Novel Inhibitors of Erm Methyltransferases
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3857
of the ErmC′ structure was partially disordered and begins
with the main chain of residues 8 and 9. The side chains of
these amino acids were disordered in the final model. The
final model was refined to a crystallographic R-factor of 19.5%
(free R-factor ) 27.0%) for data up to 3.0 Å and contains 15
solvent molecules defined as peaks greater than 4.0σ in the
Fo-Fc map.
yield; 1H NMR (DMSO-d6) δ 7.15 (4H, m), 6.78 (1H, d, J ) 7.1
Hz), 6.1 (2H, m), 4.62 (2H, m), 4.46 (2H, t, J ) 5.4 Hz), 3.22
(4H, m), 2.83 (2H, dd, J ) 7.8, 15.9 Hz), 2.75 (2H, m), 1.8-1.1
(5H, series of mult.); MS (M + H) 341. Anal. (C18H24N6O)
Theory: C 63.51, H 7.11, N 24.69. Found: C 63.83, H 7.49, N
22.48.
2-Am in o-4-(4-h ydr oxym eth ylpiper adin o)-6-(2-am in oin -
d a n yl)-1,3,5-tr ia zin e (18). In a fashion similar to 17, 18 was
prepared using 230 mg of 35 as the starting material. All other
reaction conditions are as listed for 17 to give 0.19 g of pure
In h ibition Assa ys. ErmAM (ErmC′) inhibitory activity was
determined in a reaction mixture consisting of 50 mM Tris,
pH 7.5, 100 (40) mM KCl, 4 mM MgCl2, 1 (10) mM DTT, and
95 (143) nM ErmAM (ErmC′). Test compound (1-4 µL) was
added in duplicate to 96-well v-bottom polypropylene plates
(Costar), to which 24 µL of the reaction mixture containing
the enzyme was added. This mixture was gently shaken for
15 min at room temperature, at which time 25 µL of a solution
containing 250 nM S-adenosyl-L-[methyl-3H]methionine (Am-
ersham Pharmacia Biotech) and 80 nM E. coli rRNA (Boe-
hringer-Mannheim) was added. After gently shaking for an
additional 2 h at room temperature,20 the reaction was
terminated by adding 150 µL of a 10% TCA solution containing
100 mM KCl to each well and cooling on ice for 30 min. The
samples were filtered using GF/B 96-well filter plates (Milli-
pore) presoaked with 75 µL of a solution containing 50 mM
Tris, pH 7.5, 100 mM KCl, and 100 µg/µL BSA. Wells were
rinsed five times with 200 µL of a 5% TCA solution, with a
final wash of 200 µL of 95% cold ethanol. Filters were dried
for 45 min at 310 K. TCA-precipitated counts were quantitated
in a Packard Top Count after the addition of 50 µL of
MicroScint-O (Packard). IC50 values were obtained by graphing
percent inhibition, relative to control wells without inhibitor,
as a function of inhibitor concentration. The KI value is given
by KI ) IC50/(1 + [AdoMet]/KM), with the KM for AdoMet equal
to 300 nM under the conditions of the assay. The reported KI
values are derived from this equation with an estimated error
in the KI value of (50%.
2-Am in o-4-ch lor o-6-(2-am in oin dan yl)-1,3,5-tr iazin e (33).
Powdered 2-amino-4,6-dichloro-1,3,5-triazine (31)21,22 (3.4 g,
20.9 mmol) was added to crushed ice (100 g) and 2-aminoindan
(5.4 mL, 41.7 mmol) was added in a single step with stirring.
The mixture became thick almost immediately. Stirring was
continued for 3.5 h while the reaction was allowed to warm to
room temperature. The precipitate was filtered off and was
recrystallized from EtOAc to afford 3.2 g of the product as pale
gray crystals: 58% yield; 1H NMR (DMSO-d6) δ 7.64 (1H, bs),
7.11-7.19 (4H, cm), 6.89 (2H, bs), 4.58-4.63 (1H, cm), 3.19-
3.24 (2H, dd, J ) 4.9, 15.9 Hz), 2.86-2.90 (2H, dd, J ) 7.0,
15.9 Hz). Anal. (C12H12N5Cl) C, H, N. FAB HRMS: MH+ calcd
for C12H13N5Cl, 262.086; found, 262.086.
1
alcohol: 93% yield; H NMR (DMSO-d6) δ 7.18 (4H, m), 6.85
(1H, m), 6.15 (2H, m), 4.64 (3H, m), 4.46 (1H, t, J ) 5.1 Hz),
3.24 (2H, t, J ) 5.1 Hz), 3.17 (2H, m), 2.83 (2H, dd, J ) 7.35,
15.81 Hz), 2.68 (2H, br t, J ) 12.1), 1.60 (3H, m), 1.0 (2H, m);
MS (M + H) 341. Anal. (C18H24N6O‚[0.15 CH2Cl2, 0.15EtOAc])
Theory: C 61.47, H 7.02, N 22.94. Found: C 61.50, H 7.05, N
22.94.
2-Am in o-4-(N -2,4-d ich lor o-6-h yd r oxy-2-p r op yla m i-
n op h en yl)-6-(2-a m in oin d a n yl)-1,3,5-tr ia zin e (22). A solu-
tion of amino-3-(2,4-dichloro-6-hydroxyphenyl)propyl benzyl
ether (115 mg, 0.37 mmol) and amino-3-(N-2-aminoindanyl)-
5-chlorotriazine (97 mg, 0.37 mmol) in 6 mL of THF was heated
to reflux for 16 h. TLC (75% EtOAc/hex) showed the insertion
to be complete. The reaction was evaporated and purified by
flash chromatography using 75% EtOAc/hex to give 164 mg
(83%) of the benzyl-protected insertion product. The benzyl
ether (164 mg, 0.31 mmol) was dissolved in a mixture of 10
mL of EtOAc and 5 mL of MeOH, 40 mg of 10% palladium on
carbon was added, the reaction vessel was fitted with a
hydrogen balloon, and the hydrogenation proceeded at 1 atm
and 25 °C and was complete after 1.5 h (TLC 2.5% MeOH/
EtOAc w/0.25% NH4OH). The reaction was filtered through
Celite and concentrated to give 22 as a white solid: 1H NMR
(300 MHz, CDCl3) δ 7.24-7.08 (4H, m), 6.94 (1H, d, J ) 1.84
Hz), 6.83 (1H, d, J ) 1.84 Hz), 5.2 (2H, bs), 4.8 (2H, m), 3.4-
3.2 (4H, m), 2.88-2.81 (5H, m), 1.89 (2H, bs); MS (M + H)
446.
2-Am in o-4-(th iom eth yl)-6-(2-a m in oin d a n yl)-1,3,5-tr ia z-
in e (23). To a solution of 33 (50 mg, 0.19 mmol) in THF (3
mL) was added sodium thiomethoxide (20 mg, 0.29 mmol) and
the resulting solution was stirred at 50 °C overnight. The
reaction was cooled and concentrated in vacuo. The residue
was purified by flash chromatography (100:1 CH2Cl2:MeOH)
to give 39 mg of a white powder: 75% yield; 1H NMR (DMSO-
d6) δ mixture of rotamers 7.56 and 7.47 (1H, 2d, J ) 7.0 Hz),
7.22-7.11 (4H, m), 6.83 (1H, bs), 6.65 (1H, bs), 4.69-4.55 (1H,
m), 3.18 (2H, dd, J ) 15.8, 7.4 Hz), 2.84 (2H, dd and m, J )
15.8, 7.0 Hz), 2.37 and 2.35 (3H, 2s); MS (M + H) 274. Anal.
(C13H15N5S) Theory: C 56.87, H 5.60, N 25.28. Found: C 57.12,
H 5.53, N 25.62.
2-In d a n ylbigu a n id e (36). A mixture of 2-aminoindan‚HCl
(6.0 g, 35 mM) and dicyandiamide (3.0 g, 35 mM) was heated
to 160 °C for 100 min. The reaction mixture initially melts,
then resolidifies. The reaction was cooled to room temperature.
The solid was recrystallized from EtOH/Et2O (4:3) to give 5.2
g of beige crystals: 58% yield; 1H NMR (DMSO-d6) δ 7.65 (1H,
bs), 7.25-7.13 (4H, m), 7.03-6.62 (5H, m), 4.41-4.34 (1H, m),
3.24-3.16 (2H, m), 2.86-2.79 (2H, m); MS (M + H) 218.
2-Am in o-4-cycloh exyl-6-(2-a m in oin d a n yl)-1,3,5-t r ia z-
in e (24). 2-Indanylbiguanide (36; 0.23 g, 0.90 mmol) was
suspended in 2 N NaOH (2 mL) and cooled in an ice bath. A
solution of cyclohexanecarbonyl chloride (0.12 mL, 0.90 mmol)
in acetone (2 mL) was added and the resulting solution was
allowed to warm to room temperature overnight. The acetone
was removed in vacuo and the resulting precipitate was
filtered and purified by flash chromatography (50:1 CH2Cl2:
MeOH) to give 29 mg of a clear colorless oil: 10% yield; 1H
NMR (DMSO-d6, 90 °C) δ 7.18 (2H, dd, J ) 3.3, 2.4 Hz), 7.11
(2H, dd, J ) 3.3, 1.8 Hz), 6.89 (1H, bs), 6.19 (2H, bs), 4.63
(1H, sextet, J ) 4.5 Hz), 3.19 (2H, dd, J ) 9.6, 4.5 Hz), 2.87
(2H, dd, J ) 9.6, 4.5 Hz), 2.28 (1H, tt, J ) 6.9, 2.4 Hz), 1.82
(2H, dd, J ) 8.1, 1.5 Hz), 1.73 (2H, dt, J ) 7.5, 2.1 Hz), 1.66-
1.62 (1H, m), 1.48 (2H, dq, J ) 7.8, 2.1 Hz), 1.33-1.14 (3H,
m); MS (M + H) 310.
Gen er a l Meth od F or th e Solu tion -P h a se P a r a llel Syn -
th esis of th e Tr ia m in otr ia zin es 8-16, 19-21, 34, a n d 35.21
The reactions were carried out in 25-mL screw cap vials in
batches of 25 reactions at a time. A representative procedure
is as follows for the conversion of 32:22 A mixture of 2-amino-
4-chloro-6-(piperidin-1-yl)-1,3,5-triazine (50 mg, 0.23 mmol)
and the appropriate amine (5 equiv) in H2O (1.0 mL) was
heated to 100 °C for 4 h. After cooling, 2 N aq NaOH (0.12
mL, 0.23 mmol) and 3.0 mL of H2O were added. The solution
was then extracted in the vial with 15.0 mL of ether. The
organic layer was decanted and evaporated in a speed vac and
the crude product was purified by preparative reverse-phase
HPLC. MS and analytical reverse-phase HPLC showed that
the products were obtained in purities >80%.
2-Am in o-4-(3-h ydr oxym eth ylpiper adin o)-6-(2-am in oin -
d a n yl)-1,3,5-tr ia zin e (17). A THF solution of DIBAL-H (0.8
mL, 0.8 mmol) was added dropwise to a cool (0 °C) solution of
34 (100 mg, 0.26 mmol) in THF (1 mL). Ethyl acetate (1 mL)
was added after 30 min followed by 1 N HCl (5 mL). The acidic
solution was neutralized with a saturated solution of NaHCO3
until pH ∼ 6. The aqueous phase was extracted with EtOAc
(3 × 50 mL). The organics were then combined, washed with
H2O (2 × 50 mL) and brine (25 mL), dried (Na2SO4), filtered,
and concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc) to afford 87 mg of pure alcohol: 95%