Inorganic Chemistry
Article
1
three times to obtain 260 mg of light yellow solid 2. Yield: 68%. H
CONCLUSION
■
NMR (400 MHz, DMSO-d ) δ: 12.05 (s, 1H, COOH), 6.51 (s, 6H,
NH ), 2.48−2.29 (m, 4H, 2CH ), 1.90 (s, 3H, CH ). C NMR
101 MHz, DMSO-d ) δ: 179.6 (C ), 178.2 (C ), 173.8 (C ), 30.5
C ), 29.9 (C ), 22.8 (C ). ESI-MS: m/z [M−H] calculated for
C H Cl N O Pt: 475.2, found 475.2. These spectra data are same as
6
In summary, we report the first example of a LHRH−
platinum(IV) conjugate for the purpose of targeting platinum
drug to cancer cells that overexpress LHRH receptors. The
cancer-targeting peptide LHRH is attached to compound 2
through an amide bond. The resulting LHRH−platinum(IV)
conjugate 3 is quite stable and can be reduced in the presence
of reducing agent to release the LHRH moiety and cisplatin
that binds to DNA. This conjugate with high aqueous
solubility possesses a comparable cytotoxicity but a much
higher selectivity toward LHRH receptor-positive cancer cells,
compared with cisplatin. The selectivity of the conjugate is, at
least in part, due to the selective cellular accumulation via
LHRH receptor-mediated endocytosis pathway and higher
levels of Pt in the genomic DNA of cancer cells that
overexpress LHRH receptors. Furthermore, the action
mechanism of the conjugate is proved to be similar to
cisplatin, i.e., inducing apoptosis and arresting the cell cycle at
13
2
3
2
3
(
(
6 2 3 6
−
4
5
1
6
13
2
2
6
50
that reported in the literature.
Synthesis of Compound 3. Compound 2 (100 mg, 0.21 mmol, 1
equiv) was dissolved in 6 mL of acetone. N-hydroxysuccinimide
NHS, 37 mg, 0.31 mmol, 1.5 equiv) and dicyclohexylcarbodiimide
DCC, 52 mg, 0.25 mmol, 1.2 equiv) were added. The mixture was
stirred at room temperature overnight. The formed white solid was
removed by filtration. The filtrate was concentrated under reduced
pressure. One milliliter (1 mL) of DCM and 10 mL of ethyl ether
were added to the residue to form a light yellow solid. The solid was
collected by centrifugation and washed by DCM and ethyl ether three
times to obtain 102 mg of pale yellow solid. Yield: 85%. H NMR
400 MHz, DMSO-d ) δ: 6.51 (s, 6H, 2NH ), 2.85−2.78 (m, 6H,
3CH ), 2.63 (t, J = 6.8 Hz, 2H, CH ), 1.91 (s, 3H, CH ). ESI-MS: m/
z [M−H] calculated for C H Cl N O Pt: 572.2, found 572.4.
LHRH (30 mg, 0.024 mmol, 1.1 equiv) was dissolved in 1 mL of
DMF before N,N′-diisopropylethylamine (DIPEA, 19 μL, 0.11 mmol,
equiv) was added. After 5 min, the NHS ester of compound 2 (13
mg, 0.022 mmol, 1 equiv) synthesized above was added. The solution
was stirred at room temperature for 2 h. Two milliliters (2 mL) of
DCM and 10 mL of ethyl ether were added to the reaction mixture to
form a white solid. The solid was collected by centrifugation and
washed by DCM and ethyl ether three times. The crude product was
dissolved in Milli-Q water at a concentration of 10 mg/mL and
purified via semipreparative HPLC. A quantity of 100 μL of sample
was injected into the HPLC system each time. Mobile phase A was
(
(
1
(
6
3
2
2
3
−
1
0
16
2
3
8
the G /M phase. These results indicate the possibility of
2
selectively transporting platinum anticancer drugs into LHRH-
receptor positive cancer cells while leaving the normal cells
unaffected.
5
EXPERIMENTAL SECTION
■
Materials and Instruments. All the reagents were used as
received without further purification. Cisplatin was purchased from
Shandong Boyuan Pharmaceutical Co., Ltd. LHRH peptide (Glp-His-
Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHEt) was purchased form GL
Biochem (Shanghai), Ltd. All the reactions were performed in
H O with 0.1% TFA, and mobile phase B was acetonitrile (ACN)
2
1
13
195
darkness. H, C, and Pt NMR spectra were obtained on a Bruker
AVANCE III 400 MHz NMR spectrometer or a Bruker Ascend
AVANCE III HD 600 MHz spectrometer with a BBO probe. ESI-MS
tests were performed on an Agilent API-3200 Q-Trap mass
spectrometry. High-performance liquid chromatography (HPLC)
experiments were conducted on a Shimadzu Prominence HPLC
system. For analysis, a Phenomenex column (Gemini, 5 μm, C18, 110
Å, 250 mm × 4.6 mm) was used. For semipreparation, a Waters
column (Xbridge BEH C18 OBD Prep Column, 130 Å, 5 μm, 250
mm × 10 mm) was utilized. Pt contents were measured by an
inductively coupled plasma−optical emission spectrometry (ICP-
OES) system (Optima 8000 spectrometer) or an inductively coupled
plasma mass spectrometry (ICP-MS) system (PE Nexion 2000).
DNA concentrations were determined by a NanoDrop Spectropho-
tometer (ND-1000). Apoptosis and cell cycle analyses were
conducted on a flow cytometer (BD Bioscience FACS Calibur).
Synthesis of Compound 1. Compound 1 was synthesized
with 0.1% TFA. The absorbance wavelengths were set at 254 and 360
nm. The flow rate was 2 mL/min. The HPLC program was set as
follows: 10%−40% phase B (linear increased from 0 to 30 min). The
fraction with pure product was lyophilized to obtain 10 mg of white
1
solid 3. Yield: 27%. H NMR (600 MHz, D O) δ: 8.57 (d, J = 1.2 Hz,
2
1
H), 7.61 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.25 (t, J = 7.2
Hz, 1H), 7.21 (s, H), 7.19 (s, H), 7.13 (t, J = 7.2 Hz, 1H), 7.10 (d, J =
.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 4.72 (d, J = 7.8 Hz, 1H), 4.66
8
(
dd, J = 8.4, 6.6 Hz, 1H), 4.48 (dd, J = 8.4, 5.4 Hz, 1H), 4.44−4.40
(m, 1H), 4.38−4.29 (m, 2H), 4.24−4.16 (m, 2H), 4.13 (dd, J = 9.0,
4
1
1
.8 Hz, 1H), 3.78−3.68 (m, 2H), 3.68−3.59 (m, 1H), 3.51 (dd, J =
6.8, 6.8 Hz, 1H), 3.27−3.02 (m, 10H), 2.98 (dd, J = 13.8, 7.2 Hz,
H), 2.91 (dd, J = 13.6, 8.4 Hz, 1H), 2.67 (t, J = 6.6 Hz, 2H), 2.48 (t,
J = 6.6 Hz, 2H), 2.40−2.23 (m, 3H), 2.21−2.14 (m, 1H), 2.10 (s,
3
1
H), 1.99 (dt, J = 12.0, 4.6 Hz, 1H), 1.92−1.80 (m, 2H), 1.78 (dd, J =
4.4, 6.0 Hz, 1H), 1.69−1.60 (m, 4H), 1.59−1.48 (m, 5H), 1.45−
49
1.36 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H), 1.04−0.94 (m, 2H), 0.92 (d, J
=
following the procedure reported previously. Cisplatin (1 g, 3.3
mmol, 1 equiv) was suspended in 400 mL of acetic acid before
13
6.0 Hz, 3H), 0.85 (d, J = 6.0 Hz, 3H). C NMR (151 MHz, D O)
2
δ 182.1, 181.7, 181.0, 174.9, 174.6, 174.2, 173.9, 173.7, 173.6, 173.4,
hydrogen peroxide (50% in H O, 10 mL, 176.4 mmol, 53 equiv) was
2
1
1
1
4
2
72.9, 171.2, 171.1, 170.8, 156.6, 154.7, 136.2, 133.6, 130.6, 128.3,
27.5, 127.0, 124.5, 122.0, 119.4, 118.2, 117.3, 117.2, 115.6, 115.4,
11.9, 108.7, 61.1, 60.8, 56.5, 55.7, 55.5, 54.4, 53.8, 52.3, 52.2, 51.0,
7.9, 40.6, 39.6, 39.1, 34.4, 31.4, 31.1, 30.6, 29.4, 29.2, 27.8, 27.6,
added. The mixture was stirred at room temperature for 3 h, and the
solution became clear. The reaction mixture was centrifuged to
remove unreacted cisplatin. The supernatant was concentrated under
reduced pressure. Excess ethyl ether was added to the residue to
precipitate a yellow solid. The formed solid was collected by
centrifugation and washed by acetone, dichloromethane (DCM), and
1
95
6.2, 25.1, 24.6, 24.3, 24.0, 22.3, 22.2, 21.7, 20.6, 13.5. Pt NMR
+
(129 MHz, D O) δ: 1084.8−1075.6 (m). ESI-MS: m/z [M+H]
2
calculated for C H Cl N O Pt: 1683.6, found 1683.4.
ethyl ether, respectively, to obtain 980 mg of yellow solid 1. Yield:
65 98
2
19 17
1
Synthesis of Compound 4. LHRH (5 mg, 0.004 mmol, 1 equiv)
was dissolved in 0.1 mL DMF before N,N′-diisopropylethylamine
7
1
8%. H NMR (400 MHz, DMSO-d ) δ: 6.21−5.72 (m, 6H, 2NH ),
6
3
−
.87 (s, 3H, CH ). ESI-MS: m/z [M−H] calculated for
C H Cl N O Pt: 375.1, found 375.3.
3
(DIPEA, 2.8 μL, 0.016 mmol, 4 equiv) was added. After 5 min,
2
9
2
2
3
Synthesis of Compound 2. Compound 2 was synthesized
following the procedure reported in the literature with modification.
succinic anhydride (0.8 mg, 0.008 mmol, 2 equiv) was added. The
solution was stirred at room temperature for 2 h. Ethyl ether (2 mL)
was added to the reaction mixture to form a white solid. The solid was
collected by centrifugation and washed by DCM and ethyl ether three
times. The crude product was dissolved in Milli-Q water and purified
by semipreparative HPLC. The fraction with pure product was
lyophilized to get 3 mg of white solid 4. Yield: 57%. ESI-MS: m/z [M
50
Compound 1 (300 mg, 0.8 mmol, 1 equiv) was suspended in 2 mL of
DMF. Succinic anhydride (480 mg, 4.8 mmol, 6 equiv) was then
added. The mixture was stirred at 60 °C overnight. Two milliliters (2
mL) of DCM and 90 mL of ethyl ether were added to the reaction
mixture to form a light yellow solid. The solid was collected by
centrifugation and washed by 2 mL of DCM and 10 mL of ethyl ether
+
+H] calculated for C H N O : 1325.5, found 1325.3.
6
3
90 17 15
F
Inorg. Chem. XXXX, XXX, XXX−XXX