J. Matharu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3688–3691
3691
Table 1
for the experiments. The rats were anesthetized with sodium pentobarbital
(50 mg/kg ip). The occipital and superior thyroid arteries were cauterized and
cut. A catheter (PE-60) was placed in the common carotid artery and the
perfusate (bicarbonate-buffered physiologic saline, pH 7.4) was infused into
the left common carotid artery at the rate of 20 ml/min using a pump. Prior to
the perfusion, the external carotid artery was ligated just proximal to the
bifurcation of the common carotid artery. The heart was stopped prior to
perfusion (2–3 s) by severing the left ventricle so as to eliminate potential flow
contributions from the systemic circulation. After the catheter is placed in the
common carotid artery, blood flow to the ipsilateral cerebral hemisphere is
maintained by crossover at the circle of Willis from the contralateral
circulation. The pterygopalatine artery is kept open. The cannulation
procedure of the common carotid artery is simple to perform and generally
takes <15 min. In this technique, the thoracic cavity is opened at the beginning
of the perfusion, and the cardiac ventricles are severed to stop blood flow from
the systemic circulation. The infusion rate of the perfusion into the common
carotid artery is usually sufficient to perfuse both cerebral hemispheres as well
as the brain stem. The composition of the perfusate was 128 mM sodium
chloride, 24 mM sodium bicarbonate, 4.2 mM potassium chloride, 1.5 mM
calcium chloride, 0.9 mM magnesium chloride, 2.4 mM sodium phosphate and
Relative affinities of amino acid and melphalan analogs for the system LAT1
transporter, expressed as the Ki for inhibition of the uptake of L-[14C]-leucine into
rat brain via the blood–brain barrier.
Amino acid
Nitrogen
mustard
Kia
(
lM
S.E.M)
2
55
4
L-Phenylalanine
( )-2-Amino-1,2,3,4-tetrahydro-2-naphthoic
acid
7.7 0.8
5a
5b
5c
5d
8.5 0.6
68
0.079 0.006
252 44
12.5 1.1
5.0 0.6
9
( )-2-Aminoindane-2 carboxylic acid
6
7
( )-2-Aminobenzo-bicyclo-[2.2.1]heptane-20-
exo-carboxylic acid
26
1
2.1 0.2
9 mM
D-glucose in order to supply sufficient nutrients and salts to maintain the
metabolic and structural integrity of the brain. To this was added ꢀ0.1
l
Ci
L-
a
n = 9–12.
[
14C]leucine/mL, ꢀ0.1
l
Ci [3H]-diazepam/mL (to measure cerebral perfusion
fluid flow), 0–1000
l
M
unlabelled amino acid analog (for the inhibition
studies) and/or [3H]-sucrose (for vascular volume measurements). All solutions
were filtered, warmed to 37 °C, and saturated with 95% air and 5% carbon
dioxide prior to their use. The body temperature of the rats was maintained at
36–37 °C throughout the experiments with a heating pad. Perfusions were
stopped after 15–20 s. The brain was removed from the skull and dissected on
ice. Tissue samples were then removed from the ipsilateral cerebral
hemisphere after the removal of the meninges and surface blood vessels.
Acknowledgements
This work was supported by grants from the Department of De-
fense Breast Cancer Program (W81XWH-062-0033) and NIH/
NINDS (R01 NS052484).
Two 20–30 lL aliquots of perfusion fluid were also collected for determination
of perfusate tracer concentrations. The tissue samples were weighed, digested
overnight in 1 M piperidine (1 mL/sample) and then dissolved in scintillation
cocktail (10 mL/sample: Ready Solv-MP Beckman, Fullerton, California). 3H-
and 14C dpm values were determined after correction for background, quench,
and efficiency using dual-label liquid scintillation counting.
References and notes
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Shimadzu-C-R4A™ (Columbia, MD) chromatopac integrator and printer. The
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column was a YMC Pack ODS-AMÒ column, 250 ꢁ 30 mm i.d., particle-S-5
lm;
120 A. The mobile phase was a 50:50 v/v water methanol mixture acidified to a
pH of 2.75–2.80 with concentrated HCl. The flow rate was 15 mL/min. The
fractions were collected manually, lyophilized to a dry powder and reinjected
on a comparable analytical system to confirm isomeric purity.
20. Compounds 5a and 5c have been reported elsewhere.10,11 ( )-2-Amino-(bis-2-
chloroethyl)-5-aminoindane-2-carboxylic acid (6): 1H NMR (D2O/DCl) 400 MHz
d3.26–3.33 (2H, geminalcoupling doublets, C1protons);3.44–3.49 (4H, t, N–CH2
protons); 3.66–3.73 (2H, geminal coupling doublets, C3-protons); 3.91–3.96
(4H, t, –CH2–Cl); 7.34–7.46 (3H, m, aromatic protons), GC–MS, (BSTFA
derivative) m/z 461 (M+), 425 (–Cl), 389 (–2Cl); HRMS: 316.0833, calcd for
C14H18N202Cl2 316.0745. ( )-2-Amino-(bis-2-chloroethyl)-6-amino-1,2,3,4-
tetrahydro-2-naphthoic acid dihydrochloride salt. 1H NMR (D2O/DCl) 400 MHz
d 1.90–2.0, 2.10–2.20 (2H, m, C3-protons); 2.64–2.74, 2.75–2.84 (2H, m, C4-
protons); 2.86–2.92, 3.23–3.30 (2H, doublets, C1-protons); 3.28–3.34, 3.72–3.82
(8H, m, chloroethyl protons), 7.13–7.26 (3H, m, aromatic protons), GC–MS
(BSTFA derivative) m/z 475 (M+), 440 (–Cl), 403 (–2Cl); tR HPLC19 27.91 min;
HRMS: 330. 0893, calcd for C15H20N202Cl2 330.0901. ( )-2-amino-(bis-2-
chloroethyl)-8-amino-1,2,3,4-tetrahydro-2-naphthoic acid dihydrochloride
salt. 1H NMR (D2O/DCl) 400 MHz d 1.80–1.90, 2.02–2.10 (2H, m, C2-protons);
2.55–2.64, 2.65–2.74 (2H, m, C4-protons); 2.76–2.84, 3.16–3.22 (2H, doublets,
C1-protons); 3.20–3.26, 3.66–3.74 (8H, m, chloroethyl protons); 7.08–7.14 (3H,
m, aromatic protons), GC–MS (BSTFA derivative) m/z 475 (M+), 440 (–Cl), 403 (–
2Cl); tR HPLC19 29.95 min; HRMS: 330.0951, calcd for C15H20N2O2Cl2 330.0901.
( )-20-endo-Amino-bis-2-chloroethyl-7-aminobenzobicyclo-[2.2.1]heptane-20-
exo-carboxylic acid dihydrochloride. 1H NMR (D2O/DCl) 400 MHz d 1.44–1.50
(1H, d of d, C3 axial proton); 1.80–1.88 (1H, d of d, C3 equatorial proton); 2.28–
2.34 (1H, m, C4-proton); 2.71–2.78 (1H, m, C1-proton); 3.48–3.56, 3.90–3.98
(8H, m, chloroethyl protons); 3.70–3.76 (2H, m, C9-protons); 7.34–7.38 (1H, d of
d, C6 proton); 7.44–7.46 (1H, d, C8-proton): 7.50–7.52 (1H, d, C5-proton), GC–MS
(BSTFA derivative), m/z 487 (M+), 451(–1 Cl), 417 (–2 Cl); HRMS: 342.0944, calcd
for C16H20N202Cl2 342.0901.
10. Takada, Y.; Vistica, D. T.; Grieg, N. H.; Purdon, D.; Rapoport, S. I.; Smith, Q. R.
Cancer Res. 1992, 52, 2191.
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1987, 30, 542.
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15. Brain perfusion and system L transporter affinity studies: Transport affinity was
evaluated from the concentration-dependent inhibition of
uptake into rat brain during perfusion at tracer leucine concentration
(<0.3 M) and in the absence of competing amino acids. This perfusion
L
-[14C]leucine
l
technique enabled the determination of inhibitory transport activity of the
compounds against the in vivo blood–brain barrier system LAT1 transporter in
the absence of endogenous competitors and normal plasma proteins. The
circulation to the brain is taken over for a brief period of time (<30 s) by
infusing physiologic saline into the carotid artery at a physiological rate. The
infusion rate is adjusted so that perfusion pressure matches normal systemic
blood pressure (80–110 mm Hg) and allows the delivery of a constant, defined
concentration of the test compound in the perfusion fluid to the brain in the
absence of plasma proteins, ions and other substrates that may compete for
transport. Furthermore, delivery of the test compounds to the brain occurs
without prior exposure to the peripheral organs and metabolic enzymes,
minimizing the confounding effects of radioactive metabolites. Although
metabolism may occur in the brain, due to the limited time exposure of the
perfusion, there is minimal loss of tracer products, such as 14C–carbon dioxide.
Adult male Sprague–Dawley rats weighing between 240 and 450 g were used