Synthesis and bioactivity of a Goralatide analog with antileukemic activity

Bioorganic & Medicinal Chemistry 23 (2015) 5056–5060

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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 Department of Chemistry and Physics, Georgia Regents University, 1120 15th Street SCI W3005, Augusta, GA 30912, United States 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 b c

a r t i c l e

i n f o

Article history: Received 2 February 2015 Revised 13 April 2015 Accepted 21 April 2015 Available online 14 May 2015 Keywords: Anticancer Antileukemic Goralatide Peptide Peptidomimetic

a b s t r a c t 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. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction 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 prevents 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 leukemic 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 prevents skin and hair aging,13 stimulates angiogenesis14 and shows anti-inflammatory activity.15,16 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 ⇑ Corresponding author. Tel.: +1 (352) 392 0554; fax: +1 (352) 392 9199.  

E-mail address: [email protected] (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.

are stable in the blood18–22 and inhibit primitive hematopoietic cell proliferation.17 Gaudron et al. synthesized a series of AcSDKP peptidomimetic analogs such as AcSerW(CH2-NH)Asp-LysPro, Ac-Ser-AspW(CH2NH)-LysPro, Ac-Ser-Asp-LysW(CH2-N)Pro, the C-terminus modified tetrapeptide Ac-Ser-Asp-Lys-Pro-NH217,18 (1b, Fig. 1) and the chirally distinct mimetic AcSDDKP.17 These peptidomimetics reduce in vitro the proportion of murine colony-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 analogs 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 substituted AcSDKP analogs, where R = Boc, Fmoc, Ant, Suc, coumarin (1d–h, Fig. 1). All of these analogs retained the antiproliferative activity and toxicity of Goralatide. The change of L-Ser for homo-L-Ser (1c) and L-Lys for L-Arg in the original molecule shows that polar groups in the molecule are critical for the expression of biological

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R

N H

OH n H N O

O

NH 2 n1 N

N H O COOH

1a: R = Acetyl, R 1 = OH, n = 1, n1 = 4 b: R = Acetyl , R 1 = NH 2, n = 1, n 1 = 4 c: R = Acetyl, R1 = OH, n = 2, n1 = 4 d: R = Boc, R 1 = OH, n = 1, n 1 = 4

A

O NH2 t-BuO

OH

R1 2

1

e: R = Fmoc, R = OH, n = 1, n 1 = 4 f: R = Ant, R1 = OH, n = 1, n1 = 4 g: R = Suc, R 1 = OH, n = 1, n1 = 4 h: R = Coumarin, R 1 = OH, n = 1, n 1 = 4

Figure 1. Reported synthetic structural analogs of Goralatide.

O

O

O r.t. 24 h

NH t-BuO

O

3 OH

O

Ot-Bu

O NH t-BuO

NH2 O 5

Bt 4

O

OH

Et3 N, CH3 CN

SOCl2 , BtH DCM

O

H N N

BtH =

O

NH t-BuO 6

H N

N O OH Ot-Bu

O O

activity.21 Furthermore, coumarin-SDKP (1h, Fig. 1), allows photochemical determination of angiotensin enzyme activity in plasma.22 2. Results and discussion 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 steps. First, the amino group of O-(tert-butyl)-L-serine 2 was acylated to give 3 in 88% yield. The carboxy group of N-acetyl-O(tert-butyl)-L-serine 3 was then activated with benzotriazole to afford benzotriazolide 4 (85%). Subsequent reaction with monoO-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). Coupling of N,N0 -diprotected L-lysine 7 with benzyl L-prolinate 8 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. First, the benzyl ester of L-proline and Cbz protection of L-lysine 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). 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, Nprotected 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. 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

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. 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 inhibitive 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. Goralatide 1a showed decomposition within 12 h in organic solvents (DMSO, CDCl3, MeOH) which complies with the data on the low stability of NAcSDKP in vivo caused by a rapid Asp-Lys cleavage.2 Tetrapeptide 11 showed no signs of decomposition after being treated with such solvents as DMSO, MeOH, CDCl3 for 7 days. 3. Conclusion In summary, a stable Goralatide (AcSDKP) analog has been synthesized 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 cytotoxicity towards glioblastoma U251 and kidney 293T cells. It was also effective against HL-60, Nalm-6 and HEL leukemia cells and HUVEC endothelial cells at a concentration of 50 lM and towards Nalm-6 leukemia cells at a concentration of 20 lM. The high anticancer activity profile of 11 suggests that it could be developed into a novel anti-cancer and anti-leukemic drug. The molecular mechanisms 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. 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 evaluation Goralatide analog 11 was dissolved in DMSO at 25 mM and kept at 20 °C.

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Cbz

O

HN 4

HO O

N H

OBn

HN

Boc +

HCl

8

7

O HOBt/EDCI r.t., o/n DCM

H N

+ t-BuO

HOBt/EDCI r.t., o/n

O

H N

OH Ot-Bu

O 6

DCM

NH

O

t-BuO O

O

11

NH

4

N H O

N O

O

TFA OH

DCM

Ot-Bu 12

H N

HO O

4

N H O

O H N

NH

O

H N

O

NH 2

10

Cbz

t-BuO

O

4

Cbz

Cbz 9

OBn

N

HN

O NH

NH

H 3N

DCM

NH 4

O

CF3COOH- O TFA

N

Boc

O

10

OBn

O

Pd/C, H2

N O

O

MeOH OBn

Ot-Bu CF3COO -

NH3

O

4

N H OH

N O

O

OH

O AcSDKP 1a

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

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)-L-serine (3) Acetic anhydride (0.8 mL) was added to a stirred suspension of O-tert-butyl-L-serine (1.0 g, 6.2 mmol) in THF (25 mL) and water (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,

Figure 3. The effect of tetrapeptide 11 on viability of Nalm-6 cells.

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-serine 3 (1.0 g, 4.93 mmol) was then added and after 3 h the precipitate 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. 4.2.3. Synthesis of (S)-2-((S)-2-acetamido-3-(tert-butoxy)propanamido)-4-(tert-butoxy)-4-oxobutanoic acid (6) A mixture of (S)-N-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(tertbutoxy)-1-oxopropan-2-yl)acetamide 4 (1.0 g, 3.29 mmol), (S)-2-

Figure 4. The effect of tetrapeptide 11 on viability of HEL cells.

Z. Li et al. / Bioorg. Med. Chem. 23 (2015) 5056–5060

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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

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 concentrated 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 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 filtration, 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 C12H16ClNO2: C, 59.63; H, 6.67; N, 5.79. Found: C, 59.50; H, 6.86; N, 5.64.25 4.2.5. Benzyl N6-((benzyloxy)carbonyl)-N2-(tert-butoxycarbonyl)(9) A solution of N0 -((benzyloxy)carbonyl)-N-(tert-butoxycarbonyl)-L-lysine 7 (0.76 g, 2 mmol), benzyl L-prolinate 8 (0.345 g, 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 anhydrous 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, 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). 13 C NMR (75 MHz, CDCl3): d 170.2, 171.2, 156.6, 155.6, 136.8, L-lysyl-L-prolinate

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 concentrated 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 4.2.7. Benzyl N2-((S)-2-((S)-2-acetamido-3-(tert-butoxy)propanamido)-4-(tert-butoxy)-4-oxobutanoyl)-N6-(2-phenoxyacetyl)L-lysyl-L-prolinate (11) A solution of benzyl N0 -((benzyloxy)carbonyl)-L-lysyl-L-prolinate 10 (0.23 g, 0.5 mmol) and (S)-2-((S)-2-acetamido-3-(tertbutoxy)propanamido)-4-(tert-butoxy)-4-oxobutanoic acid 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. 4.2.8. Acetyl-L-seryl-L-aspartyl-L-lysyl-L-proline trifluoroacetate (1a) To a solution of benzyl N2-((S)-2-((S)-2-acetamido-3-(tert-butoxy)propanamido)-4-(tert-butoxy)-4-oxobutanoyl)-N6-(2-phenoxyacetyl)-L-lysyl-L-prolinate 11 (0.25 g, 0.5 mmol) in MeOH (10 mL) 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 concentrated 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 concentrated 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 4.3. Cell lines 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,

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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. 4.4. Viability assay Cells were treated with peptides for 24 h at different concentrations. DMSO was added as a negative control. The 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium compound from Promega 96 well Viability kit (Madison, IL) was added, and the cells were incubated at 37 °C for 1–2 h. To determine cell viability the optical density at 490 nm (OD 490 nm) of cells on a 96-well plate was analyzed using a microplate reader. All experiments were performed in triplicate. 4.5. Clonogenicity assay The 1000 cells were plated on 6 well plates and incubated with or without tested compound for 1–2 weeks. Then cells were fixed in 25% MeOH and stained with Crystal Violet. 4.6. Statistical analysis Student’s t-test was performed to determine significance. The difference between data with p <0.05 was considered significant. Acknowledgments We thank the University of Florida for the support of this project. 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 technical 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. 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.

References and notes 1. Lenfant, M.; Wdzieczak-Bakala, J.; Guittet, E.; Prome, J. C.; Sotty, D.; Frindel, E. Proc. Nat. Acad. Sci. U.S.A. 1989, 86, 779. 2. Masse, A.; Ramirez, L. H.; Bindoula, G.; Grillon, C.; Wdzieczak-Bakala, J.; Raddassi, K.; Deschamps de Paillette, E.; Mencia-Huerta, J. M.; Koscielny, P.; Potier, P.; Sainteny, F.; Carde, P. Blood 1998, 91, 441. 3. Aidouli, S.; Guignon, M.; Caen, J.; Han, Z. C. Br. J. Hematol. 1996, 94, 443. 4. Watanable, T.; Kelsey, L. S.; Yan, Y.; Brown, G. S.; Jackson, J. D.; Ewel, C.; Talmadge, J. E. Br. J. Haematol. 1996, 94, 619. 5. Jackson, J. D.; Ozerol, E.; Yan, Y.; Ewel, C.; Talmadge, J. E. J. J. Hematother. Stem Cell Res. 2000, 9, 489. 6. Liu, J.-M.; Garcia-Alvarez, M.-C.; Bignon, J.; Kusinski, M.; Kuzdak, K.; Riches, A.; Wdzieczak-Bakala, J. Ann. N.Y. Acad. Sci. 2006, 30, 514. 7. Rousseau, A.; Michaud, A.; Chauvet, M.-T.; Lenfant, M.; Corvol, P. J. Biol. Chem. 1995, 270, 3656. 8. Huang, W. Q.; Wang, B. H.; Wang, Q. R. Cell Biol. Int. 2006, 30, 514. 9. Bonnet, D.; Césaire, R.; Lemoine, F.; Aoudjhane, M.; Najman, A.; Guigon, M. Exp. Hematol. 1992, 20, 251. 10. Cashman, J. D.; Eaves, A. C.; Eaves, C. J. Blood 1994, 84, 1534. 11. Bogden, A. E.; Moreau, J.-P.; Gamba-Vitalo, C.; Deschamps de Paillette, E.; Tubiana, M.; Frindel, E.; Carde, P. Int. J. Cancer 1998, 76, 38. 12. Wierenga, P. K.; Konings, A. W. Exp. Hematol. 1996, 24, 246. 13. Najem, N.; Chapelle, A.; Bingon, J.; Pinault, A.; Liu, J. M.; Salah-Mohellibi, E.; Lati, J.; Wdzieczak-Bakala, J. Int. J. Cosmet. Sci. 2013, 35, 286. 14. Kanasaki, M.; Nagai, T.; Kitada, M.; Koya, D.; Kanasaki, K. Fibrogenesis Tissue Repair 2011, 4, 425. 15. Sharma, U.; Rhaleb, N.-E.; Pokharel, S.; Harding, P.; Rasoul, S.; Peng, H.; Carretero, O. A. Am. J. Physiol. Heart Circ. Physiol. 2008, 294, H1226. 16. González, G. E.; Rhaleb, N.-E.; Nakagawa, P.; Liao, T. D.; Liu, Y.; Leung, P.; Dai, X.; Yang, X.-P.; Carretero, O. A. Clin. Sci. 2014, 126, 85. 17. Gaurdon, S.; Grillon, C.; Thierry, J.; Riches, A.; Wierenga, P. K.; WdzieczakBakala, J. Stem Cells 1999, 17, 100. 18. Junot, C.; Theodoro, F.; Thierry, J.; Clement, G.; Wdzieczak-Bakala, J.; Ezan, E. J. Immunoassay Immunochem. 2001, 22, 15. 19. Gaudron, S.; Adeline, M.-T.; Potier, P.; Thierry, J. J. Med. Chem. 1997, 40, 3963. 20. Thierry, J.; Papet, M. P.; Saez-Servent, N.; Plissonneau-Haumont, J.; Potier, P.; Lenfant, M. J. Med. Chem. 1990, 33, 2122. 21. Thierry, J.; Grillon, C.; Gaudron, S.; Potier, P.; Riches, A.; Wdzieczak-Bakala, J. J. Pept. Sci. 2001, 7, 284. 22. Cheviron, N.; Rousseau-Plasse, A.; Lenfant, M.; Adeline, M. T.; Potier, P.; Thierry, J. Anal. Biochem. 2000, 280, 58. 23. Zikos, C.; Livaniou, E.; Leondiadis, L.; Ferderigos, N.; Ithakissios, D. S.; Evangelatos, G. P. J. Pept. Sci. 2003, 9, 419. 24. Kang, X.; Szallies, A.; Rawer, M.; Echner, H.; Duszenko, M. J. Cell Sci. 2002, 115, 2529. 25. Sellanes, D.; Campot, F.; Núñez, I.; Lin, G.; Espósito, P.; Dematteis, S.; Saldaña, J.; Domínguez, L.; Manta, E.; Serra, G. Tetrahedron 2010, 66, 5384.