Journal of Medicinal Chemistry
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4H), 2.58 (t, J = 16.0 Hz, 1H), 2.94 (t, J = 16.0 Hz, 1H), 3.01−3.27
(m, 4H), 3.59−3.70 (m, 4H), 4.05 (d, J = 5.6 Hz, 2H), 6.81 (t, J = 7.2
Hz, 1H), 6.94 (d, J = 7.2 Hz, 2H), 7.23 (t, J = 7.2 Hz, 2H), 8.49 (t, J =
5.6 Hz, 1H).
for 2 h, cooled to RT, and concentrated in vacuo. The residue was
partitioned between 2 N NaOH (aq) (10 mL) and CH2Cl2 (2 × 30
mL), and the combined organic fractions were dried (Na2SO4),
concentrated in vacuo, and adsorbed onto silica for purification by
flash column chromatography (0−10% MeOH/CH2Cl2). This gave 45
as a pale yellow gum which was used crude in the next reaction (7.70
( 1 R , 2 R ) - N - ( C y a n o m e t h y l ) - 2 - ( 8 - fl u o r o - 1 , 3 , 4 , 5 -
t e t r a h y d r o p y r i d o [ 4 , 3 - b ] i n d o l e - 2 - c a r b o n y l ) -
cyclohexanecarboxamide (13). 8-Fluoro-1,3,4,5-tetrahydropyrido-
[4,3-b]indole 13a (162 mg, 0.87 mmol) was utilized with 1-
aminoacetonitrile hydrochloride (69.0 mg, 0.75 mmol) (13c) to
yield 13, after purification via prep HPLC, as a solid gum (134 mg,
57%). HRMS (ES+) for C21H24O2N4F (M+ + H): calcd 383.1878;
found, 383.1880. 1H NMR (500 MHz, DMSO) δ 1.13−1.50 (m, 4H),
1.62−1.92 (m, 4H), 2.58 (t, J = 10.4 Hz, 1H), 2.75−3.09 (m, 4H),
3.48−3.75 (m, 3H), 4.00−4.16 (m, 2H), 6.88 (d, J = 3.8 Hz, 2H), 6.97
1
g, 100% yield). H NMR (400 MHz, DMSO) δ 2.34 (d, J = 5.6 Hz,
2H), 2.62−2.36 (m, 6H), 3.52 (s, 2H), 6.68 (ddd, J = 7.1, 4.9, 0.8 Hz,
1H), 7.04 (s, 1H), 7.51−7.06 (m, 5H), 7.59−7.51 (m, 1H), 8.05 (dd, J
= 4.9, 1.1 Hz, 1H), 9.47 (s, 1H).
N-Benzyl-2,3,4,5-tetrahydro-1-benzylpyrido[4,3-b]-7-azain-
dole (46). Polyphosphoric acid (60 g) was added to 45 (7.69 g, 27.5
mmol) and the mixture stirred gently at 150 °C for 24 h. The mixture
was cooled to RT, and ice (50 g) was added to break up the gum. The
reaction mixture was made basified with 2 M NaOH (aq) and
extracted with EtOAc (3 × 300 mL). The combined organic extracts
were treated with brine (90 mL), dried (Na2SO4), concentrated in
vacuo, and adsorbed onto silica for purification by flash column
chromatography (0−10% MeOH/CH2Cl2). The mustard colored
solid obtained (3.80 g) was triturated with a small volume of CH2Cl2,
filtered, and dried to furnish the desired compound as a sand colored
solid 57 (3.00 g, 42% yield). LCMS (+ve ESI): tR = 0.97 min, 264 (M
1
(dt, J = 13.7, 5.9 Hz, 2H), 8.48 (t, J = 5.5 Hz, 1H). H NMR (500
MHz, DMSO) δ 1.18−1.48 (m, 4H), 1.62−1.91 (m, 4H), 2.79 (t, J =
11.7 Hz, 1H), 2.75−2.93 (m, 2H), 3.03(t, J = 11.7 Hz, 1H), 3.80 (s,
1H), 3.87−4.06 (m, 2H), 4.64 (t, J = 16.6 Hz, 2H), 6.83 (td, J = 9.2,
2.4 Hz, 1H), 7.20 (d, J = 7.0 Hz, 1H), 7.25 (dd, J = 8.7, 4.5 Hz, 1H),
8.08 (s, 1H), 10.64 (s, 1H).
(1R,2R)-N-(1-Cyanocyclopropyl)-2-[(8-fluoro-1,3,4,5-tetrahy-
d r o - 2 H - p y r i d o [ 4 , 3 - b ] i n d o l - 2 - y l ) c a r b o n y l ] -
cyclohexanecarboxamide (14). 13a (798 mg, 4.20 mmol) was
utilized with 14c (585 mg, 5.04 mmol) to yield 14, after purification
silica chromatography, as a solid gum (212 mg, 12%). HRMS (ES+)
1
+ H)+. H NMR (400 MHz, DMSO) δ 2.80 (m, 4H), 3.57 (s, 2H),
3.73 (s, 2H), 6.94 (dd, J = 7.8, 4.7 Hz, 1H), 7.21−7.42 (m, 5H), 7.67
(dd, J = 7.8, 1.2 Hz, 1H), 8.07 (dd, J = 4.7, 1.5 Hz, 1H), 11.32 (s, 1H).
2,3,4,5-Tetrahydro-1-benzylpyrido[4,3-b]-7-azaindole (39a).
46 (2.90 g, 11.0 mmol), NH4CO2H(s) (2.78 g, 44.0 mmol), and 20%
PdOH on carbon (290 mg) were suspended in EtOH (200 mL) and
stirred under reflux. After 1 h, more NH4CO2H(s) (695 mg, 11.0
mmol) was added and refluxing continued for 1 h. The catalyst was
filtered off through Celite and washed with a small volume of CH2Cl2,
and the combined filtrate was concentrated in vacuo and dried under
vacuum to furnish the desired compound as off-white solid 39a (1.90
1
for C23H26O2N4F (M+ + H): calcd 409.2034; found, 409.2036. H
NMR (400 MHz, CDCl3) δ 0.77−0.94 (m, 1H), 0.95−1.54 (m, 7H),
1.75−1.91 (m, 4H), 2.61 (“t”, J = 11.4 Hz, 1H), 2.68−2.94 (m, 3H),
3.64−3.80 (m, 1H), 3.88−4.03 (m, 0.5H), 4.20−4.33 (m, 0.5H), 4.60
(d, J = 15.7 Hz, 0.5H), 4.69 (s, 1H), 4.89 (d, J = 15.8 Hz, 0.5H), 6.53
(s, 0.5H), 6.61 (s, 0.5H), 6.89 (td, J = 11.3, 2.4 Hz, 1H), 7.08 (td, J =
9.4, 2.3 Hz, 1H), 7.20 (td, J = 8.8, 2.3 Hz, 1H), 7.93 (s, 1H). 1H NMR
(500 MHz, DMSO) δ 0.88 (s, 1H), 1.01 (s, 1H), 1.19−1.45 (m, 6H),
1.70−1.85 (m, 4H), 2.79−2.90 (m, 2H), 3.01 (td, J = 11.9, 3.6 Hz,
1H), 3.81 (s, 1H), 3.88 (dt, J = 11.3, 5.7 Hz, 1H), 4.63 (s, 2H), 6.82
(dt, J = 9.2, 2.6 Hz, 1H), 7.22 (d, J = 9.2 Hz, 1H), 7.26 (dd, J = 8.8, 4.6
Hz, 1H), 8.26 (s, 1H), 10.65 (s, 1H).
1
g, 100% yield). H NMR (400 MHz, DMSO) δ 2.46−2.55 (m, 2H),
2.67 (t, J = 5.6 Hz, 2H), 3.02 (t, J = 5.6 Hz, 2H), 3.83 (s, 2H), 6.95
(dd, J = 7.7, 4.7 Hz, 1H), 7.70 (dd, J = 7.7, 1.4 Hz, 1H), 8.07 (dd, J =
4.7, 1.4 Hz, 1H), 11.25 (s, 1H).
6-Methoxy-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole (34a).
Piperidin-4-one hydrochloride (41) (4.66 g, 34.5 mmol) was added to
a suspension of (2-methoxyphenyl)hydrazine hydrochloride (6.00 g,
34.4 mmol) in ethanol (80 mL) and concd HCl (6.0 mL), and the
resulting solution was heated at 80 °C for 3 h. The reaction was
allowed to cool to room temperature overnight, and the resulting
precipitated solid was filtered, washed with chilled ethanol (20 mL),
and dried under vacuum to give a salt. This was stirred in water (100
mL) and made basic with 2 M NaOH(aq) (∼5 mL), and the resulting
solid was filtered, azeotroped with toluene (30 mL, rotary evaporator),
and dried under vacuum to yield 34a (3.84 g, 55%) as an off-white
solid. LCMS (+ve ESI): tR = 0.85 min, 203.3 (M + H)+. 1H NMR (400
MHz, DMSO) 2.63−2.66 (m, 2H), 2.99−303 (m, 2H), 3.83 (‘s’, 2H),
3.89 (s, 3H), 6.59 (d, J = 7.5 Hz, 1H), 6.83 (“t”, J = 7.8 Hz, 1H), 6.93
(d, J = 7.8 Hz, 1H), 10.72 (s, 1H).
(1R,2R)-N-(1-Cyanocyclopropyl)-2-[1,3,4,5-tetrahydro-1H-
p y r i d o [ 4 , 3 - b ] - 7 - a z a i n d o l - 2 - y l ) c a r b o n y l ] -
cyclohexanecarboxamide (39). 39a (200 mg, 1.15 mmol) was
utilized with 14c (177 mg, 1.20 mmol) to furnish 39, after purification,
as an off-white solid (40.0 mg, 9% yield). HRMS (ES+) for
1
C22H26O2N5 (M+ + H): calcd 392.2081; found, 392.2084. H NMR
(400 MHz, DMSO, 303 K) δ 0.72 (s, 1H), 0.69−0.80 (dd, J = 41.8,
7.8 Hz, 0.5H), 0.93−1.30 (m, 1H), 1.15−1.45 (m, 6H), 1.62−1.85 (m,
4H), 2.67−2.75 (m, 0.5H), 2.78−2.84 (m, 0.5H), 2.95−3.05 (m, 1H),
3.69−3.82 (m, 0.5H), 3.84−3.94 (m, 1.5H), 4.52 (d, J = 15.8 Hz,
0.5H), 4.65 (d, J = 15.8 Hz, 0.5H), 4.75 (s, 1H), 7.02 (“ddd”, J = 16.6,
7.7, 4.8 Hz, 1H), 7.83 (d, J = 7.6 Hz, 0.5H), 7.93 (d, J = 7.6 Hz, 0.5H),
8.13 (dd, J = 8.5, 4.4 Hz, 1H), 8.71 (d, J = 3.7 Hz, 1H), 11.44 (s, 1H).
1H NMR (500 MHz, DMSO, 373 K) δ 0.84−0.86 (m, 1H), 0.94 (d, J
= 62.4 Hz, 1H), 1.16−1.53 (m, 6H), 1.70−1.92 (m, 4H), 2.81 (m,
1H), 3.02 (td, J = 11.8, 3.5 Hz, 1H), 3.83 (b, 1H), 3.89 (dt, J = 13.3,
5.6 Hz, 1H), 4.66 (s, 2H), 6.98 (dd, J = 7.2, 4.7 Hz, 1H), 7.80 (d, J =
7.2 Hz, 1H), 8.12 (dd, J = 4.7, 1.5 Hz, 1H), 8.27 (s, 1H), 11.04 (s,
1H).
Assay for the Identification of Cat K Inhibitors. QFRET
Technology (quenched fluorescent resonance energy transfer) was
used to measure the inhibition by test compounds of Cat K-mediated
cleavage of the synthetic peptide Z-Phe-Arg-AMC. Compounds were
screened at twelve concentrations (0.0003−10 μM), on two separate
occasions, and the mean IC50 values reported.
A concentration of 0.5 nM [final] rhuman Cat K (prepared in-
house) in phosphate buffer was added to a 384-well black microtiter
plate containing investigative compounds. The enzyme and compound
were preincubated at room temperature for 30 min before the addition
of 50 μM [final] Z-Phe-Arg-AMC synthetic substrate in sodium
acetate/EDTA buffer at pH 5.5. The plates were covered and
incubated for 1 h at room temperature and protected from light.
Following the incubation, the reaction was stopped with 7.5% [final]
(1R,2R)-N-(1-Cyanocyclopropyl)-2-[(6-methoxy-1,3,4,5-tetra-
h y d r o - 2 H - p y r i d o [ 4 , 3 - b ] i n d o l - 2 - y l ) c a r b o n y l ] -
cyclohexanecarboxamide (34). 34a (3.84 g, 19.0 mmol) was
utilized with 1-aminocyclopropanecarbonitrile hydrochloride 14c
(6.76 g, 57.0 mmol) to yield 34 after purification via flash silica
chromatography and crystallization slurry stirring over 48 h (6.27 g,
78%) as a white powder. HRMS (ES+) for C24H29O3N4 (M+ + H):
1
calcd 421.2234; found, 421.2233. H NMR (400 MHz, DMSO) δ
0.73−0.83 (m, 1H), 0.87−0.91 (m, 0.5H), 0.87−1.03 (m, 1H), 1.17−
1.40 (m, 6H), 1.61−1.82 (m, 5H), 2.63−2.70 (m, 1H), 2.75−2.82 (m,
0.5H), 2.84−3.05 (m, 1.5H), 3.64−3.72 (m, 0.5H), 3.94−3.73 (m,
5H), 4.71 (s, 1H), 6.64 ("t", J = 8.2 Hz, 1H), 6.90 (qn, J = 7.8 Hz, 1H),
6.97 (d, J = 7.8 Hz, 0.5H), 7.10 (d, J = 7.8 Hz, 0.5H), 8.64 (s, 1H),
10.91 (s, 1H).
1-Benzylpiperidin-4-one Pyridin-2-yl Hydrazone (45). 2-
Hydrazinopyridine dihydrochloride (5.00 g, 27.5 mmol) and 1-
benzylpiperidin-4-one (6.18 g, 27.5 mmol) were suspended in EtOH
(70 mL). AcOH (2 mL) was added and the mixture stirred at reflux
6371
dx.doi.org/10.1021/jm3007257 | J. Med. Chem. 2012, 55, 6363−6374