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NaOH (1.50 g), compound 9b was obtained as a colorless oil
(15.1 g, 119 mmol, 79%); purity: 92% (at 220 nm); 1H NMR
([D6]DMSO): d=1.20 (t, J=7.1 Hz, 3H), 2.68 (s, 4H), 4.11 ppm (q,
J=7.1 Hz, 2H); 13C NMR ([D6]DMSO): d=12.4, 14.0, 29.2, 60.5, 119.8,
170.6 ppm; IR (ATR): n˜ =2250 (w), 1729 cmÀ1 (s); HRMS (ESI) m/z
[M+H+] calcd for C6H10NO2+: 128.0712, found: 128.0701.
zymes incorporating the same catalytic metal, active site struc-
ture, and functionality, and was more common among early-
stage lead compounds than in fully developed inhibitors.
Almost all approved drugs showed no off-target inhibition, re-
gardless of the complexing warhead, suggesting that inhibitor
selectivity is a combination of metal binding and the suppor-
tive backbone interactions. Therefore, metal chelating inhibi-
tors of metalloproteins should no longer be automatically as-
sociated with high risk for indiscriminate off-target inhibition
of metalloenzymes, and their development poses no more risk
for nonspecific activity than small-molecule inhibitors of metal-
independent enzymes. After all, the relative selectivity of our
fragment-like lead 11 a for KDM4A, and the lack of activity
against HDAC1 seem to support these findings.
Ethyl 4-cyanobutanoate (9c): With halogenated ester 8c (50.0 g,
332 mmol), NaCN (23.9 g, 498 mmol), DMSO (113 mL) and NaOH
(3.00 g), compound 9c was obtained as a colorless oil (38.9 g,
276 mmol, 83%); purity: 100% (at 220 nm); 1H NMR ([D6]DMSO):
d=1.19 (t, J=7.1 Hz, 3H), 1.81 (qu, J=7.3 Hz, 2H), 2.40 (t, J=
7.3 Hz, 2H), 2.53 (t, J=7.3 Hz, 2H), 4.07 ppm (q, J=7.1 Hz, 2H);
13C NMR ([D6]DMSO): d=14.0, 15.6, 20.5, 32.2, 60.0, 120.2,
171.8 ppm; IR (ATR): n˜ =2245 (w), 1727 cmÀ1 (s); HRMS (ESI) m/z
[M+H+] calcd for C7H11NO2+: 142.0868, found: 142.0869.
General procedure for the synthesis of compounds 10a–c: The
appropriate cyano acid ester (1.0 equiv), NaN3 (1.1 equiv) and
NH4Cl (0.2 equiv) were suspended in DMF. The reaction mixture
was heated at 95–1258C for 8–24 h. Subsequently the solvent was
removed under reduced pressure. The resulting residue was dilut-
ed with H2O and adjusted to pH 1 with concentrated hydrochloric
acid (CAUTION: Release of HN3!). The desired product was ob-
tained by precipitation or extraction.
Experimental Section
General. All starting materials (aldehydes and ketones, halogenat-
ed acid esters, aromatic heterocycles) and solvents, unless other-
wise noted, were obtained from Sigma Aldrich or ABCR GmbH &
Co. KG and used without further purification. Medium pressure
liquid chromatography (MPLC) was done on silica gel from Macher-
ey–Nagel (particle size 50–100 mm, 140–270 mesh ASTM) with
Büchi devices C-630, C-601 and C-660 (column length 40 cm,
column diameter 3.5 cm). Melting points were determined on
a hot stage microscope by Kofler PHMK 81/3035 “Botius” (VEB
Wägetechnik Rapido) with 16-fold amplification or Büchi melting
point apparatus M-565 and are uncorrected. Microwave-assisted
synthesis was performed using a Discover LabMate (‘closed vessel’
mode, 10 mL total capacity vessel, temperature control via IR
sensor) from CEM. The parameter ‘PowerMax’ indicates a perma-
nent radiation of microwaves simultaneously to intense cooling.
The specified purities were determined by the 100% method of
the DAD chromatogram at wavelengths as indicated. Acylhydra-
zones are subject to isomerism at the amide moiety; when both
isomers could be resolved chromatographically, purity values apply
for both isomers together. NMR spectra were recorded using an
Avance III instrument with Ultrashield 400 (1H: 400.2 MHz, 13C:
100.6 MHz) from Bruker at 258C with tetramethylsilane (TMS) as in-
ternal standard, using the ppm scale. High-resolution mass spectra
(HRMS) were obtained after high-performance liquid chromatogra-
phy (HPLC) with a mass spectrometer (LC-IT-TOF) from Shimadzu
based on a deviance tolerance limit ꢀ5 ppm. Mid-infrared spectra
were recorded on a Nicolet IR200 FT-IR from Thermo Electron Cor-
poration with diamond ATR accessory. Figures 2 and 5 were pre-
pared using LigPlot.[42]
Ethyl 2-(1H-tetrazol-5-yl)acetate (10a): With cyano acid ester 9a
(23.0 g, 200 mmol), NaN3 (14.3 g, 220 mmol), NH4Cl (2.30 g,
43 mmol), DMF (220 mL) and H2O (220 mL) for 8 h at 958C. The re-
action was worked up by filtering off the precipitate, followed by
recrystallization from propan-2-ol, to obtain compound 10a as
a colorless solid (17.6 g, 113 mmol, 56%); purity: 70% (at 220 nm);
mp: 1258C; 1H NMR ([D6]DMSO): d=1.21 (t, J=7.0 Hz, 3H), 4.12–
4.17 (q, J=7.0 Hz, 2H), 4.17 (s, 2H), 16.34 ppm (s, 1H); 13C NMR
([D6]DMSO): d=14.0, 29.5, 61.3, 150.4, 167.7 ppm; IR (ATR): n˜ =
+
1741 cmÀ1 (s); HRMS (ESI) m/z [M+H+] calcd for C5H9N4O2
:
157.0726, found: 157.0727.
Ethyl 3-(1H-tetrazol-5-yl)propanoate (10b): With cyano acid ester
9b (17.8 g, 140 mmol), NaN3 (10.0 g, 154 mmol), NH4Cl (1.80 g,
34 mmol), DMF (70 mL) and H2O (70 mL) for 8 h at 1058C. The
crude solution was worked up by diluting with brine and extract-
ing exhaustively with EtOAc, drying the combined organic phases
over Na2SO4 and concentrating under reduced pressure. The result-
ing colorless oil was cooled to À188C, filtering off the resulting
precipitate and washing with Et2O gave compound 10b as a color-
less solid (9.47 g, 55.7 mmol, 42%); mp: 84–858C; 1H NMR
([D6]DMSO): d=1.16 (t, J=7.0 Hz, 3H), 2.83 (t, J=7.2 Hz, 2H), 3.12
(t, J=7.2 Hz, 2H), 4.06 (q, J=7.0 Hz, 2H), 16.04 ppm (s, 1H);
13C NMR ([D6]DMSO): d=14.0, 18.5, 30.7, 60.2, 155.2, 171.4 ppm; IR
(ATR): n˜ =1719 cmÀ1 (s); HRMS (ESI) m/z [M+H+] calcd for
C6H11N4O2+: 171.0882, found: 171.0878.
General procedure for the synthesis of compounds 9a–c: NaCN
(1.5 equiv) was suspended in DMSO at room temperature. Subse-
quently, the appropriate chlorinated acid ester (1.0 equiv) was
added dropwise while the temperature of the resulting mixture
was kept at 408C. The suspension was then heated at 508C for 3 h
and stirred for another 15 h at room temperature. NaOH was
added and the reaction mixture was diluted with H2O until all
solids had dissolved. The solution was exhaustively extracted with
Et2O, dried over Na2SO4 and concentrated under reduced pressure
to obtain a colorless oil.
Ethyl 4-(1H-tetrazol-5-yl)butanoate (10c): With cyano acid ester
9c (38.0 g, 270 mmol), NaN3 (19.3 g, 297 mmol), NH4Cl (3.80 g,
72 mmol), DMF (135 mL) and H2O (135 mL) for 24 h at 1258C. To
work up the solution, it was exhaustively extracted with EtOAc, the
collected organic phases dried over Na2SO4 and concentrated
under reduced pressure. The orange oil was cooled to À188C, fil-
tering off the resulting precipitate and recrystallization from Et2O
gave compound 10c as a colorless solid (7.50 g, 40.8 mmol, 15%);
Ethyl 2-cyanoacetate (9a): This compound was commercially
available.
1
mp: 49–528C; H NMR ([D6]DMSO): d=1.18 (t, J=7.1 Hz, 3H), 1.96
(qu, J=7.4 Hz, 2H), 2.39 (t, J=7.4 Hz, 2H), 2.92 (t, J=7.4 Hz, 2H),
4.06 (q, J=7.1 Hz, 2H), 16.04 ppm (s, 1H); 13C NMR ([D6]DMSO): d=
14.0, 21.9, 22.2, 32.4, 59.8, 155.4, 172.2 ppm; IR (ATR): n˜ =
Ethyl 3-cyanopropanoate (9b): With halogenated ester 8b
(20.0 g, 150 mmol), NaCN (10.5 g, 220 mmol), DMSO (45 mL) and
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