.1016/j.bmc.2015.04.061
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 5056–5060
5057
NH2
n1
O
O
O
OH
O
n
H
O
NH2
NH
R
N
N
SOCl2, BtH
DCM
r.t. 24 h
N
H
N
H
t-BuO
t-BuO
OH
t-BuO
Ot-Bu
OH
R1
O
O
COOH
A
O
O
H
N
3
2
: R = Fmoc, R1 = OH, n = 1, n1 = 4
: R = Acetyl, R1 = OH, n = 1, n1 = 4
e
1a
1
N
OH
BtH
=
b: R = Acetyl , R1 = NH2, n = 1, n1 = 4
c: R = Acetyl, R1 = OH, n = 2, n1 = 4
d: R = Boc, R1 = OH, n = 1, n1 = 4
f
: R = Ant, R = OH, n = 1, n1 = 4
O
N
: R = Suc, R1 = OH, n = 1, n1 = 4
O
g
O
h: R = Coumarin, R1 = OH, n = 1, n1 = 4
NH
O
H
NH
O
NH2
N
5
t-BuO
Bt
OH
Ot-Bu
Figure 1. Reported synthetic structural analogs of Goralatide.
Et3N, CH3CN
O
O
4
6
O
activity.21 Furthermore, coumarin-SDKP (1h, Fig. 1), allows photo-
chemical determination of angiotensin enzyme activity in
plasma.22
Scheme 1. Synthesis of an O- and N-protected L-Ser-L-AspOH 6.
Section 4. The inhibitory activity of 11 on HL-60 cells was also
observed by MTT assay in Nalm-6 and HEL cell lines.
2. Results and discussion
Next, we tested the effect of 11 on the viability of endothelial
HUVEC cells. It significantly decreased viability of HUVEC cells in
a dose-dependent manner (Fig. 5). Tetrapeptide 11 was incubated
at different doses with HUVEC cells for 24 h to perform MTT assay
*p<0.05, Students’ t-test.
We ran analogous studies on Goralatide 1a and it had no inhi-
bitive effect on the cancer cell lines employed in the study
(Fig. S2, SI). These results are in good agreement with previous
reports9,10 on selectivity towards inhibition of hematopoietic stem
cells and effects on cancer cells.
In this work we synthesized an analog of natural tetrapeptide
Goralatide23 (1a) which showed significant antileukemic activity
and stability in a number of organic solvents (DMSO, MeOH,
CDCl3). The activity of Goralatide analog 11 was studied on human
myeloid HL-60, HEL and Nalm-6 leukemia cells, U251 glioblastoma
astrocytoma, 293T kidney and HUVEC endothelial cells.
2.1. Chemistry
N- and O-protected tetrapeptide 11 was synthesized in six
Goralatide 1a showed decomposition within 12 h in organic sol-
vents (DMSO, CDCl3, MeOH) which complies with the data on the
low stability of NAcSDKP in vivo caused by a rapid Asp-Lys cleav-
age.2 Tetrapeptide 11 showed no signs of decomposition after
being treated with such solvents as DMSO, MeOH, CDCl3 for 7 days.
steps. First, the amino group of O-(tert-butyl)-
lated to give 3 in 88% yield. The carboxy group of N-acetyl-O-
(tert-butyl)- -serine 3 was then activated with benzotriazole to
L-serine 2 was acy-
L
afford benzotriazolide 4 (85%). Subsequent reaction with mono-
O-protected aspartic acid 5 gave dipeptide 6 (64%, Scheme 1).
Intermediate dipeptide 6 was then used as follows to construct
the core of protected Goralatide tetrapeptide precursor 11
(Scheme 2).
3. Conclusion
Coupling of N,N0-diprotected
L-lysine 7 with benzyl L-prolinate 8
In summary, a stable Goralatide (AcSDKP) analog has been syn-
thesized and its anticancer activity studied. In vitro cytotoxicity
assays demonstrated that N-, O-deprotected AcSDKP analog 11
effectively kills a range of cancer cells. It showed highest cytotox-
icity towards glioblastoma U251 and kidney 293T cells. It was also
effective against HL-60, Nalm-6 and HEL leukemia cells and HUVEC
gave dipeptide benzyl ester 9 in 75% yield. After the Boc group of 9
was removed, trifluoroacetate dipeptide 10 was obtained and was
subsequently reacted with dipeptide 6 (HOBt/EDCI, rt 12 h, 80%) to
give O- and N-protected NAcSDKP precursor 11 which, after two
deprotection steps, gave final product 1a.
endothelial cells at a concentration of 50
leukemia cells at a concentration of 20
l
M and towards Nalm-6
First, the benzyl ester of L-proline and Cbz protection of L-lysine
lM. The high anticancer
were removed by Pd/C 10% wt under H2. Intermediate 12 showed
low stability in organic solvents and therefore acidolysis of 12 was
performed rapidly after filtering and drying the reaction mixture to
afford 1a23 (Scheme 2).
activity profile of 11 suggests that it could be developed into a
novel anti-cancer and anti-leukemic drug. The molecular mecha-
nisms responsible for the antileukemic activity of 11 are currently
under investigation and further novel Goralatide analogs with
antileukemic mode of action are also being developed.
2.2. Evaluation of cytotoxicity in cell lines
In order to explore the bioactivity potential of Goralatide analog
11, we tested it on the viability of human leukemia cells HL-60,
HEL, Nalm-6 and HUVEC endothelial cells. The effect of the O, N-
protected tetrapeptide 11 on survival of glioblastoma U251 and
kidney 293T cells by clonogenicity assay was also studied and it
was found that 11 decreased the clonogenicity of U251 and 293T
cancer cells (Fig. S1, SI).
To test the effect of 11 on cell viability, we performed MTT assay
on HL-60 cancer cells (Fig. 2). Tetrapeptide 11 showed a significant
decrease in the survival of leukemia cells, compared to the DMSO
control.
4. Experimental section
4.1. Materials and methods
All reagents were purchased from commercial sources and used
as received unless otherwise indicated. The products were purified
by column chromatography on silica gel (300–400 mesh). Melting
points were determined on a capillary point apparatus equipped
with a digital thermometer. NMR spectra were recorded in CDCl3,
DMSO-d6 or CD3OD on Mercury or Gemini NMR spectrometers
operating at 300 MHz for 1H (with TMS as an internal standard)
and 75 MHz for 13C. Elemental analyses were performed on a
Carlo Erba-EA1108 instrument. High Resolution Mass Spectra were
recorded using Thermo Scientific LCQ Ion Trap. For biological eval-
uation Goralatide analog 11 was dissolved in DMSO at 25 mM and
kept at ꢀ20 °C.
An analogous toxicity effect for 11 was observed by a viability
assay on Nalm-6 cells (Fig. 3) and HEL cells (Fig. 4).
Tetrapeptide 11 decreases HL-60 viability in a dose-dependent
manner. The HL-60 cells were treated with different doses of 11
for 24 h and
a MTT assay was performed as described in

5058
Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 5056–5060
O
Cbz
HN
HO
CF3COOH-
H3N
O
O
N
OBn
OBn
O
OBn
HCl
O
HOBt/EDCI
r.t., o/n
H
N
N
TFA
4
HN
Boc
+
DCM
Boc
DCM
4
HN
Cbz
N
H
4 NH
Cbz
O
8
10
7
9
Cbz
NH
O
O
HOBt/EDCI
r.t., o/n
NH
O
NH
O
H
N
4
H
N
Pd/C, H2
MeOH
10
+
t-BuO
t-BuO
N
O
OH
N
H
O
DCM
O
Ot-Bu
O
OBn
O
6
O
11
Ot-Bu
O
O
NH3
CF3COO-
OH
NH2
O
NH
NH
O
4
H
N
4
H
N
N
N
t-BuO
TFA
HO
N
H
O
N
H
OH
DCM
O
OH
O
O
O
O
O
Ot-Bu
O
AcSDKP 1a
12
Scheme 2. Synthesis of N-, O-protected bioactive AcDKP precursor 11 and AcSDKP 1a.
1H), 6.54 (d, J = 7.8 Hz, 1H), 4.72 (ddd, J = 7.8 Hz, 4.1 Hz, 3.2 Hz,
1H), 3.89 (dd, J = 9.2, 3.2 Hz, 1H), 3.58 (dd, J = 9.2, 4.1 Hz, 1H),
2.08 (s, 3H), 1.17 (s, 9H). 13C NMR (75 MHz, CDCl3): d 173.7,
171.1, 74.2, 61.6, 52.8, 27.4, 23.1. Anal. Calcd for C9H17NO4: C,
53.19; H, 8.43; N, 6.89. Found: C, 53.44; H, 8.56; N, 7.01.24
4.2.2. Synthesis of (S)-N-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-3-
(tert-butoxy)-1-oxopropan-2-yl)acetamide (4)
Thionyl chloride (0.45 mL) was added to a stirred solution of
benzotriazole (BtH) (2.35 g, 19.8 mmol) in DCM (30 mL) at 25 °C,
and the mixture was stirred 10 min. N-Acetyl-O-(tert-butyl)-L-ser-
ine 3 (1.0 g, 4.93 mmol) was then added and after 3 h the precipi-
tate was filtered off and filtrate was washed with aqueous Na2CO3
(2 ꢁ 200 mL), dried over MgSO4, and concentrated to afford 4 as
yellow oil (1.3 g, 85%, 4.20 mmol). 1H NMR (300 MHz, CDCl3): d
8.27 (d, J = 8.1 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.69 (t, J = 8.1 Hz,
1H), 7.53 (t, J = 8.1 Hz, 1H), 6.62 (d, J = 7.8 Hz, 1H), 6.05 (dt,
J = 8.1 Hz, 3.3 Hz 1H), 4.22 (dd, J = 9.6 Hz, 3.3 Hz, 1H), 3.87 (dd,
J = 9.6 Hz, 3.3 Hz, 1H), 2.15 (s, 3H), 1.01 (s, 9H). 13C NMR
(75 MHz, CDCl3): d 170.4, 169.3, 146.0, 131.4, 130.9, 126.6, 120.5,
114.5, 74.2, 62.9, 54.4, 27.3, 23.3. Anal. Calcd for C15H20N4O3: C,
59.20; H, 6.62; N, 18.41. Found: C, 59.44; H, 6.70; N, 18.81.
Figure 2. The effect of tetrapeptide 11 in the viability of HL-60 leukemia cells. Bars
show average and standard deviations of three independent experiments. *p<0.05
versus untreated and DMSO treated, Student’s t-test.
4.2. General synthetic procedures
4.2.1. Synthesis of N-acetyl-O-(tert-butyl)-
Acetic anhydride (0.8 mL) was added to a stirred suspension of
O-tert-butyl- -serine (1.0 g, 6.2 mmol) in THF (25 mL) and water
L-serine (3)
L
(4 mL) over 15 min at 20 °C; stirring was continued for 24 h. The
solvent was evaporated from the reaction mixture at 50 °C, toluene
(50 mL) was added and then evaporated. The crude residue was
dissolved in dichloromethane (DCM), and then the solution was
concentrated to afford 3 as a white solid (88%, 1.1 g, 5.5 mmol);
mp 158.0–160.0 °C. 1H NMR (300 MHz, CDCl3): d 9.80–9.50 (br s,
4.2.3. Synthesis of (S)-2-((S)-2-acetamido-3-(tert-butoxy)propan-
amido)-4-(tert-butoxy)-4-oxobutanoic acid (6)
A mixture of (S)-N-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(tert-
butoxy)-1-oxopropan-2-yl)acetamide 4 (1.0 g, 3.29 mmol), (S)-2-
Figure 3. The effect of tetrapeptide 11 on viability of Nalm-6 cells.
Figure 4. The effect of tetrapeptide 11 on viability of HEL cells.

Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 5056–5060
5059
135.6, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 79.7, 67.0, 66.5,
58.9, 51.6, 47.0, 40.6, 32.3, 29.2, 29.0, 28.4, 25.0, 21.8. Anal. Calcd
for C31H41N3O7: C, 65.59; H, 7.28; N, 7.40. Found: C, 65.66; H,
7.63; N, 7.23.19
4.2.6. Benzyl N6-((benzyloxy)carbonyl)-
L-lysyl-L-prolinate
trifluoroacetate (10)
TFA (15 mL) was added to a solution of benzyl N0-((benzyloxy)-
carbonyl)-N-(tert-butoxycarbonyl)-L-lysyl-L-prolinate 9 in DCM
(15 mL) at rt, and the mixture was stirred for 3 h. It was then con-
centrated in vacuum to give 10 (0.4 g, 85%) as yellow oil. 1H NMR
(300 MHz, CD3OD): d 7.41–7.21 (m, 10H), 5.25–5.01 (m, 4H), 4.57
(q, J = 4.6 Hz, 1H), 4.19 (t, J = 6.2 Hz, 1H), 3.10 (t, J = 6.3 Hz, 2H),
2.42–2.16 (m, 1H), 2.12–1.90 (m, 3H), 1.90–1.70 (m, 2H), 1.54–
1.32 (m, 4H). 13C NMR (75 MHz, CD3OD): d 173.0, 169.1, 158.9,
138.4, 137.1, 129.7, 129.6, 129.5, 129.4, 129.1, 128.8, 68.1, 67.4,
60.7, 52.9, 48.4, 41.2, 38.4, 31.2, 30.4, 30.0, 26.0, 22.4. Anal. Calcd
for C28H34F3N3O7: C, 57.83; H, 5.89; N, 7.23. Found: C, 57.99; H,
6.01; N, 7.43.19
Figure 5. The effect of tetrapeptide 11 on viability of HUVEC cells by MTT assay.
amino-4-(tert-butoxy)-4-oxobutanoic acid 5 (0.65 g, 3.44 mmol)
and Et3N (0.35 g, 3.5 mmol) in CH3CN/H2O (3:1, 20 mL) was stirred
at rt for 48 h. The solvent was removed under reduced pressure,
the residue was dissolved in DCM, washed with 4 N HCl
(3 ꢁ 100 mL) and brine (3 ꢁ 100 mL), dried over MgSO4 and con-
centrated to give 6 as yellow solid (64%, 0.78 g, 2.2 mmol); mp
112.0–114.0 °C. 1H NMR (300 MHz, CDCl3): d 7.80 (d, J = 7.5 Hz,
1H), 6.63 (d, J = 6 Hz, 1H), 4.82–4.75 (m, 1H), 4.55–4.43 (m, 1H),
3.77 (q, J = 4.3 Hz, 1H), 3.39 (t, J = 8.4 Hz, 1H), 3.02–2.88 (m, 1H),
2.73 (dd, J = 17.1, 5.0 Hz, 1H), 2.04 (s, 3H), 1.43 (s, 9H), 1.22 (s,
9H). 13C NMR (75 MHz, CDCl3): d 173.6, 171.0, 170.8, 170.2, 81.8,
74.5, 61.4, 53.0, 49.0, 37.2, 28.0, 27.2, 23.0. Anal. Calcd for
4.2.7. Benzyl N2-((S)-2-((S)-2-acetamido-3-(tert-butoxy)propan-
amido)-4-(tert-butoxy)-4-oxobutanoyl)-N6-(2-phenoxyacetyl)-
L
-lysyl-
A solution of benzyl N0-((benzyloxy)carbonyl)-
nate 10 (0.23 g, 0.5 mmol) and (S)-2-((S)-2-acetamido-3-(tert-
butoxy)propanamido)-4-(tert-butoxy)-4-oxobutanoic acid
L-prolinate (11)
L
-lysyl-L-proli-
6
(0.21 g, 0.55 mmol), HOBt (0.1 g, 0.74 mmol) and EDCI (0.15 g,
0.74 mmol) in DCM (50 mL) was stirred at rt o/n. Reaction mixture
was washed with 5% NaHCO3, 0.1 N HCl and brine, then dried over
anhydrous MgSO4 and concentrated. The crude oil was purified by
flash column chromatography (5–40% EtOAc in n-Hexane, then 5–
10% MeOH in DCM) to afford 11 as an yellow oil (0.33 g, 0.4 mmol,
80%). 1H NMR (300 MHz, CDCl3): d 7.8 (d, J = 8.4 Hz, 1H), 7.58 (td,
J = 21.3 Hz, 8.3 Hz, 1H), 7.30–7.10 (m, 10H), 6.65 (dd, J = 13.5 Hz,
6.6 Hz, 1H), 5.54 (dt, J = 18.0 Hz, 5.1 Hz, 1H), 5.01 (dd, J = 18.0 Hz,
5.1 Hz, 2H), 5.00 (s, 2H), 4.72–4.55 (m, 2H), 4.52–4.30 (m, 2H),
3.75–3.25 (m, 4H), 3.07 (s, 2H), 2.92–2.40 (m, 2H), 2.00–1.75 (m,
6H), 1.92–1.24 (m, 13H), 1.40–0.96 (m, 12H). 13C NMR (75 MHz,
CDCl3): d 171.7, 170.9, 170.5, 170.4, 169.9, 169.7, 156.4, 136.7,
135.4, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 81.2, 66.7, 66.2,
61.2, 60.2, 58.7, 53.3, 50.4, 49.4, 46.8, 40.3, 36.7, 31.4, 28.7, 27.8,
27.2, 24.7, 22.8, 21.5, 20.8. HRMS (ESI) for C43H61N5O11 [M+H]+:
calcd 824.4440, found 824.4435. Anal. Calcd for C43H61N5O11: C,
62.68; H, 7.46; N, 8.50. Found: C, 62.54; H, 7.32; N, 8.51.
C17H30N2O7: C, 54.53; H, 8.08; N, 7.48. Found: C, 54.32; H, 8.24;
N, 7.69.
4.2.4. (S)-2-((Benzyloxy)carbonyl)pyrrolidin-1-ium chloride (8)
Benzyl alcohol (70 mL, 651 mmol) was cooled to 0 °C under
nitrogen and 7.0 mL thionyl chloride (91.2 mmol) was added.
L-Proline (5.0 g, 43.4 mmol) was then added and the mixture was
stirred at 0 °C under nitrogen for 2 h. The mixture was warmed
to room temperature and stirring continued for 48 h. The reaction
mixture was then poured into 300 mL diethyl ether and stored at
ꢀ20 °C for 7 days. The precipitate formed was collected by filtra-
tion, washed with diethyl ether, and dried under vacuum to give
8 as white solid (9.88 g, 93% yield); mp 142.1–144.0 °C; lit: 143–
144 °C. 1H NMR (300 MHz, CDCl3): d 7.41–7.21 (m, 5H), 5.16 (s,
2H), 3.80 (dd, J = 3.83, 5.9 Hz, 1H), 3.15–3.01 (m, 1H), 3.00–2.82
(m, 1H), 2.42–2.21 (m, 1H), 2.13 (dd, J = 12.9 Hz, 7.5 Hz, 1H),
1.92–1.62 (m, 3H). 13C NMR (75 MHz, CDCl3): d 175.5, 136.0,
128.8, 128.5, 128.3, 66.9, 59.9, 47.2, 30.4, 25.6. Anal. Calcd for
4.2.8. Acetyl-L-seryl-L-aspartyl-L-lysyl-L-proline trifluoroacetate
(1a)
C
12H16ClNO2: C, 59.63; H, 6.67; N, 5.79. Found: C, 59.50; H, 6.86;
To a solution of benzyl N2-((S)-2-((S)-2-acetamido-3-(tert-bu-
toxy)propanamido)-4-(tert-butoxy)-4-oxobutanoyl)-N6-(2-phenoxy-
N, 5.64.25
acetyl)-L-lysyl-L-prolinate 11 (0.25 g, 0.5 mmol) in MeOH (10 mL)
4.2.5. Benzyl N6-((benzyloxy)carbonyl)-N2-(tert-butoxycarbonyl)-
10% Pd/C (0.03 g) was added and the resulting mixture was stirred
under H2. When TLC showed no starting materials remained, the
Pd/C was filtered on a pad of celite and the solution was then con-
centrated under reduced pressure to give a crude residue 12 which
was dissolved in DCM (5 mL) followed the addition of TFA (5 mL) at
0 °C. The reaction mixture was stirred at rt for 3 h, then concen-
trated under vacuum to give the 1a as a white solid (0.24 g,
0.47 mmol, 93%); mp 163.9–165.0 °C. The obtained data matched
those reported in literature.23
L
-lysyl-
solution of N0-((benzyloxy)carbonyl)-N-(tert-butoxycar-
bonyl)- -lysine 7 (0.76 g, 2 mmol), benzyl -prolinate 8 (0.345 g,
L-prolinate (9)
A
L
L
2 mmol), HOBt (0.3 g, 2.2 mmol) and EDCI (0.47 g, 2.4 mmol) was
stirred at rt for 12 h in DCM (50 mL). The reaction mixture was
washed with 5% NaHCO3, 0.1 N H2SO4 and brine, dried over anhy-
drous MgSO4 and concentrated. The crude oil was purified by flash
column chromatography (5–40% EtOAc in n-hexane) to afford the
desired product as a colorless oil (0.8 g, 75%). 1H NMR (300 MHz,
CDCl3):
d
7.31 (s, 11H), 5.33 (d, J = 8.4 Hz, 1H), 5.19 (d,
4.3. Cell lines
J = 10.8 Hz, 1H), 5.13 (s, 1H), 5.09–5.00 (m, 2H), 4.58 (t, J = 4.8 Hz,
1H), 4.43 (dd, J = 13.1 Hz, 7.4 Hz, 1H), 3.70 (t, J = 6.3 Hz, 1H),
3.62–3.49 (m, 1H), 3.25–3.01 (m, 2H), 2.28–2.11 (m, 1H), 2.02–
1.82 (m, 3H), 1.78–1.64 (m, 3H), 1.61–1.46 (m, 2H), 1.41 (s, 12H).
13C NMR (75 MHz, CDCl3): d 170.2, 171.2, 156.6, 155.6, 136.8,
Human myeloid HL-60 leukemia cell line was obtained from
ATCC and maintained in RPMI medium, with addition of 10% FBS,
1
lg/ml penicillin/streptomycin, 1% dextrose, 1% HEPES, 1% Na
pyruvate. NAlm-6 and HEL-6 were obtained from Dr. Jian Liao,

5060
Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 5056–5060
Roswell Park Cancer Center. Human endothelial HUVEC cell line
was obtained from ATCC and maintained in Medium 200 with
low serum growth supplement. Human glioblastoma U251 and
embryonic kidney SV40 transformed 293T cell lines were obtained
from ATCC and maintained according to manufacturer’s protocol.
References and notes
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tions. DMSO was added as
a negative control. The 3-(4,
5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium compound from Promega 96 well Viability
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microplate reader. All experiments were performed in triplicate.
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We thank the University of Florida for the support of this pro-
ject. This project was also supported by the NSTIP Strategic
Technologies Program in the Kingdom of Saudi Arabia-Project No.
12-ADV2732-03. The authors also, acknowledge with thanks
Science and Technology Unit, King Abdulaziz University for techni-
cal support. The authors are grateful to Dr. Rachel A. Jones, Mr. Z.
Wang for helpful discussions and Dr. M.C.A. Dancel for HRMS
analyses.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.04.061.