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complete data acquisition from the second HTS assay, we compared the results
obtained from the two HTS systems and correlated the restoration of the yeast
growth with the selective elimination of cancer cells. This cross analysis enabled us
to successfully identify E260 as the best candidate for inhibiting the Fer-kinase
activity and for selectively killing cancer cells. Notably, we found a direct
correlation between the successful restoration of growth in Fer-expressing yeast
cells and the effective and selective cytotoxic properties of the compounds in cancer
cells. E260 was also computationally analyzed using the ChemSoft software
8.82 (s, 1H), 4.61–4.03 (m, 2H), 3.65–3.25 (m, 5H), 3.20–2.82 (m, 6H), 2.74 (s, 3H),
1.84–1.65 (m, 4H); 13C-NMR (CD3OD) δ: 174.13 (C5′′), 54.51 (C8′′a), 54.20 (C8′′b),
44.27 (C2′′), 43.69 (C10′′), 43.65 (C7′′a), 40.06 (C7′′b), 36.30(C4′′), 26.65 (C3′′b), 26.43
(C3′′a); HRMS (m/z): [M + H]+ calcd. for C11H21N3O, 212.17574; found, 212.1762.
MP: 298 °C.
Compound IV (Supplementary Fig. 1C): 1-Methyl-4-(piperidin-4-ylmethyl)
piperazine. 1H-NMR (DMSO-d6) δ: 3.30–2.92 (br, 2H), 2.90 (m, 2H), 2.39
(t, J = 12Hz, 2H), 2.35–2.17 (m, 6H), 2.15 (s, 3H), 2.05 (d, 2H), 1.57 (m, 2H), 1.51
(m, 1H), 0.92 (m, 2H); 13C-NMR (CDCl3) δ: 65.28 (C5′′), 55.19 (C8′′), 53.64 (C7′′),
46.64 (C2′′), 46.08 (C10′′), 33.80 (C4′′), 32.30 (C3′′); HRMS (m/z): [MH]+ calcd. for
(ChemDiv, USA) and proven to possess drug-likeness according to the Lipinski’s
61
rule of 5
.
Chemical analyses of the newly developed compounds. E260 was synthesized
(Supplementary Fig. 1C) and used as a tartrate salt (compound X in Supplementary
Fig. 1C) for the in vitro mammalian cell-cultures and in vivo experiments. The
chemical identities of the synthetic intermediates were determined using 1H-NMR,
HRMS, and LC–MS analyses (Supplementary Fig. 2 and below). The chemical
identity and sample purity of the final synthesis product and the main SMC used in
this study, E260-tartrate, (X) were determined using 1H-NMR, 13C-NMR, LC–MS,
FTIR, and UPLC integrated with HRMS (Supplementary Fig. 1D–I and below).
NMR spectra were recorded, in Bruker instruments, for the following materials:
II (in CDCl3, 1H at 300 MHz, and 13C at 176.1 MHz at 300 K); III (in DMSO-d6,
C
11H23N3, 198.19647; found, 198.19623.
Compound Va (Supplementary Fig. 1C): 2-Amino-1,3,4-thiadiazol. 1H-NMR
(DMSO-d6) δ: 8.55 (s, 1H), 7.18 (s, 2H);); 13C-NMR (Acetone-d6) δ: 168.98 (C5),
143.16 (C2); HRMS (m/z): [M + H]+ calcd. for C2H3N3S, 102.01204; found,
102.01207. MP: 185–187 °C (ref. 62; 191 and 192 °C).
Compound VI (Supplementary Fig. 1C): 2-Amino-5-bromo-1,3,4-thiadiazole.
1H-NMR (DMSO-d6) δ: 7.51 (s, 2H); 13C-NMR (acetone-d6) δ: 171.57 (C5), 125.23
(C2); HRMS (m/z): [M + H]+ calcd. for C15H18BrNO, 179.92256 and 181.92042;
found, 179.92289 and 181.92066 (1:1 doublet). MP: 192–193 °C (ref 63;180 and
181 °C).
1H at 300 MHz, and in CD3OD, 13C at 176.1 MHz at 300 K); IV (in DMSO-d6, 1
at 300 MHz, and in CDCl3, 13C at 176.1 MHz at 300 K); Va (in DMSO-D6, 1H
H
at 300 MHz, and in Acetone-d6, 13C at 176.1 MHz. at 300 K); VI (in DMSO-D6, 1H
at 300 MHz, and in Acetone-d6 13C at 176.1 MHz at 300 K; VIII (in DMSO-d6, 1
at 400.4 MHz, and in CDCl3, 13C at 176.1 MHz at 300 K); IX (in CDCl3, 1H at
H
400.4 MHz, and in CDCl3, 13C at 176.1 MHz at 300 K); and X (in DMSO-d6, 1H
and 13C at 700.5 and 176.1 MHz, respectively, at 318 K). For X, a series of
2D spectra were also obtained (COSY-1H × 1H correlation; HMQC-one-bond
13C × 1H correlation; and HMBC-long-range 13C × 1H correlation), which allowed
the full attribution of all carbon and proton signals and confirms the chemical
structure as shown.
LC–MS (HR) information: UPLC (Agilent 1260) integrated with HRMS were
obtained on a 6545 QTOF instrument (Agilent) using the following parameters:
HPLC-Agilent 1260: Column: Agilent Zorbax HD-C18, 1.8-Micron, 2.1 × 50
mm, mobile phase A: water/0.1% formic acid, mobile phase B: acetonitrile/0.1%
formic acid, gradient: 15–95% B in 10 min, hold 95% B to 2 min, 95% B to 5% in
2 min, 5% B for 2 min, then stop. Flow rate: 0.5 ml/min. Column temperature: 30 °
C. Sample preparation: dissolved in water/acetonitrile (1:1). Detector: 210, 254, and
355 nm
Compound VIII (Supplementary Fig. 1C): 2-Bromo-6-(4-isopropylphenyl)
imidazo[2,1-b][1,3,4]thiadiazole. 1H-NMR (DMSO-d6) δ: 8.68 (s, 1H, H5),
7.77 (d, J = 8 Hz, 2H, H2′), 7.28 (d, J = 8 Hz, 2H, H3′), 2.90 (m, 1H, H5′), 1.22
(d, J = 7 Hz, 6H, H6′); 13C-NMR (CDCl3) δ: 149.32 (C4′), 145.39 (C6), 145.31 (C8),
133.82 (C2), 129.76 (C1′), 127.03 (C3′), 125.28 (C2′), 109.54 (C5), 33.95 (C5′),
23.90 (C6′) MS (ESI+) (m/z) [MH]+:calcd. for C13H12BrN3S 322.2, 323.9; found
322.2, 324.0; HRMS (m/z): [MH]+ calcd. for C13H12BrN3S, 322.00081; found,
322.00047. MP: 139–141 °C.
Compound IX (Supplementary Fig. 1C): 6-(4-isopropylphenyl)-2-{4-[(4-
methylpiperazine-1-yl)methyl]piperidin-1-yl}imidazo[2,1-b][1,3,4]thiadia-
zole. 1H-NMR (CDCl3) δ: 7.71 (m, 3H, H5, H2′), 7.26 (d, J = 8 Hz, 2H, H3′),
3.87 (d, J = 12 Hz, 2H, H2′′), 3.09 (t, J = 12 Hz, 2H, H2′′), 2.93 (sept, J = 6.5 Hz,
1H, H5′), 2.50 (br, 8H, H7′′, H8′′), 2.34 (s, 3H, H10′′), 2.25 (d, J = 6.8 Hz, 2H, H5′′),
1.91 (d, J = 12.2 Hz, 2H, H3′′), 1.79 (m, 1H, H4′), 1.33 (d, J = 12.2 Hz, 2H, H3′′),
1.29 (m, 6H, H6′); 13C-NMR (CDCl3) δ: 164.59 (C2), 147.50 (C4′), 143.56 (C6),
141.30 (C8), 132.09 (C1′), 126.64 (C3′), 124.57 (C2′), 108.58 (C5), 64.05 (C5′′),
55.15 (C8′′), 53.63 (C7′′), 48.45 (C2′′), 46.05 (C10′′), 33.83 (C5′), 33.15 (C4′), 29.76
(C3′), 23.98 (C6′); MS (ES+) (m/z): [MH]+ calcd. for C24H34N6S 439.0; found,
439.5;); HRMS (m/z): [MH]+ calcd. for C24H34N6S 439.26384; found, 439.26346.
MP: 190–192 °C.
HRMS parameters: Interface:. ESI (positive), SheathGasFlow 12,
SheathGasTemp (°C) 400, Nebulizer (psig) 40, Gas Flow (l/min) 8, Gas Temp (°C)
300, OctopoleRFPeak 750, Skimmer1 45, Fragmentor 180, Nozzle Voltage (V) 0,
VCap 3500.
LC–MS information: HPLC-Shimadzu Analytical 10Avp series,
autosampler: Gilson 215, ELSD: Sedex 75(55), mass spectrometer: PE SCIEX
API 165(100). Column: Synergi 2 μ Hydro-RP Mercury, 20 × 2.0 mm. Mobile
phase A: water/0.05% TFA. Mobile phase B: acetonitrile/0.05% TFA. Gradient:
0.01 min—5% B, 3.6 min—95% B, 4.5 min—95% B, 5.00 min—controller stop.
Flow rate: 0.7 ml/min. Pressure range: 0–4500 psi. Sample preparation: dissolved
in water/acetonitrile (1:1). Detector: 210, 254 nm. MS parameters: Interface: ESI
(positive).
Fourier transform infrared spectroscopy: FTIR spectrum was collected using a
Thermo Scientific Nicolet iS10 FTIR spectrometer equipped with a Smart iTR
attenuated total reflectance sampler containing a single bounce diamond crystal.
Data were collected and analyzed using OMNIC software. Spectra were collected in
the 650–4000 per cm range at a spectral resolution of 4.
Melting point determination: The melting point (MP) of Compound III was
determined using the Buchi melting point apparatus M-560 (BUCHI Corporation,
UK) set to default parameters. A quick capillary-thermometer-hotplate based
method was used for all other compounds. Briefly, small amount of finely
powdered crystals of the test compound was placed in a thin-walled capillary tube.
The tube was placed in very close proximity to a thermometer and capillary tube
was heated in a small electric hotplate (IKA, Germany) in a rate of heating no more
than 1° or 2° rise per minute. The capillary was closely observed until the crystals
were completely melted to determine the MP. Differences in measured versus
literature reported MP values could result from differences in determination
methods and compounds purity.
Compound X (Supplementary Fig. 1C): 6-(4-isopropylphenyl)-2-{4-[(4-
methylpiperazine-1-yl)methyl]piperidin-1-yl}imidazo[2,1-b][1,3,4]thiadiazole
tartrate (salt). 1H-NMR (DMSO-d6) δ: 8.17 (s, 1H, H5), 7.68 (d, J = 8 Hz, 2H,
H2′), 7.22 (d, J = 8 Hz, 2H, H3′), 4.14 (s, 2H, CHOH-tartrate), 3.87 (d, J = 13 Hz,
2H, H2′′), 3.14 (t, J = 13 Hz, 2H, H2′′), 2.88 (sept, J = 7 Hz, 1H, H5′), 2.69 (m, 4H,
H
8′′) 2.49 (m, 4H, H7′′), 2.42 (s, 3H, H10′′), 2.22 (d, J = 7 Hz, 2H, H5′′), 1.81
(d, J = 12 Hz, 3H, H3′′, H4′′), 1.29 (m, 2H, H3′′), 1.21 (d, J = 7 Hz, 6H, H6′)
13C-NMR (DMSO-d6) δ: 173.36 (COOH), 164.14 (C2), 146.68 (C4′), 142.48
(C6), 140.40 (C8), 132.01 (|C1′), 126.29 (C3′), 124.14 (C2′), 109.35 (C5), 71.69
(CHOH-tartrate), 62.72 (C5′′), 53.75 (C8′′), 51.58 (C7′′), 44.13 (C10′′), 48.22
(C2′′), 33.03 (C5′), 32.07 (C4′′), 29.05 (C3′′), 23.74 (C6′); HRMS (m/z): [MH]+
calcd. for C24H34N6S,439.263; found, 439.263. MP: 206–207 °C.
Synthesis process of E260 as a tartrate salt. The following compounds were
synthesized according to the scheme in Supplementary Fig. 1C.
Compound II: Tert-butyl 4-[(4-methylpiperazin-1-yl)carbonyl]piperidine-1-
carboxylate. 218 mmol (50.00 g) of compound I were stirred with 218 mmol
(35.36 g) of CDI in 500 ml chloroform for 1 h at room temperature. Then,
218 mmol (21.84 g) 1-methylpiperazine were added and the reaction mixture was
stirred under reflux for 2 h. When the reaction mass was cooled down to RT,
200 ml water were added, phases were separated, organic phase was washed with
water (2 × 100 ml), combined organic layers were dried under anhydrous Na2SO4,
and the solvent was removed under reduced pressure to provide 66.55 g (98%) of
compound II, as a colorless oil.
Compound II (Supplementary Fig. 1C): Tert-butyl 4-[(4-methylpiperazin-1-yl)
carbonyl]piperidine-1-carboxylate. 1H-NMR (CDCl3) δ: 4.23–4.05 (m, 2H), 3.65
(m, 2H), 3.54 (m, 2H), 2.77 (m, 2H), 2.62 (m, 1H), 2.41 (m, 4H), 2.33 (s, 3H),
1.80–1.60 (m, 4H), 1.46 (s, 9H); 13C-NMR (CDCl3) δ: 172.81 (C5′), 154.64
(C = O BOC), 79.48 (C-BOC), 55.38 (C8′′a), 54.74 (C8′′b), 45.99 (C10′′), 45.33
(C7′′a), 43.56 (C2′′a), 42.83(C2′′b), 41.61(C7′′b), 38.32 (C4′′), 28.42 (Me-BOC), 28.39
(C3′′); HRMS (m/z): [MNa]+ calcd. for C16H29N3O3, 334.21011; found, 334.20986.
MP: 98 °C.
Compound III: 1-Methyl-4-(piperidin-4-ylcarbonyl)piperazine bis hydro-
chloride. 217 mmol (67.57 g) of II were dissolved in 400 ml EtOH and 36 ml of
concentrated hydrochloric acid were added. The reaction mass was then stirred
under reflux for 2 h. When cooled down, the formed precipitate was removed by
Compound III (Supplementary Fig. 1C): 1-Methyl-4-(piperidin-4-ylcarbonyl)
piperazine bis hydrochloride. 1H-NMR (DMSO-d6) δ: 11.45 (s, 1H), 9.15 (s, 1H),
N
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