S. Biselli, M. Bresinsky, K. Tropmann et al.
European Journal of Medicinal Chemistry 214 (2021) 113190
and TFA (4.0 mL) according to the general procedure (method A)
4.2.18. 1-(Amino((2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)
amino)methylene)-3-(1-hexyl)urea (185)
and obtained as a colorless foamlike solid (210 mg, 74%): RP-HPLC:
20
99%, (tR ¼ 11.14, k ¼ 2.53), ee ¼ 99%, [
a
]
D þ26.54 (c 0.31, MeOH).
136 was prepared from 12 (0.07 g, 0.44 mmol), 50 (0.14 g,
0.44 mmol), HgCl2 (0.24 g, 0.88 mmol) and TEA (0.18 mL,
1.32 mmol) in DCM (25 mL) conforming to the general procedure
for the guanidinylation reaction. Subsequently, 185 was prepared
from the Boc-protected intermediate 136 in DCM (4.0 mL) and TFA
(1.0 mL) according to the general procedure (method A) and ob-
tained as a colorless foamlike solid (46 mg, 18%): RP-HPLC: 95%,
1H NMR (300 MHz, CD3OD, di-trifluoroacetate)
d
(ppm) 7.59e6.96
(m, 5H), 4.88e4.84 (m, 1H), 3.34e3.25 (m, 2H), 2.69 (t, J ¼ 7.5 Hz,
2H), 2.16 (s, 3H), 1.87 (quint, J ¼ 7.1 Hz, 2H), 1.47 (d, J ¼ 7.0 Hz, 3H).
13C NMR (75 MHz, CD3OD, di-trifluoroacetate)
d (ppm) 170.38,
155.99, 154.73, 144.89, 132.62, 129.68, 128.38, 127.03, 118.40, 51.17,
41.44, 29.89, 23.58, 22.70, 11.45. HRMS (ESI-MS): m/z [M þ Hþ]
calculated for C17H25N6OSþ: 361.1805, found 361.1802; C17H24N6OS
x 2 TFA (588.53).
(tR
¼
17.10,
k
¼
4.90). 1H NMR (600 MHz, DMSO‑d6, di-
(ppm) 10.17 (s, 1H), 9.07 (s,1H), 8.67 (br s, 4H),
trifluoroacetate):
d
7.50 (s, 1H), 4.05e4.04 (m, 1H), 3.09e3.06 (m, 2H), 2.89e2.86 (m,
1H), 2.60e2.51 (m, 3H), 1.97e1.83 (m, 2H), 1.42e1.39 (m, 2H),
1.29e1.24 (m, 6H), 0.86e0.84 (m, 3H). 13C NMR (150 MHz,
4.2.15. 1-(Amino((3-(2-amino-4-methylthiazol-5-yl)propyl)amino)
methylene)-3-(1-(3-fluorophenyl)ethyl)urea (157)
157 was prepared from 108 (0.21 g, 0.36 mmol) in DCM (8.0 mL)
and TFA (2.0 mL) according to the general procedure (method A)
and obtained as a colorless foamlike solid (100 mg, 45%): RP-HPLC:
99%, (tR ¼ 9.50, k ¼ 2.56). 1H NMR (400 MHz, CD3OD, di-
DMSO‑d6, di-trifluoroacetate):
136.50, 112.00, 46.90, 39.49, 31.30, 29.30, 28.40, 26.80, 26.30, 22.50,
21.70, 14.30. HRMS (ESI-MS): m/z [M
Hþ] calculated for
15H27N6OSþ: 339.1962, found 339.1962; C15H26N6OS x 2 TFA
(566.52).
d (ppm) 168.90, 154.20, 153.70,
þ
C
trifluoroacetate)
d (ppm) 7.38e7.28 (m, 1H), 7.16e6.94 (m, 3H),
4.91 (q, 1H), 2.69 (t, J ¼ 7.6 Hz, 2H), 2.15 (s, 3H), 1.94e1.81 (m, 2H),
1.47 (d,
7.0 Hz, 3H). 13C NMR (101 MHz, CD3OD, di-
trifluoroacetate)
J
¼
4.3. Biological assays: experimental protocols
d
(ppm) 168.92, 162.98 (d, J ¼ 244.6 Hz), 162.07
(q, J ¼ 34.6 Hz, TFA), 154.54, 153.34, 146.50 (d, J ¼ 6.8 Hz), 131.26,
129.98 (d, J ¼ 8.2 Hz), 121.44 (d, J ¼ 2.9 Hz), 116.95, 116.59 (q,
J ¼ 291.7 Hz, TFA), 113.50 (d, J ¼ 21.3 Hz), 112.42 (d, J ¼ 22.2 Hz),
49.34 (d, J ¼ 1.2 Hz), 48.03, 40.00, 28.43, 22.13, 21.03,10.02. 19F NMR
Cell culture. Cells were cultured in 75 cm2 flasks (Sarstedt,
Nümbrecht, Germany) in a humidified atmosphere (95% air, 5%
CO2) at 37 ꢁC. HEK293T-
ElucN-
b
-Arr2-hH2R cells [43e45], HEK293T
b
arr2 hD2longR-ELucC cells [54] and HEK293T ElucN- arr2
b
(377 MHz, CD3OD, di-trifluoroacetate)
d
(ppm) ꢀ75.42
hD3R-ELucC cells [54] were cultured as described previously and
regularly monitored for mycoplasma infection using the Venor
GeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany).
Radioligand binding assays. Histamine H1-4 receptors. Radio-
ligand competition binding experiments using membranes of Sf9
(TFA), ꢀ113.47. HRMS (ESI-MS): m/z [M þ Hþ] calculated for
C
17H24FN6OSþ: 379.1711, found 379.1711; C17H23FN6OS x 2 TFA
(606.52).
4.2.16. (R)-1-(Amino((3-(2-amino-4-methylthiazol-5-yl)propyl)
amino)methylene)-3-(1-phenylethyl)thiourea (174)
insect cells co-expressing hH1R and RGS4, expressing hH2R-Gs ,
aS
co-expressing hH3R, Gai2 and Gb1g2 or co-expressing hH4R, Gai2
and Gb1g2 were performed according to published protocols
[14,39]. The following radioligands were used: [3H]mepyramine
(hH1R, specific activity: 20 Ci/mmol, Hartmann Analytics,
174 was prepared from 125 (0.05 g, 0.09 mmol) in DCM (4.0 mL)
and TFA (1.0 mL) according to the general procedure and obtained
as a colorless solid (30 mg, 57%), mp 75e78 ꢁC, RP-HPLC: 95%,
20
(tR ¼ 14.18, k ¼ 3.49), ee ¼ 99%, [
a
]
þ20.30 (c 0.34, MeOH). 1H
Braunschweig, Germany or specific activity: 75e87 Ci/mmol,
D
3
€
€
NMR (300 MHz, CD3OD, di-trifluoroacetate)
d
(ppm) 7.42e7.19 (m,
Novandi Chemistry AB, Sodertalje, Sweden), [ H]UR-DE257 [38]
(hH2R, specific activity: 63.0 Ci/mmol, was synthesized in our lab-
oratories), [3H]Na-methylhistamine (specific activity: 85.3 Ci/
mmol, Hartmann Analytics) or [3H]UR-PI294 [40] (hH3R, specific
activity: 41.8 Ci/mmol, was synthesized in our laboratories) and
[3H]histamine (hH4R, specific activity: 25 Ci/mmol Hartmann An-
alytics). Modifications were made as follows: the washing steps
were performed with PBS (8 g NaCl, 0.2 g KCl,1.0 g Na2HPO4 x 2H2O,
0.15 g NaH2PO4 x H2O, 0.1 g KH2PO4 in 1 L Millipore H2O; pH 7.4;
5H), 5.45 (q, J ¼ 7.5, 7.0 Hz, 1H), 3.36 (t, J ¼ 6.7 Hz, 2H), 2.74 (t,
J ¼ 7.6 Hz, 2H), 2.17 (s, 3H), 1.91 (quint, J ¼ 6.9 Hz, 2H), 1.54 (d,
J ¼ 7.0 Hz, 3H). 13C NMR (75 MHz, CD3OD, di-trifluoroacetate)
d
(ppm) 179.76, 170.37, 156.02, 143.45, 132.68, 129.70, 128.58,
127.48, 118.35, 54.90, 41.74, 29.71, 23.71, 21.84, 11.53. HRMS (ESI-
MS): m/z [M þ Hþ] calculated for C17H25N6Sþ2 : 377.1577, found
377.1578; C17H24N6S2 x 2 TFA (604.59).
4.2.17. 1-(Amino((3-(2-aminothiazol-4-yl)phenyl)amino)
4
ꢁC) instead of binding buffer.
methylene)-3-(1-propyl)urea (179)
Dopamine D2long, receptors. Radioligand competition binding
3
130 was prepared from 11 (0.06 g, 0.29 mmol), 48 (0.08 g,
0.29 mmol), HgCl2 (0.16 g, 0.58 mmol) and TEA (0.12 mL,
0.87 mmol) in DCM (25 mL) conforming to the general procedure
for the guanidinylation reaction. Subsequently, 179 was prepared
from the Boc-protected intermediate 130 in DCM (4.0 mL) and TFA
(1.0 mL) according to the general procedure (method A) and ob-
tained as a colorless foamlike solid (40 mg, 25%): RP-HPLC: 99%,
experiments using homogenates of HEK293T-CRE-Luc cells
expressing hD2longR or hD3R were performed as described in detail
by Forster et al. [54] [3H]N-methylspipirone (specific activity: 77 Ci/
mmol) was from Novandi Chemistry AB.
[
35S]GTP S binding assay. The [35S]GTP
g gS binding assay was
performed using membranes of Sf9 insect cells expressing the
hH2R-Gs S fusion protein as described in detail by Kagermeier et al.
a
(tR
¼
13.87,
k
¼
3.78). 1H NMR (600 MHz, DMSO‑d6, di-
g
S was from PerkinElmer Life Science (Boston, USA) or
Hartmann Analytics.
-Arrestin recruitment assay. The
says using HEK293T- -Arr2-hH2R cells [43,45], HEK293T ElucN-
arr2 hD2longR-ELucC cells [54] or HEK293T ElucN- arr2 hD3R-
trifluoroacetate):
d
(ppm) 10.74 (s, 1H), 10.15 (s, 1H), 8.97 (br s,
1H), 8.62 (br s, 1H), 7.80e7.78 (m, 1H), 7.73e7.72 (m, 1H), 7.60 (t, 1H,
J ¼ 5.56 Hz), 7.49 (t, J ¼ 7.74 Hz, 1H), 7.24e7.23 (m, 1H), 7.15 (s, 1H),
b
b-arrestin recruitment as-
b
3.10e3.06 (m, 2H), 1.49e1.43 (m, 2H), 0.86 (t, J ¼ 7.50 Hz, 3H). 13
C
b
b
NMR (150 MHz, DMSO‑d6, di-trifluoroacetate):
d
(ppm) 168.70,
ELucC cells [54] were performed as described previously.
Histamine H2 receptor assay on the isolated guinea pig right
atrium (spontaneously beating). This functional assay was per-
formed as previously described in detail [39].
153.60, 153.50, 147.00, 135.50, 134.00, 130.10, 124.84, 124.81, 123.00,
103.00, 41.00, 22.20,11.20. HRMS (ESI-MS): m/z [M þ Hþ] calculated
for C14H19N6OSþ: 319.1336, found 319.1346; C14H18N6OS x 2 TFA
(546.45).
Data processing. Compound purities were calculated as the
17