Synthesis, drug release and biological evaluation of
new anticancer drug-bioconjugates containing
somatostatin backbone cyclic analog as a targeting
moiety
Redko B.,1,3 Ragozin E.,1 Bazylevich A.,1 Tuchinsky H.,2 Albeck A.,3 Shekhter Zahavi T.,4 OronHerman M.,5 Kostenich G.5 and Gellerman G.1*
1
Department of Biological Chemistry, Ariel University, Ariel, 40700, Israel
2
Department of Molecular Biology, Ariel University, Ariel, 40700, Israel
3
The Julius Spokojny Bioorganic Chemistry Laboratory, Department of Chemistry, Bar Ilan University, Ramat Gan
52900, Israel
4
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv
University, Tel-Aviv, 69978, Israel
5
The Advanced Technologies Center, Sheba Medical Center, Tel Hashomer 52621, Israel
Address for correspondence
Prof. Gary Gellerman,
Dept. Biological Chemistry,
Ariel University, Ariel 40700, Israel
Email: garyg@ariel.ac.il
General
All HPLC purifications were done via reverse phase on ECOM prep. system, with dual UV
detection at 254 nm and 230 nm. Phenomenex Gemini® 10 µm C18 110 Å, LC 250 x 21.2 mm
prep column was utilized. The column was kept at rt. Elution solvents were A (0.3% TFA in H2O)
and B (0.3% TFA in ACN). A typical elution was a gradient of 100% A to 100% B over 45 min at
a flow rate of 40 mL/min. Electron spray mass spectra (ESI-MS) were obtained using an Autoflex
III smart-beam (MALDI, Bruker), Q-TOF micro (Waters) or an LCQ FleetTM ion trap mass
spectrometer (Finnigan/Thermo). HPLC/LC-MS analyses were made using Agilent infinity 1260
connected to Agilent quadruple LC-MS 6120 series equipped with ZORBAX SB-C18, 2.1 x
50mm, 1.8µm column. In all cases the eluent solvents were A (0.1% FA in H2O) and B (0.1% FA
in ACN) and the elution gradient profile was: 100% A for first 4 min, followed by 8 min (from
min 4 to min 12) during which it reached 100% B, followed by 4 min (from min 12 to min 16) of
100% B, followed by two min (from min 16 to min 18) during which it returned back to A,
followed by 2 min (from min 18 to min 20) of 100% A. The UV detection was at 254 nm. The
column temperature was kept at 500C. The flow rate was of 0.4 ml /min. The MS fragmentor was
tuned on 100V on positive or negative mode.
Abbreviations
(NMP) -N- methylpyrollidone, (Fmoc)-Fluorenylmethoxy carbonyl, (PyBroP) -Bromo-trispyrrolidino-phosphonium
hexafluorophosphate,
(DIEA)-diisopropylethylamine,
(Rt)-room
temperature, (DCM)-Dichloromethane, (DMF)-N,N-Dimethylformamide, (TFA)-Trifluoroacetic
acid, (TIS)-Triisopropylsilane, (FITC)-Fluoresceinisothiocyanate, (Acm)-Acetamidomethyl
Synthesis of PTR-86 peptide
Materials. Rink amide MBHA resin, amino acids, FITC, TIS and PyBroP were purchased from
Tzamal D-Chem Laboratories Ltd. Petah-Tikva, Israel. DIEA, Et2O and TFA, DMF, NMP and
DCM were purchased from BioLab Ltd. (Israel).
Procedure
In a reaction vessel equipped with a sintered glass bottom, rink amide MBHA resin, (substitution
level 0.56mmol/g, 1g) was swelled in NMP by agitation overnight. The Fmoc group was removed
from the resin upon treatment with 20% piperidine in NMP ( 10ml) for 15 min. This action was
repeated twice. After washing the resin with NMP (7 times, 10 ml, 2 min each time), FmocGlyS2(Acm)-OH (3 eq, 10.5 mmol, 0.64 g) dissolved in NMP (7 ml) was activated with PyBroP
(3 eq, 10.5 mmol, 0.7 g) and DIEA (6 eq, 21 mmol, 0.521 ml) for 4 min at room temperature,
transferred to the reaction vessel and allowed to react for 1 h at rt. Following coupling, the peptidyl
resin was washed with NMP (5 times, 7 ml, 2 min each time). Completion of reaction was
monitored by ninhydrin test (Kaiser test, yellow).
Peptides were synthesized under standard Fmoc protocols, with 3 equivalents of each amino acid
and 3 equivalents of PyBrop as coupling reagent. The deblock mixture was a mixture of 80:20
DMF/piperidine (v/v).
Cyclization step
After coupling of Fmoc-Cys(Acm)-OH and NMP wash, the resin was washed with 4:1 DMF/water
(3 times, 6.5 ml, 2min each time). A solution of I2 (10eq, 35 mmol, 1.29 g) in 4:1 DMF/water (10
ml) was added to the peptidyl – resin followed by agitation at rt for 1h to afford the disulfide
bridge cyclization. The peptidyl – resin was filtered and washed extensively with 4:1 DMF/water
(7 times, 10 ml, 2 min each time), DMF ( 6 times, 10 ml, 2 min each time), DCM (6 times, 10 ml,
2 min each time), CHCl3 (4 times, 10 ml, 2 min each time), 2% ascorbic acid in DMF (6 times, 10
ml, 2 min each time) and last wash with DMF (6 times, 10 ml, 2 min each time). Finally, coupling
of last animo acid Fmoc-D-Phe-OH after cyclization, give cyclic peptide.
Coupling of Fmoc--aminobutyric acid (linker)
Fmoc--aminobutyric acid (3eq, 10.5mmol, 0.49 g) dissolved in NMP (7 ml) was activated with
PyBroP (3 eq, 10.5 mmol, 0.7 g) and DIEA (6 eq, 21 mmol, 0.521 ml) for 4 min at room
temperature, transferred to the reaction vessel and allowed to react for 1 h at rt. After, post
coupling wash and Fmoc-deprotection.
Cleavage of the peptide from the resin.
After coupling of linker and NMP wash, the resin was washed with DCM ( 4 times, 10 ml, 2 min
each time), MeOH ( 4 times, 10 ml, 2 min each time), DCM ( 4 times, 10 ml, 2 min each time)
and dried under vacuum for 20 min. The peptide was cleaved from the resin using cocktail solution
of 95:2.5:2.5 TFA/TIS/H2O (13 ml) for 5min at 00C under argon and then 1h at rt under argon. The
resin was filtered and washed with the cocktail (10 ml) and TFA (2 ml). The filtrate solution was
evaporated to give an oily residue. Upon the addition of cold Et2O, the oily residue solidified.
Centrifugation and decantation of the Et2O layer and repeated treatment with additional cold Et2O
afforded the crude as an orange solid which was purified by SP-HPLC.
The peptide was kept in dark at -200C under argon.
Synthesis of Fmoc-GlyS2(Acm)-OH building unit
Following building unit 4 was synthesized according procedure of Gellerman et al ., J. Peptide Res.
57, 2001/277-291, with minor modifications.
Acetamidomethyl – thioethylamine (2)
Cysteamine hydrochloride (15g, 0.132mol) was dissolved in 105ml of TFA. The solution was
stirred at rt for 30min and a solution of acetamidomethanol (12.3g, 0.138mol) in 30ml TFA was
then added. The stirring was continued for additional 2.5h. After evaporation most of the TFA,
600ml of distilled water were added. The solution was cooled by ice bath and brought to pH= 9
using concentrated NH4OH and then saturated with NaCl. The product was extracted with
CHCl3/isopropanol 3:1 (6 x 300ml). The organic layer was dried over Na2SO4, evaporated to
dryness and placed in vacuum desiccator overnight, yielding 8.82g (45%) of yellow oil (2). This
crude substance was used without purification in the next step.
Fmoc-GlyS2(Acm)-OH (4)
Acm-thioethylamine (8.45g, 0.057mol) and NaBH3CN (3.58g, 0.057mol) were dissolved in 115ml
MeOH. Glyoxilic acid monohydrate (4.42g, 0.048mol) was then added. The solution was stirred
overnight and the MeOH was evaporated. The oily residue was dissolved in 140ml of distilled
water and Et3N (14ml, 0.1mol) was added. To this mixture, a solution of Fmoc-OSu (12.3g, 0.0365
mol) in 215ml CH3CN was added. The mixture was stirred for 4h. Part of the CH3CN was
evaporated and the pH found to be 9. The solution was washed with petroleum ether (40-60) (3 x
250ml) followed by ether/petroleum ether (40-60) 7:3 (3 x 250ml). The solution was brought to
pH=3-4 using 2N HCl under cooling (an ice bath) and extracted with ethyl acetate (4 x 400ml).
The organic layer was washed by 1N HCl (3 x 145ml) and saturated KHSO4 solution (10 x
145ml), dried over Na2SO4 and evaporated to yield 5.6g Fmoc-GlyS2(Acm)-OH as a white solid
(23%) . m.p. 139 – 1450C, Mw = 428.51. ESI-MS: m/z calcd: 428.14 found: 429.2 (MH+).
Compound Comb A4-CO-OPh-(p)NO2
4-Nitrophenyl carbonochloridate (1.59 g, 7.89 mmol) was added dropwise to a solution of
Combretastatin A4 (1.66 g, 5.26 mmol), excess of Et3N and catalytic amount of DMAP in THF
(25 mL) at 0 °C. The cooling bath was removed, and the solution stirred at rt for 2 h. The solvents
were removed under reduced pressure. The solid was dissolved in EtOAc (100 mL) and washed
with saturated aqueous NH4Cl (3 ✕ 75 mL). The organic layer was dried over Na2SO4 and filtered.
The solvent was removed under reduced pressure and the crude product was purified by flash
chromatography, eluting with EtOAc : petroleum ether (20:80). Comb A4-CO-OPh-(p)NO2 was
obtained as a beige powder. Yield 82%. This crude substance was used without purification in the
next step. Progress of the reaction was monitored by LCMS.
ESI-MS: m/z calcd: 481.14 found: 482 (MH+).
Compound ABT-751-CO-OPh-(p)NO2
4-Nitrophenyl carbonochloridate (1.59 g, 7.89 mmol) was added dropwise to a solution of ABT751 (1.95 g, 5.26 mmol), excess of Et3N and catalytic amount of DMAP in THF (25 mL) at 0 °C.
The cooling bath was removed, and the solution stirred at rt for 2 h. The solvents were removed
under reduced pressure. The solid was dissolved in EtOAc (100 mL) and washed with saturated
aqueous NH4Cl (3 ✕ 75 mL). The organic layer was dried over Na2SO4 and filtered. The solvent
was removed under reduced pressure and the crude product was purified by flash chromatography,
eluting with EtOAc : petroleum ether (20:80). ABT-751-CO-OPh-(p)NO2 was obtained as a beige
powder. Yield 75%. This crude substance was used without purification in the next step. Progress
of the reaction was monitored by LCMS.
ESI-MS: m/z calcd: 536.10 found: 537.2 (MH+).
Compound CPT-CO-OPh-(p)NO2
4-Nitrophenyl carbonochloridate (1.59 g, 7.89 mmol) was added dropwise to a solution of CPT
(1.83 g, 5.26 mmol), excess of Et3N and catalytic amount of DMAP in THF (25 mL) at 0 °C. The
cooling bath was removed, and the solution stirred at rt for 2 h. The solvents were removed under
reduced pressure. The solid was dissolved in EtOAc (100 mL) and washed with saturated aqueous
NH4Cl (3 ✕ 75 mL). The organic layer was dried over Na2SO4 and filtered. The solvent was
removed under reduced pressure and the crude product was purified by flash chromatography,
eluting with EtOAc : petroleum ether (20:80). CPT-CO-OPh-(p)NO2 was obtained as a beige
powder. Yield 89%. This crude substance was used without purification in the next step. Progress
of the reaction was monitored by LCMS.
ESI-MS: m/z calcd: 513.12 found: 514.2 (MH+).
Compound AM-Glut
To solution of 10 ml of DCM and Amonofide ( 1 g, 3.5 mmol) was add glytaric anhydride (0.45 g,
3.88 mmol) and stirred at rt. After 3 hours, additional amount of glytaric anhydride (1.1 eq) was
added and left stirred overnight. The precipitate solids was collected and dried under vacuum to
give orange powder. Yield 78%. This crude substance was used without purification in the next
step. Progress of the reaction was monitored by LCMS.
ESI-MS: m/z calcd: 397.16 found: 398.2 (MH+).
Compound 1a
To PTR on rink amide resin with Fmoc-γ-aminobutyric acid treatment with 20% piperidine in
NMP (10ml) for 15 min. This action was repeated twice. After washing the resin with NMP (7
times, 10 ml, 2 min each time), CLB (3 eq) dissolved in NMP (7 ml) was activated with PyBroP (3
eq) and DIEA (6 eq) for 4 min at room temperature, transferred to the reaction vessel and allowed
to react for 1 h at rt. Following coupling, the peptidyl resin was washed with NMP (5 times, 7 ml,
2 min each time). Completion of reaction was monitored by ninhydrin test (Kaiser test, yellow).
Finally the conjugate was cleaved by treating the resin with TFA/H2O/ TIPS (95:2.5:2.5) cocktail
for 2 h. The solvents were removed under gentle nitrogen flow and the crude was precipitated out
from ether. The pellets were re-dissolved in ACN:H2O and purified via preparative HPLC column.
1a was obtained as white crystals. Yield 52%. HRMS: ESI-MS m/z calcd: 1649.6479, found:
1650.6483 (M+H), calcd: 1672.6409, found: 1672.6432 (M+Na).
Compound 1b
To PTR on rink amide resin with Fmoc-γ-aminobutyric acid, after Fmoc deprotection, was add 2
eq of CPT-CO-OPh-(p)NO2 and 4 eq of DIEA. This solution allowed to react for 3 h at rt.
Following coupling, the peptidyl resin was washed with NMP (5 times, 7 ml, 2 min each time).
Completion of reaction was monitored by ninhydrin test (Kaiser test, yellow). Finally the
conjugate was cleaved by treating the resin with TFA/H2O/ TIPS (95:2.5:2.5) cocktail for 2 h. The
solvents were removed under gentle nitrogen flow and the crude was precipitated out from ether.
The pellets were re-dissolved in ACN:H2O and purified via preparative HPLC column. 1b was
obtained as white crystals. Yield 42%. HRMS: m/z calcd: 1759.6759 found: 1759.6792 (M+Na).
Compound 1c
To PTR on rink amide resin with Fmoc-γ-aminobutyric acid treatment with 20% piperidine in
NMP ( 10ml) for 15 min. This action was repeated twice. After washing the resin with NMP (7
times, 10 ml, 2 min each time), AM-Glut (3 eq) dissolved in NMP (7 ml) was activated with
PyBroP (3 eq) and DIEA (6 eq) for 4 min at room temperature, transferred to the reaction vessel
and allowed to react for 1 h at rt. Following coupling, the peptidyl resin was washed with NMP (5
times, 7 ml, 2 min each time). Completion of reaction was monitored by ninhydrin test (Kaiser
test, yellow). Finally the conjugate was cleaved by treating the resin with TFA/H2O/ TIPS
(95:2.5:2.5) cocktail for 2 h. The solvents were removed under gentle nitrogen flow and the crude
was precipitated out from ether. The pellets were re-dissolved in ACN:H2O and purified via
preparative HPLC column. 1c was obtained as white crystals. Yield 48%. HRMS: m/z calcd:
1742.7674, found: 1742.7693 (M+H), calcd: 1764.7276, found: 1764.7291 (M+Na).
Compound 1d
To PTR on rink amide resin with Fmoc-γ-aminobutyric acid, after Fmoc deprotection, was add 2
eq of ABT-751-CO-OPh-(p)NO2 and 4 eq of DIEA. This solution allowed to react for 3 h at rt.
Following coupling, the peptidyl resin was washed with NMP (5 times, 7 ml, 2 min each time).
Completion of reaction was monitored by ninhydrin test (Kaiser test, yellow). Finally the
conjugate was cleaved by treating the resin with TFA/H2O/ TIPS (95:2.5:2.5) cocktail for 2 h. The
solvents were removed under gentle nitrogen flow and the crude was precipitated out from ether.
The pellets were re-dissolved in ACN:H2O and purified via preparative HPLC column. 1d was
obtained as white crystals. Yield 41%. HRMS: ESI-MS m/z calcd: 1759.6774 found: 1759.6758
(M+H).
Compound 1e
To PTR on rink amide resin with Fmoc-γ-aminobutyric acid, after Fmoc deprotection, was add 2
eq of Comb A4-CO-OPh-(p)NO2 and 4 eq of DIEA. This solution allowed to react for 3 h at rt.
Following coupling, the peptidyl resin was washed with NMP (5 times, 7 ml, 2 min each time).
Completion of reaction was monitored by ninhydrin test (Kaiser test, yellow). Finally the
conjugate was cleaved by treating the resin with TFA/H2O/ TIPS (95:2.5:2.5) cocktail for 2 h. The
solvents were removed under gentle nitrogen flow and the crude was precipitated out from ether.
The pellets were re-dissolved in ACN:H2O and purified via preparative HPLC column. 1e was
obtained as white crystals. Yield 38%. HRMS: ESI-MS m/z calcd: 1705.7385 found: 1705.7391
(M+H), calcd: 1727.7143, found: 1727.7164 (M+Na).
Spectra
Fmoc-GlyS2(Acm)-OH
ES-MS:
Compound Comb A4-CO-OPh-(p)NO2
ES-MS:
Compound ABT-751-CO-OPh-(p)NO2
ES-MS:
Compound CPT-CO-OPh-(p)NO2
ES-MS:
Compound AM-Glut
ES-MS:
Compound 1a
HRMS:
Compound 1b
HRMS:
Compound 1c
HRMS:
Compound 1d
ES-MS:
Compound 1e
HRMS:
Free peptide carrier cytotoxicity study:
PTR-86-3207
Control
0.1 uM
1 uM
10uM
% Survival
100
50
0
HEK293
24h
HCT116
HEK293
HCT116
72h
All the cell lines were seeded and allowed to adhere in the wells, after which they were treated with the
different compounds in increasing concentrations for 24 and 72 hr. After the treatment, growth inhibition
was measured using the XTT assay: The culture medium was replaced by a new one, and then the XTT
reagent was added. The wells were incubated for 2-4 hr after which the optical density (OD) was measured
at 480 and 680 nm. Percentage of growth inhibition for each compound was calculated by comparison of
the treated culture vs a control culture (free of any compound). The result shown for each concentration
point represents the mean ± standard error calculated from two different experiments. In each experiment
the compounds were tested in quadruplicates.
Cell penetration capabilities of 3207-86 linked to FITC vs free fluorescein:
PTR-86-3207
A
Control: Fluorescein
B
o
HCT116 human colorectal cancer cells were incubated at 37 C with 100nM solutions of fluorescently
labelled PTR-86-3207 (A), or 100nM free fluorescein (B). After 4 hrs the cells were washed 3 times, and
photographed using inverted fluorescence microscopy system (Olympus IX81, X100 magnification,
camera DP71). Fluorescently-labelled PTR-86-3207 demonstrated efficient binding to human cancer
cells, while free fluorescein was washed out.
Panc-1