Z. Omran et al. / Bioorg. Med. Chem. 19 (2011) 3492–3496
3495
37.83 (2C), 77.76 (2C), 155.47 (2C), 171.84 (4C). HR-MS calculated
for C32H61N6O8S6 (M+H)+: 849.2870, found: 849.2863.
37.23 (2C), 37.82 (2C), 37.90 (2C), 37.99 (2C), 53.48 (2C), 127.14
(2C), 128.50 (4C), 129.54 (4C), 134.69 (2C), 167.96 (2C), 171.93
(2C), 171.96 (2C). HR-MS calculated for C40H63N8O6S6 (M+H)+:
943.3189, found: 943.3185.
6.1.2.2.3. (1-{2-[2-(3-{2-[2-(3-{2-[2-(2-(S)-tert-Butoxycarbonyla-
mino-3-phenyl-propionylamino)-ethyldisulfanyl]-ethylcarbamoyl}-
propionylamino)-ethyldisulfanyl]-ethylcarbamoyl}-propionylamino)-
ethyldisulfanyl]-ethylcarbamoyl}-2-(S)-phenyl-ethyl)-carbamic acid
tert-butyl ester 8a. Yield: 55%. 1H NMR (400 MHz, DMSO-d6) d
(ppm): 1.28 (s,18H), 2.31 (s, 8H), 2.73 (m, 12H), 2.92 (m, 4H),
3.34 (m, 12H), 4.10 (m, 2H), 6.90 (d, J = 8.4 Hz, 2H), 7.23 (m,
10H), 8.04 (t, J = 2.8 Hz, 4H), 8.11 (t, J = 4.8 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6) d (ppm): 28.15 (6C), 30.68 (4C), 37.01 (2C),
37.19 (4C), 37.67 (2C), 37.95 (2C), 37.99 (4C), 55.77 (2C), 77.98
(2C), 126.18 (2C), 128.10 (4C), 129.19 (4C), 138.15 (2C), 155.19
(2C), 171.47 (4C), 171.73 (2C). HR-MS calculated for C48H75N8O10S6
(M+H)+: 1115.3925, found: 1115.3905.
6.1.2.2.4. (1-{2-[2-(4-{2-[2-(4-{2-[2-(2-(S)-tert-Butoxycarbonyla-
mino-3-phenyl-propionylamino)-ethyldisulfanyl]-ethylcarbamoyl}-
butyrylamino)-ethyldisulfanyl]-ethylcarbamoyl}-butyrylamino)-eth-
yldisulfanyl]-ethylcarbamoyl}-2-(S)-phenyl-ethyl)-carbamic acid tert-
butyl ester 8b. Yield: 47%. 1H NMR (400 MHz, DMSO-d6) d (ppm):
1.35 (s,18H), 1.77 (quint, J = 7.2 HZ, 4H), 2.12 (t, J = 7.2 Hz, 8H), 2.81
(m, 12H), 2.98 (m, 4H), 3.41 (m, 12H), 4.18 (m, 2H), 6.98 (d,
J = 8.8 Hz, 2H), 7.31 (m, 10H), 8.07 (t, J = 5.0 Hz, 4H), 8.18 (t,
J = 5.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 21.36
(2C), 28.09 (6C), 34.65 (4C), 36.93 (2C), 37.15 (4C), 37.66 (2C),
37.83 (4C), 37.89 (2C), 55.72 (2C), 77.91 (2C), 126.12 (2C), 127.96
(4C), 129.13 (4C), 138.10 (2C), 155.14 (2C), 171.68 (2C), 171.84
(4C). HR-MS calculated for C50H79N8O10S6 (M+H)+: 1143.4238,
found: 1143.4218.
6.2. Biology
6.2.1. General
Human cystinotic fibroblasts (GM00008) were purchased from
Coriell Cell Repositories (NJ, USA) and cultured in Eagle’s minimum
essential media supplemented with 15% FBS, 200 U/ml penicillin,
200 lg/ml streptomycin and 2 mM glutamine at 37 °C in 5% CO2.
Alamar blue reagent was purchased from (Serotech, UK). Thiol
and sulphide quantification kit (Molecular Probes T6060) was pur-
chased from FisherSicentific (UK). Bradford reagent was purchased
from Sigma (UK).
6.2.2. Alamar blue assay
Cystinotic fibroblasts cultured in 96 well plates were incubated
for 0–72 h in the presence of 50 lM either cysteamine, or 1a–d in
media supplemented with 10% Alamar blue. Cell growth was mea-
sured over a 72 h period on a multiwell plate reader Biotek FL6000
and is presented as the net change in absorbance at 570 nm rela-
tive to the reading at time 0 h.
6.2.3. Thiol assay
Cystinotic fibroblasts were seeded in a 25 cm3 vented flask
and allowed to reach approximately 80% confluence before the
addition of the test compounds; 50
1a–d in 4 cm3 Eagles minimum essential media supplemented
with 15% FBS, 200 U/ml penicillin, 200 g/ml streptomycin and
lM either cysteamine, or
6.1.2.3. General procedures for the synthesis of the pro-drugs
l
1a–d.
To a solution of the compounds 6a-b or 8a-b
2 mM glutamine. This was then incubated at 37 °C and 5% CO2
for 24 h. The cells were harvested, frozen in liquid nitrogen
and stored at ꢁ80 °C until the cysteine concentration was deter-
mined per quantity of protein. The cells were recovered from
(0.04 mmol) was added 5 ml of 4 M hydrogen chloride solution
in dioxane, at 0 °C under a nitrogen atmosphere. The reaction mix-
ture was stirred at 0 °C for 30 min and then for further 30 min at
room temperature. The white precipitate was filtered and washed
by 3 ꢀ 50 ml of AcOEt and 3 ꢀ 50 ml of CH2Cl2. The product was
then dried in the vacuum oven at 70 °C.
storage at ꢁ80 °C and suspended in 100
ll 1 mM N-ethylmali-
mide prepared in phosphate buffer (pH 7.6) followed by sonica-
tion for 10 s which was repeated three times with 20 s cooling
intervals on ice. The solution was centrifuged at 800 g for
10 min at 40 °C (Biofuge primo R Heraeus centrifuge). Cell super-
6.1.2.3.1. Pro-drug 1a. Yield: 96%, 1H NMR (400 MHz, DMSO-
d6) d (ppm): 2.31(s, 8H), 2.76 (m, 8H), 2.94 (m, 4H), 3.07 (m, 4H),
3.31 (m, 8H), 8.11 (m, 10H). 13C NMR (100 MHz, DMSO-d6) d
(ppm): 30.61 (4C), 34.00 (2C), 36.98 (2C), 37.14 (2C), 37.79 (2C),
37.87 (2C), 37.94 (2C), 171.48 (2C), 171.54 (2C). HR-MS calculated
for C20H41N6O4S6 (M+H)+: 621.1508, found: 621.1503.
natant (40
NaOH/DMSO. After 5 min incubation at room temperature;
800 l of sodium acetate buffer (pH 4.7) was added. A 5 l vol-
ume of the diluted solution was then added to 100 l of 0.6 mg/
ml solution of Papain-SSCH3 in 96 wells plate and incubated for
1 h at room temperature. A 100 l volume of 4.9 mM L-BAPNA
ll) was then added; 4 ll of 4 M NaBH4 in 7:3 0.1 M
l
l
l
6.1.2.3.2. Pro-drug 1b. Yield: 92%, 1H NMR (400 MHz, DMSO-
d6) d (ppm): 1.70 (quint, J = 7.2, 4H), 2.07 (t, J = 7.2, 8H), 2.78 (m,
8H), 2.95 (m, 4H), 3.08 (m, 4H), 3.32 (m, 8H), 8.09 (m, 10H). 13C
NMR (100 MHz, DMSO-d6) d (ppm): 21.42 (2C), 34.04 (2C), 34.09
(4C), 37.09 (2C), 37.22 (2C), 37.77 (2C), 37.82 (2C), 37.89 (2C),
171.90 (2C), 171.96 (2C). HR-MS calculated for C22H45N6O4S6
(M+H)+: 649.1821, found: 649.1819.
l
solution in sodium acetate buffer (pH 4.0) was then added to
each well of the 96 well plate, gently mixed and incubated for
further 1 h at room temperature. The absorption at 410 nm
was measured and the cysteine levels were calculated by com-
parison to known cysteine standards.
6.1.2.3.3. Pro-drug 1c. Yield: 95%, 1H NMR (400 MHz, DMSO-
d6) d (ppm): 2.31 (s, 8H), 2.63 (m, 4H), 2.74 (t, J = 6.4 Hz, 8H),
3.05 (m, 4H), 3.29 (m, 8H), 3.43 (m, 4H), 4.00 (m, 2H), 7.26 (m,
10 H), 8.14 (m, 4H), 8.36 (m, 6H), 8.80 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) d (ppm): 30.70 (4C), 36.70 (2C), 36.95 (2C),
37.05 (2C), 37.21 (2C), 37.81 (2C), 38.00 (2C), 38.11 (2C), 53.47
(2C), 127.13 (2C), 128.49 (4C), 129.54 (4C), 134.97 (2C), 167.95
(2C), 171.47 (2C), 171.53 (2C). HR-MS calculated for C38H59N8O6S6
(M+H)+: 915.2876, found: 915.2878.
The protein concentration in every sample was determined
according to Bradford method14 at the same time of the thiol assay.
Briefly, 200 ll of Bradford reagent were added to 5 ll of the previ-
ously obtained cell supernatant in each well of a 96 well plate and
incubated for 5 min at room temperature and the absorption at
595 nm was measured. The protein concentrations were calculated
using a range of concentrations of bovine serum albumin as a stan-
dard. The cysteine levels are presented following normalisation to
lM cysteine per mg of protein.
6.1.2.3.4. Pro-drug 1d. Yield: 90%, 1H NMR (400 MHz, DMSO-
d6) d (ppm): 1.70 (quint, J = 7.6 Hz, H-7, 4H), 2.06 (t, J = 7.6 Hz,
8H), 2.64 (m, 4H), 2.74 (t, J = 6.8 Hz, 8H), 3.02 (m, 4H), 3.30 (m,
8H), 3.43 (m, 4H), 3.96 (m, 2H), 7.30 (m, 10 H), 8.04 (m, 4H),
8.20 (m, 6H), 8.64 (m, 2H). 13C NMR (100 MHz, DMSO-d6) d
(ppm): 21.46 (2C), 35.81 (4C), 36.69 (2C), 36.96 (2C), 37.08 (2C),
Acknowledgment
The authors acknowledge financial support from Cystinosis
Foundation, Ireland.