Journal of Medicinal Chemistry
Article
(m, 1H), 7.50−7.42 (m, 6H), 7.30 (s, 4H), 7.25 (tt, 1H, J = 7.1, 1.6
Hz), 6.45 (s, 1H), 6.05 (td, 1H, J = 9.1, 1.0 Hz), 5.79 (d, 1H, J = 6.5
Hz), 5.64 (d, 1H, J = 7.2 Hz), 5.44 (d, 1H, J = 6.5 Hz), 5.13 (d, 1H, J
= 14.9 Hz), 5.03 (dd, 1H, J = 9.4, 1.6 Hz), 4.91−3.86 (m, 1H), 4.75
(dd, 1H, J = 6.5, 4.7 Hz), 4.44−4.36 (m, 3H), 4.30−4.22 (m, 2H),
4.20 (br s, 2H, J = 4.2 Hz), 4.16 (ddd, 1H, J = 12.0, 8.7, 3.6 Hz), 4.09−
4.08 (m, 2H), 3.90 (d, 1H, J = 6.0 Hz), 3.82 (d, 1H, J = 7.1 Hz), 3.74−
3.68 (m, 2H), 3.61 (d, 1H, J = 17.2 Hz), 3.54 (dt, 1H, J = 11.7, 2.8
Hz), 3.42 (dd, 1H, J = 14.6, 6.4), 3.27−3.16 (m, 2H), 2.80−2.75 (m,
2H), 2.72−2.51 (m, 7H), 2.37 (s, 3H), 2.18−2.12 (m, 4H), 2.09−2.01
(m, 1H), 1.92 (s, 3H), 1.86−1.76 (m, 3H), 1.68−1.63 (m, 5H), 1.14
(s, 3H), 1.13 (s, 3H); 13C NMR (101 MHz, CD3OD) δ 205.5, 174.0,
173.60, 173.46, 173.0, 171.63, 171.54, 171.46, 171.2, 170.5, 167.7,
142.4, 140.2, 138.4, 135.5, 134.80, 134.63, 132.9, 131.39, 131.22,
130.1, 129.72, 129.60, 129.56, 129.3, 128.6, 100.0, 85.9, 82.3, 79.0,
77.5, 76.9, 76.2, 75.9, 72.9, 72.4, 61.6, 60.6, 59.2, 55.4, 54.4, 53.2, 50.5,
48.0, 44.6, 43.76, 43.69, 42.2, 39.9, 37.6, 36.49, 36.36, 31.1, 29.8, 27.7,
26.9, 26.5, 25.1, 23.3, 22.3, 20.8, 15.1, 10.5; IR (film) 3360, 3075, 2940,
1729, 1714, 1693, 1665, 1537, 1421, 1241, 1135, 1071 cm−1. MS (ESI)
m/z calcd for [C78H92N11O24]+, 1566.63 [M + H]+; found, 1566.6.
cyclo[DKP-f4-RGD]-PTX 12. Compound 12 was synthesized
according to general procedure C (42 mg, 63% yield). 1H NMR
(400 MHz, DMSO-d6) δ 9.22 (d, 1H, J = 8.5 Hz), 8.95 (s, 1H), 8.79
(s, 1H), 8.44−8.40 (m, 1H), 8.35 (t, 1H, J = 5.7 Hz), 8.20 (s, 1H),
7.98 (dd, 2H, J = 7.1, 1.3 Hz), 7.86 (dd, 2H, J = 7.2, 1.3 Hz), 7.76−
7.69 (m, 1H, J = 1.5 Hz), 7.69−7.63 (m, 2H), 7.59−7.53 (m, 1H),
7.49 (d, 1H, J = 7.6 Hz), 7.46−7.42 (m, 5H), 7.23−7.17 (m, 5H), 6.30
(s, 1H), 5.83 (t, 1H, J = 8.9 Hz), 5.54 (t, 1H, J = 8.7 Hz), 5.42 (d, 1H,
J = 7.1 Hz), 5.36 (d, 1H, J = 8.9 Hz), 5.21 (d, 1H, J = 14.4 Hz), 4.92
(d, 2H, J = 10.6 Hz), 4.62 (s, 1H), 4.27−4.07 (m, 5H), 4.04−3.99 (m,
3H), 3.94−3.87 (m, 1H), 3.83−3.79 (m, 1H), 3.70 (br s, 2H), 3.58 (d,
1H, J = 7.1 Hz), 3.43−3.26 (m overlapped with water signal, 2H), 3.07
(br s, 2H), 2.89 (br s, 2H), 2.69−2.56 (m, 3H), 2.45 (t, 2H, J = 6.8
Hz), 2.38−2.30 (m, 2H), 2.24−2.20 (m, 4H), 2.10 (s, 3H), 1.84−1.76
(m, 5H), 1.64 (t, 1H, J = 12.4 Hz), 1.54−1.41 (m, 7H), 1.02 (s, 3H),
1.00 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 202.3, 173.9, 172.13,
171.94, 170.8, 170.2, 169.63, 169.50, 169.1, 168.84, 168.74, 168.3,
167.9, 166.4, 165.2, 157.3, 139.4, 138.7, 137.3, 134.8, 134.3, 133.45,
133.33, 132.7, 131.4, 129.95, 129.93, 129.56, 129.54, 128.73, 128.65,
128.55, 128.29, 128.14, 127.83, 127.71, 127.65, 127.4, 83.6, 80.2, 76.7,
75.3, 74.68, 74.54, 74.50, 72.5, 70.7, 70.4, 57.4, 56.1, 53.98, 53.86,
52.17, 52.13, 46.1, 44.9, 42.9, 42.1, 41.9, 40.1, 39.9, 38.2, 36.5, 35.7,
34.4, 29.5, 28.7, 27.6, 26.3, 25.3, 22.5, 21.4, 20.63, 20.52, 13.9, 9.7; IR
(film) 3370, 3071, 2941, 1731, 1714, 1699, 1667, 1538, 1421, 1243,
1135, 1071 cm−1, MS (ESI) m/z calcd for [C78H92N11O24]+, 1566.63
[M + H]+; found, 1566.6.
cyclo[DKP-f6-RGD]-PTX 13. Compound 13 was synthesized
according to general procedure C (43 mg, 65% yield). 1H NMR
(400 MHz, DMSO-d6) δ 9.42 (s, 1H), 9.25 (d, 1H, J = 8.5 Hz), 8.74
(s, 1H), 8.62 (s, 1H), 8.45 (s, 1H), 8.37 (t, 1H, J = 5.7 Hz), 7.99−7.97
(m, 2H), 7.87−7.84 (m, 3H), 7.73 (t, 1H, J = 7.3 Hz), 7.66 (t, 2H, J =
7.4 Hz), 7.56 (tt, 1H, J = 7.3, 2.0 Hz), 7.50−7.44 (m, 7H), 7.25−7.15
(m, 5H), 6.30 (s, 1H), 5.83 (t, 1H, J = 8.8 Hz), 5.53 (t, 1H, J = 8.7
Hz), 5.41 (d, 1H, J = 7.2 Hz), 5.35 (d, 1H, J = 9.0 Hz), 5.11 (d, 1H, J
= 14.9 Hz), 4.98−4.90 (t, 2H), 4.64 (s, 1H), 4.29−4.20 (m, 3H),
4.17−4.08 (m, 2H), 4.04−3.95 (m, 3H), 3.93−3.79 (m, 3H), 3.74−
3.64 (m, 1H), 3.59 (d, 1H, J = 6.8 Hz), 3.53−3.43 (m, 1H), 3.26−3.19
(m, 1H), 3.07 (br s, 1H), 2.97 (br s, 1H), 2.72−2.58 (m, 4H), 2.56−
2.52 (m, 1H), 2.45 (t, 2H, J = 6.8 Hz), 2.40−2.28 (m, 2H), 2.23 (s,
3H), 2.10 (s, 3H), 1.82−1.60 (m, 7H), 1.52−1.46 (m, 6H), 1.02 (s,
3H), 1.00 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 202.7, 172.0,
171.8, 170.3, 169.72, 169.57, 169.2, 168.79, 168.72, 166.8, 166.4,
165.2, 157.3, 139.5, 137.4, 135.2, 134.3, 133.5, 133.32, 133.28, 132.7,
131.5, 129.9, 129.6, 128.7, 128.38, 128.21, 128.0, 127.7, 127.5, 83.6,
80.3, 76.7, 75.3, 74.69, 74.62, 74.49, 70.7, 70.4, 57.2, 54.4, 54.1, 51.98,
51.92, 46.10, 45.97, 43.0, 41.9, 40.9, 39.7, 37.8, 36.6, 34.4, 29.5, 28.7,
28.0, 26.4, 24.4, 22.6, 21.4, 20.7, 14.0, 9.8; IR (film) 3365, 3071, 2940,
1732, 1716, 1699, 1665, 1537, 1421, 1243, 1135, 1071 cm−1. MS (ESI)
m/z calcd for [C78H92N11O24]+, 1566.63 [M + H]+; found, 1566.6.
Plasma Stability Assays. A 10 mM stock solution of cyclo[DKP-
f 3-RGD]-PTX 11 (MW = 1566.62) was obtained by dissolving 2 mg
of compound in 127.66 μL of DMSO. A further dilution 1:50 in pH
7.5 phosphate buffer (PBS) was performed (10 μL of stock solution
into 490 μL of PBS) to obtain a 200 μM solution. From this last
solution, an amount of 25 μL was spiked into 475 μL of plasma
(murine or human) to obtain the final concentration of 10 μM.
Standards (lidocaine and 2-piperidinoethyl-4-amino-5-chloro-2-me-
thoxybenzoate) were tested at 2.5 μM final concentration starting from
a 500 μM stock solution in DMSO, further diluted 1:10 into PBS and
1:20 into plasma.
Aliquots of 50 μL volume were taken at 0, 15, 30, 60, 120, 180, and
300 min of incubation at 37 °C and immediately quenched with 200
μL of a solution of verapamil, 250 ng/mL (internal standard) in
acetonitrile. Samples were centrifuged for 20 min at 3000 rpm and
supernatants analyzed by UPLC (Waters) interfaced with a Premiere
XE triple quadrupole (Waters). Eluents were the following: phase A,
95% H2O, 5% CH3CN + 0.1% HCOOH; phase B, 5% H2O, 95%
CH3CN + 0.1% HCOOH. Waters UPLC parameters were the
following: flow of 0.6 mL/min, column BEH C18, 50 mm × 2.1 mm,
1.7 μm, at 50 °C, vol injection of 5 μL. Samples were analyzed in
multiple reaction monitoring (MRM) conditions: ESI positive,
desolvation temperature 450 °C, desolvation gas 900 L/h, cone gas
90 L/h, collision gas 0.2 L/h. Results are presented as the mean SD,
n = 2 for standards, n = 3 for cyclo[DKP-f 3-RGD]-PTX 11.
Cell Sensitivity Studies. The human ovarian carcinoma IGROV-1
cell line,46 the cisplatin-resistant IGROV-1/Pt1 subline,46 the human
ovarian carcinoma cell line SKOV3, and the human pancreatic
carcinoma cell lines PANC-1 and MIA-PaCa2 were cultured in
Dulbecco’s modified Eagle medium (DMEM) medium. The human
osteosarcoma U2-OS cell line was grown in McCoy’s 5A medium.
HDFC cells were cultured in DMEM-F12 medium. In all cases, the
medium was supplemented with 10% fetal calf serum. The cell
sensitivity to drugs was measured by using the growth inhibition assay
based on cell counting. Cells were seeded in duplicate into six-well
plates and exposed to drug 24 h later. Paclitaxel and the studied
compounds were dissolved in DMSO and then added to culture
medium. DMSO concentration in medium never exceeded 0.25%.
After 72 h of drug incubation, cells were harvested for counting with a
cell counter (Z1 Beckman Coulter counter). IC50 is defined as the
drug concentration producing 50% decrease of cell growth. At least
five independent experiments were performed.
Analysis of Integrin Levels. The expression of integrins was
measured by flow cytometry, following optimization of antibody
concentration. Exponentially growing cells were harvested and
incubated for 30 min at 4 °C with anti-human αvβ3 or αvβ5 antibodies
or isotypic controls (Millipore, Temecula, CA; Chemicon Interna-
tional). Cells were than washed, and samples were immediately used
for flow cytometric analysis (FACScan, Becton-Dickinson). Expression
of integrins was expressed as the ratio between the mean fluorescence
intensity obtained in cells incubated with anti-integrin antibodies
divided by that of cells incubated with isotypic control.
In Vivo Antitumor Activity Studies. All experiments were
carried out using female athymic Swiss nude mice, 8−10 weeks old
(Charles River, Calco, Italy). Mice were maintained in laminar flow
rooms, keeping temperature and humidity constant. Mice had free
access to food and water. Experiments were approved by the Ethics
Committee for Animal Experimentation of the Istituto Nazionale
Tumori of Milan, Italy, according to institutional guidelines. The
IGROV-1/Pt1 human tumor xenograft, derived from cultures of the
corresponding ovarian carcinoma cell line,46 was used. Exponentially
growing cells (107/mouse) were sc injected into the right flank of
athymic nude mice, and the tumor line was achieved by serial sc
passages of fragments of regrowing tumors into healthy mice. Groups
of four mice bearing bilateral sc tumors were employed. Tumor
fragments were implanted on day 0, and tumor growth was followed
by biweekly measurements of tumor diameters with a Vernier caliper.
Tumor volume (TV) was calculated according to the formula TV
(mm3) = d2D/2, where d and D are the shortest and the longest
diameter, respectively. Compounds were delivered iv and administered
L
dx.doi.org/10.1021/jm301058f | J. Med. Chem. XXXX, XXX, XXX−XXX