F. Bianchini et al. / Bioorg. Med. Chem. 23 (2015) 1112–1122
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Analyzer. ESI mass spectra were recorded on a Thermo LCQ-Fleet. All
commercially available reagents and solvents were purchased from
Sigma–Aldrich and used as received, unless otherwise specified.
Analytical HPLC purity tests and semipreparative HPLC purifications
of radioligands were performed on a Beckman-Coulter System
allowed us to obtain the final product 8 as a white solid in quantita-
tive yield. 1H NMR (300 MHz, D2O) d 7.62 (s, 1H); 7.60–7.56 (m, 1H),
7.52–7.48 (m, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.71–6.66 (m, 3H), 6.65–
6.61 (m, 1H), 5.09–4.95 (m, 3H), 3.16 (t, J = 6.9 Hz, 2H), 2.77–2.73
(m, 2H), 2.67 (t, J = 6.9 Hz, 2H), 1.91–1.81 (m, 2H). 13C NMR
(50 MHz, D2O) d 173.6, 165.5, 154.1, 151.5, 145.6, 142.3, 133.6,
130.9, 127.9, 126.9, 124.1, 114.6, 112.1, 111.3, 51.5, 49.3, 44.7,
39.7, 39.0, 25.8, 20.6. ESI-MS m/z 425.2 [(M+H)+, 100]. Anal. Calcd
for C21H24N6O4: C, 59.42; H, 5.70; N, 19.80. Found: C, 59.55; H,
5.76; N, 19.66. HPLC: tR = 12.80 min, 99% purity.
GOLD (column Grace Discovery Sciences Alltima C18 10 lm;
250 ꢁ 10 mm), using H2O/CH3CN gradient eluant buffered with
0.1% TFA (flow rate 2.5 mL/min, k = 254 nm, gradient eluant:
CH3CN 10%/5 min, CH3CN 10–90%/25 min). HPLC analysis was used
to determine purity and all tested compounds possessed >95%
purity.
4.6. (S)-3-(2-(4-(3-(3-(4-hydroxyphenethyl)guanidino)propyl)-
1H-1,2,3-triazol-1-yl)acetamido)-3-phenylpropanoic acid
hydrochloride (9)
4.2. General procedure (A) for the synthesis of RGD mimetic
ligands 6–9
Azide (1.1 equiv) was dissolved in THF (0.15 M solution), then
alkyne (1 equiv) and CuP(OEt)3I (0.11 equiv) were added. The reac-
tion was performed under microwave irradiation at 120 °C for 1 h,
then THF was evaporated, the crude residue dissolved in EtOAc or
DCM, and washed with concentrated ammonia solution and brine.
The organic layer was dried over Na2SO4, evaporated under vac-
uum and purified via flash column chromatography. The protected
adduct was suspended in 3 M HCl (10 mL/mmol) and stirred at
30 °C overnight. The solution was evaporated to dryness in vacuo
to afford the corresponding product in pure form.
Following the general procedure (A), Pg-9 was obtained as a
colorless oil in 82% yield. The hydrolysis of Pg-9 in 3 M HCl allowed
us to obtain the final product 9 as a white solid in quantitative
yield. 1H NMR (300 MHz, D2O) d 7.47 (s, 1H), 7.22–7.14 (m, 5H),
6.90 (d, J = 8.4 Hz, 2H), 6.58 (d, J = 8.4 Hz, 2H), 5.15–5.05 (m, 1H),
5.01 (d, J = 3.6 Hz, 2H), 3.19 (t, J = 6.6 Hz, 2H), 2.87 (t, J = 6.6 Hz,
2H), 2.77 (d, J = 6.6 Hz, 2H), 2.55 (t, J = 6.6 Hz, 2H), 2.45 (t,
J = 7.2 Hz; 2H), 1.60–1.52 (m, 2H). 13C NMR (50 MHz, CDCl3) d
174.4, 166.5, 155.5, 154.1, 139.9, 130.2, 129.4, 128.9, 128.1,
127.0, 126.3, 115.4, 52.4, 50.7, 42.3, 40.1, 39.8, 33.5, 27.1, 21.3.
ESI-MS m/z 494.4 [(M+H+, 100]. Anal. Calcd for C25H32ClN7O4: C,
56.65; H, 6.09; N, 18.50. Found: C, 56.73; H, 6.18; N, 18.24. HPLC:
tR = 23.34 min, 98% purity.
4.3. (S)-3-(2-(4-(3-Guanidinopropyl)-1H-1,2,3-triazol-1-yl)aceta
mido)-3-(4-hydroxyphenyl)propanoic acid hydrochloride (6)
Following the general procedure (A), the side chain-protected
(Pg) Pg-6 was obtained as a pale yellow oil in 89% yield. The
hydrolysis of Pg-6 allowed us to obtain the final product 6 as a
white solid in quantitative yield. 1H NMR (300 MHz, D2O) d
7.63 (s, 1H), 7.10 (d, J = 7.8 Hz, 2H), 6.72 (d, J = 7.8 Hz, 2H), 5.2–
4.9 (m+s, 3H), 3.02 (t, J = 6.6 Hz, 2H), 2.78–2.75 (m, 2H), 2.63 (t,
J = 6.6 Hz, 2H), 1.78 (t, J = 6.6 Hz, 2H). 13C NMR (50 MHz, D2O) d
173.6, 165.2, 155.8, 154.3, 130.9, 127, 114.7, 51.9, 49.4, 39.4,
39.1, 26.3, 20.4. ESI-MS m/z 412.3 [(M+Na)+, 66], 390.33
[(M+H)+, 100]. Anal. Calcd for C17H24ClN7O4: C, 47.94; H, 5.68;
N, 23.02. Found: C, 48.11; H, 5.73; N, 22.90. HPLC: tR = 10.22 min,
98% purity.
4.7. General procedure (B) for the synthesis of 2–4
Deprotected RGD mimetic ligand was dissolved in 1:1 H2O–
MeOH mixture (0.05 M), and NaI (1 equiv), dissolved in the mini-
mum quantity of water, was added to the mixture. Then a solution
of chloramine-T, dissolved in the minimum quantity of water, was
added and the resulting mixture was stirred for 20 min at rt. After-
wards, an aqueous solution of sodium bisulfite (mixture with meta-
bisulfite, 1 equiv) was added, followed by water addition (starting
volume ꢁ 4) and the solution was washed with DCM. The aqueous
layer was evaporated and aliquots of the mixture were purified
via semipreparative HPLC giving suitable quantities of iodinated
product for 1H NMR and biological assays.
4.4. (S)-2-((3-(1-(2-((2-Carboxy-1-(4-hydroxyphenyl)ethyl)
amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)propyl)amino)-1H-
benzo[d]imidazol-3-ium trifluoroacetate (7)
4.8. (S)-Amino((3-(1-(2-((2-carboxy-1-(4-hydroxy-3-iodophenyl)
ethyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)propyl)amino)
methaniminium trifluoroacetate (2)
Following the general procedure (A), Pg-7 was obtained as a
colorless oil in 57% yield. Following a different hydrolysis proce-
dure, Pg-7 (45 mg, 0.06 mmol) was dissolved in 2 mL of a 1:1 mix-
ture of DCM–TFA and stirred at room temperature 2 h. The solvent
was evaporated to yield the deprotected product 7 in quantitative
yield. 1H NMR (300 MHz, D2O) d 7.62 (s, 1H), 7.08–7.03 (s+d,
4+2H), 6.64 (dd, J = 6.6, 2.1 Hz, 2H), 5.08–5.00 (m, 3H), 3.19 (t,
J = 6.9 Hz, 2H), 2.76–2.66 (m, 4H), 1.90 (t, J = 6.9 Hz, 2H). 13C
NMR (50 MHz, D2O) d 173.7, 165.7, 154.1, 149.5, 146.0, 130.9,
128.1, 126.8, 123.9, 122.4, 114.5, 110, 51.3, 49.3, 44.6, 41.0, 39.1,
26.4, 20.7. ESI-MS m/z 464.3 [(M+H)+, 100]. Anal. Calcd for
Iodination of compound 6 was achieved according to general
procedure (B) as above to give pure 2 after HPLC purification. 1H
NMR (300 MHz, D2O) d 7.66 (s, 1H), 7.59 (s, 1H), 7.11 (d,
J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 5.10–5.03 (m, 3H), 3.04 (t,
J = 6.9 Hz, 2H), 2.78 (bd, J = 6.9 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H),
1.81 (t, J = 7.2 Hz, 2H). ESI-MS m/z 537.5 [(M+Na)+, 100], 516.1
[(M+H)+, 18]. HPLC: tR = 13.22 min, 97% purity.
4.9. (S)-2-((3-(1-(2-((2-Carboxy-1-(4-hydroxy-3-iodophenyl)
ethyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)propyl)amino)-
1H-benzo[d]imidazol-3-ium trifluoroacetate (3)
C25H26F3N7O6: C, 51.99; H, 4.54; N, 16.98. Found: C, 52.25; H,
4.64; N, 16.79. HPLC: tR = 15.91 min, 98% purity.
Iodination of compound 7 was achieved according to general
procedure (B) as above to give pure 3 after HPLC purification.
1H NMR (300 MHz, D2O) d 7.63 (s, 1H), 7.45 (d, J = 2.1 Hz, 1H),
7.05–6.99 (m, 5H), 6.62 (d, J = 8.7 Hz; 1H), 5.10–4.93 (m, 3H),
3.23–3.10 (m, 2H), 2.71 (t, J = 6.9 Hz, 4H), 1.92 (t, J = 6.9 Hz,
2H). ESI-MS m/z 612.14 [(M+Na)+, 24], 590.16 [(M+H)+, 100]. HPLC:
tR = 17.96 min, 97% purity.
4.5. (S)-3-(4-Hydroxyphenyl)-3-(2-(4-(3-(pyridin-2-ylamino)
propyl)-1H-1,2,3-triazol-1-yl)acetamido)propanoic acid
hydrochloride (8)
Following the general procedure (A), Pg-8 was obtained as a col-
orless yellow oil in 55% yield. The hydrolysis of Pg-8 in 3 M HCl