4
M. Gao et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
flow rate 1.0 mL/min; UV (254 nm) and
preparative RP HPLC was performed using a Prodigy (Phenomenex) 5
column, 10 Â 250 mm; mobile phase 70%CH CN/30%H O; flow rate 4 mL/min;
UV (254 nm) and -ray (PIN diode) flow detectors. C18 Light Sep-Pak cartridges
were obtained from Waters Corporation (Milford, MA). Sterile Millex-FG
.2 m filter units were obtained from Millipore Corporation (Bedford, MA).
b). 6-Amino-2-(benzylthio)pyrimidin-4-ol (1): Benzyl bromide (32.23 g,
89 mmol) was added dropwise into a solution of 4-amino-6-hydroxy-2-
mercaptopyrimidine (25.77 g, 180 mmol) in 1 M NaOH (36 mL) and water
6 mL). The reaction was kept stirring at 40–50 °C for 4 h and was left at RT for
2 h. Then the mixture was neutralized with acetic acid to obtain a white solid,
which was collected by filtration, washed with water and hexanes, and dried in
air to give 1 as a white solid (38.70 g, 92%). R = 0.70 (1:9 MeOH/CH Cl ), mp
): d 4.32 (s, 2H, CH ), 6.52 (s, 2H, NH ), 7.24 (t,
J = 7.5 Hz, 1H, Ph-H), 7.30 (t, J = 7.5 Hz, 2H, Ph-H), 7.41 (d, J = 7.5 Hz, 2H, Ph-H),
c
-ray (PIN diode) flow detectors. Semi-
2
1.71–1.76 (m, 2H, CH ), 1.77–1.79 (m, 1H, CH), 4.32 (d, J = 13.5 Hz, 1H, SCHH),
l
m C-18
4.47 (d, J = 13.5 Hz, 1H, SCHH), 4.75–4.77 (m, 1H, NCH), 7.18 (t, J = 7.5 Hz, 1H,
Ph-H), 7.26 (t, J = 7.0 Hz, 2H, Ph-H), 7.40 (d, J = 7.5 Hz, 2H, Ph-H). 13C NMR
3
2
c
4
(MeOD-d ): d 22.33, 23.70, 26.28, 36.10, 43.03, 55.86, 127.87, 129.38, 130.00,
+
139.85, 155.98, 157.13, 168.53, 169.67, 173.64, 180.60. MS (ESI): 404 ([M+H] ,
À
0
(
1
l
100%); MS (ESI): 402 ([MÀH] , 10%). HRMS (ESI) calcd for C18
21 5 2 2
H N O S H,
+
11
404.1215 ([M+H] ); found 404.1223. (h). ([ C]methyl (2-amino-5-(benzylthio)
11
thiazolo[4,5-d]pyrimidin-7-yl)-
D
-leucinate ([11C]5): [ C]CO
2
was produced by
3
the 14N(p,
a) C nuclear reaction in the small volume (9.5 cm ) aluminum gas
11
(
1
target provided with the Siemens RDS-111 Eclipse cyclotron. The target gas
consisted of 1% oxygen in nitrogen purchased as a specialty gas from Praxair,
Indianapolis, IN. Typical irradiations used for the development were 58
lA
f
2
2
beam current and 15 min on target. The production run produced approxi-
1
11
2
30–232 °C. H NMR (DMSO-d
6
2
2
mately 25.9 GBq of [ C]CO
dissolved in CH CN (300
2
at EOB. The acid precursor (6, 0.1–0.3 mg) was
L). To this solution was added aqueous NaOH (2 N,
3
l
+
À
1
1.46 (s, 1H, OH). MS (ESI): 234 ([M+H] , 100%); MS (ESI): 232 ([MÀH] , 14%).
2
l
L). The mixture was transferred to a small reaction vial. No-carrier-added
11
(
(
c). 6-Amino-2-(benzylthio)-5-thiocyanatopyrimidin-4-ol (2): Compound
1
(high specific activity)
production method within 12 min from [ C]CO
[
C]CH
3
OTf that was produced by the gas-phase
13
11
11
11
37.36 g, 160.3 mmol) and KSCN (65.44 g, 673 mmol) were suspended in
2 4
through [ C]CH and [ C]
DMF (760 mL) and heated to 60 °C. Pidine (22.8 g, 288.5 mmol) was added, and
the solution was cooled to 5 °C. Bromine (25.62 g, 160.3 mmol) was added
dropwise, and the solution was stirred at 5–10 °C for 2 h and then at RT
overnight. The mixture was poured into ice water and stirred for 1 h. The
CH Br with silver triflate (AgOTf) column was passed into the reaction vial at
RT until radioactivity reached a maximum (2 min), and then the reaction vial
was isolated and heated at 80 °C for 3 min. The contents of the reaction vial
3
3
were diluted with aqueous NaHCO (0.1 M, 1 mL). The reaction vial was
resulting solid was filtered, washed with cold water, and dried in air to afford 2
connected to a 3-mL HPLC injection loop. The labeled product mixture solution
was injected onto the semi-preparative HPLC column for purification. The
product fraction was collected in a recovery vial containing 30 mL water. The
diluted tracer solution was then passed through a C-18 Sep-Pak Light cartridge,
and washed with water (3 Â 10 mL). The cartridge was eluted with EtOH
(3 Â 0.4 mL) to release the labeled product, followed by saline (10–11 mL). The
1
as a white solid (38.45 g, 83%). R
NMR (DMSO-d ): d 4.38 (s, 2H, CH
2.5 Hz, 2H, Ph-H), 7.46 (t, J = 6.0 Hz, 2H, Ph-H), 7.60 and 7.80 (2 s, 2H, NH
f
= 0.82 (1:9 MeOH/CH
2
Cl
2
), mp 235–237 °C.
H
6
2
), 7.25–7.27 (m, 1H, Ph-H), 7.31 (dd, J = 7.0,
),
1
1
2
+
À
2.34 (s, 1H, OH). MS (ESI): 291 ([M+H] , 100%); MS (ESI): 289 ([MÀH] , 90%).
(
(
d). 2-Amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-ol (3): Compound
2
20.3 g, 70 mmol) was suspended in water (45 mL) and DMF (140 mL) and
eluted product was then sterile-filtered through a Millex-FG 0.2 lm membrane
heated at 120 °C for 27 h. The reaction mixture was poured onto ice, and
resulting pale-yellow precipitate was collected by filtration and washed with
cold water. The solid material was suspended in water (250 mL) and heated to
into a sterile vial. Total radioactivity was assayed and total volume (10–11 mL)
was noted for tracer dose dispensing. The overall synthesis time including
HPLC-SPE purification and reformulation was ꢀ40 min from EOB. The decay
corrected radiochemical yield was 40–50%. Retention times in the analytical
7
5 °C, and NaOH (10 M, 17 mL) was added. The resulting suspension was
11
filtered, and the product was precipitated by addition of concentrated HCl until
HPLC system were:
t
R
6 = 3.08 min,
t
R
5 = 5.64 min,
t
R
[
R
C]5 = 5.73 min.
6 = 3.75 min, t
C]5 = 8.48 min. (i). Radioligand depletion experiments: Radi-
oligand depletion experiments were performed using CX CR1 cell membrane
pH 4. The solid was filtered, and dried in vacuo to give 3 as a white solid
Retention times in the preparative HPLC system were: t
R
1
11
(
4
19.28 g, 95%). R
.41 (s, 2H, CH
f
= 0.63 (1:9 MeOH/CH
2
Cl
2
), mp > 330 °C. H NMR (DMSO-d
6
): d
R
5 = 8.36 min, t [
2
), 7.23–7.26 (m, 1H, Ph-H), 7.31 (t, J = 7.5 Hz, 2H, Ph-H), 7.43 (d,
3
+
11
J = 7.5 Hz, 2H, Ph-H), 8.17 (s, 2H, NH
1
2
), 12.54 (s, 1H, OH). MS (ESI): 291 ([M+H] ,
preps (Millipore) to measure the bound radioligand [ C]5 concentration. Four
À
00%); MS (ESI): 289 ([MÀH] , 20%). (e). 5-(Benzylthio)-7-chlorothiazolo[4,5-d]
replicates of membranes (0.004–4.5 mg protein/mL assay medium) were
11
pyrimidin-2-amine (4): Compound 3 (10.0 g, 34.5 mmol) was suspended in
POCl (100 mL), and the mixture was heated at reflux for 5 h. After the reaction
incubated with 1 nM or 10 nM [ C]5 in a final volume of 0.2 mL in assay
buffer (25 mM HEPES, 10 mM MgCl 1 mM CaCl 1 mM EDTA, pH 7.4)
for30 min at 25 °C. Unlabeled compound 5 was added at 100 fold excess of
3
2
,
2
,
mixture was concentrated in reduced pressure, the ice water was slowly added
into above residue. The resulting precipitate was filtered, washed with cold
11
[
C]5 to determine non-specific binding. In some experiments 10% Solutol
water, and dried in air to give 4 as a yellow solid (9.67 g, 91%). R
f
= 0.70 (1:1
): d 4.38 (s, 2H, CH ), 7.24
t, J = 7.5 Hz, 1H, Ph-H), 7.31 (d, J = 7.5 Hz, 2H, Ph-H), 7.49 (d, J = 7.5 Hz, 2H, Ph-
(Sigma) was added to increase the solubility of the radioligand. To measure the
total radioactivity added to the experiment, 20 lL of stock radioligand [ C]5
1
11
EtOAc/hexanes), mp 217–219 °C. H NMR (acetone-d
6
2
(
(amount added to each experimental well) was spotted onto a unifilter GF/B
plate then allowed to air dry. An additional aliquot was added to a scintillation
vial and counted on a Beckman LSC to determine CPMs (counts per minute)
added. For termination of the binding reaction, the samples were filtered onto
GF/B unifilter plates (Perkin Elmer) that had been pre-soaked in 20 mM
tetrasodium pyrophosphate for 30 min using a unifilter-96 cell harvester
(Perkin-Elmer). Plates were washed 6 times with ice cold saline, dried under a
vacuum, and exposed to a TR2025 phosphorscreen (GE Healthcare) for 20–
60 min. Exposed phophorscreens were then read on a Typhoon FLA-7000IP (GE
+
À
H), 8.13 (s, 2H, NH
2
). MS (ESI): 309 ([M+H] , 100%); MS (ESI): 307 ([MÀH] ,
-leuci-
nate (5): To a solution of compound 4 (0.93 g, 3.0 mmol) in anhydrous CH CN
-leucinate (1.45 g, 10.0 mmol) and DIPEA
0.78 g, 6.0 mmol). The reaction mixture was stirred and heated at 100 °C for
days. Then the reaction mixture was concentrated in vacuo, and the residue
was purified by column chromatographyon silica gel with eluent (1:99 to 5:95
6
0%). (f). Methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-D
3
(
(
4
120 mL) were added methyl D
MeOH/CH
MeOH/CH
2
Cl
Cl
2
) to afford 5 as a yellowish solid (138 mg, 11%). R
f
= 0.38 (1:14
): d 0.92 (d, J = 6.5 Hz, 3H,
), 1.62–1.68 (m, 1H, CH), 1.72–1.84 (m, 2H,
), 4.33 (dd, J = 5.5, 13.5 Hz, 1H, SCHH), 4.41 (dd,
1
11
2
2
), mp 143–145 °C. H NMR (MeOD-d
4
Healthcare) along with [ C]5 calibration standards. CPMs were determined by
CH
CH
3
), 0.95 (d, J = 6.5 Hz, 3H, CH
), 3.65 (d, J = 5.5 Hz, 3H, CH
3
calibrating the image to the CPMs in the calibration standards via MCID
analysis Software. (j). Ligand competitive binding assays: To compare the affinity
of the unlabeled compound 5 (ligand) with the native ligand, fracktalkine,
competitive binding assays were performed using the above cell membrane.
2
3
J = 5.5, 13.5 Hz, 1H, SCHH), 4.83 (dd, J = 5.0, 10.0 Hz, 1H, NCH), 6.68 (s, 1H, NH),
.20–7.24 (dd, J = 2.0, 8.0 Hz, 1H, Ph-H), 7.29 (dd, J = 2.0, 8.5 Hz, 2H, Ph-H), 7.44
7
1
(
s, 2H, NH
2
), 7.47 (t, J = 8.5 Hz, 2H, Ph-H); H NMR (CDCl
3
): d 0.88 (d, J = 6.5 Hz,
), 1.62 (t, J = 7.0 Hz, 2H, CH ), 1.75–1.80 (m,
), 4.77–4.78 (m, 1H, NCH), 5.96 (s,
Approximately 0.5 lg per well of membrane protein was incubated with 11
3
1
1
H, CH
3
), 0.92 (d, J = 6.5 Hz, 3H, CH
3
2
different concentrations of the compound over a six log unit range. Assays were
run with 0.025 nM [1 I]fractalkine (Perkin Elmer) in assay buffer as above with
the addition of 0.5% BSA (bovine serum albumin) to reduce non-specific
binding. Triplicate determinations were done at each concentration of the test
compound. Unlabeled fractalkine (Peprotech) was used to determine non-
specific binding. Each assay plate included a positive control (fractalkine) and a
negative control (buffer). Assays were incubated at 25 °C for 2 h. After
equilibrium was reached, bound radioligand was separated from free radioli-
gand using a Perkin Elmer 96-well filtration apparatus. Plates were washed at
least 3 times with ice cold saline. Filters were quickly dried under a vacuum
and radioactive counts collected on a Perkin Elmer TopCount microplate
scintillation counter using a counting protocol for 1 I. Data was analyzed with
Prism 7 (GraphPad Software Inc.) to calculate Ki values.
25
H, CH), 3.71 (s, 3H, OCH
H, NH), 7.18 (t, J = 7.0 Hz, 1H, Ph-H), 7.23 (d, J = 7.5 Hz, 2H, Ph-H), 7.38 (d,
3
), 4.35 (s, 2H, SCH
2
+
À
J = 7.5 Hz, 2H, Ph-H). MS (ESI): 417 ([M+H] , 100%); MS (ESI): 416 ([MÀH] ,
1
5%). (g). 2-Amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)- -leucine (6): To
D
a solution of compound 5 (83 mg, 0.2 mmol) in methanol (40 mL) was added
KOH (0.5 g, 8.9 mmol). The reaction mixture was stirred at RT for 21 h. Then the
reaction mixture was concentrated in vacuo, and the mixture was neutralized
with 1 N HCl. The mixture was extracted with EtOAc (3 Â 60 mL), and the
4
combined organic layers were washed with brine, dried over MgSO , and
concentrated. The resulting residue was purified by column chromatography
on silica gel with eluent (5:95 to 30:70 MeOH/CH Cl ) to give 6 as a white off
solid (40 mg, 50%). R = 0.24 (1:5 MeOH/CH Cl ), mp 245 °C (decomposed).
3
NMR (MeOD-d ): d 0.92 (d, J = 6.5 Hz, 3H, CH ), 0.94 (d, J = 6.5 Hz, 3H, CH ),
25
2
2
1
f
2
2
H
4
3
Gao, Mingzhang
