Journal of Medicinal Chemistry
Article
was added dropwise. After stirring for approximately 0.5 h, the
reaction was removed to room temperature and stirred for 3−5 h.
When the reaction was complete according to thin-layer chromatog-
raphy (TLC) detection, the reaction mixture was filtered and distilled
to give 3D−I, which was directly used for the next step. Subsequently,
to a solution of 3D−I and K2CO3 (15.0 mmol, 2.1 g) in acetone, an
aqueous solution of dimethylamine (15.0 mmol, 676.3 mg) was
added. After stirring at 25 °C for 12−18 h, the reaction mixture was
filtered and concentrated. The concentrate was isolated by silica-gel
column chromatography (CC) to obtain compounds 4D−I.
Synthesis of Compounds 5a−c, 5e−r, 6c−f, 6i−l, and 6o−r.
Compounds 1A−C or 2A−C (0.2 mmol) and compounds 4D−I (0.6
mmol) were dissolved in MeOH, and then the mixture was stirred at
reflux. After the reaction was completed, the mixture was evaporated
under reduced pressure, and the residue was purified by PTLC or
flash chromatography [eluting solvents: CH2Cl2/MeOH (v/v) =
25:1−10:1] to afford the title compounds 5a−c, 5e−r, 6c−f, 6i−l,
and 6o−r in yields of 18−97%. The spectral data of some
representative compounds 5i, 5o, 6i, and 6o are listed below; the
3,3′-((3′,5-Diallyl-[1,1′-biphenyl]-2,4′-diyl)bis(oxy))bis(N-(2-(bu-
tylamino)-2-oxoethyl)-N,N-dimethylpropan-1-aminium) Bromide
(5i). A mixture of compound 1B (0.2 mmol, 101.7 mg) and
intermediate 4F (0.6 mmol, 94.9 mg) was dissolved in MeOH, and
then stirred under reflux. After the reaction was completed, the
mixture was concentrated under reduced pressure and purified by
PTLC to obtain 5i as a pale-yellow liquid in 37% yield. 1H NMR (600
MHz, DMSO-d6) δ: 8.69 (t, J = 6.0 Hz, 1H, −NH−), 8.61 (t, J = 4.8
Hz, 1H, −NH−), 7.33 (dd, J = 8.4, 2.4 Hz, 1H, −Ph), 7.24 (d, J = 2.4
Hz, 1H, −Ph), 7.08−7.11 (m, 2H, −Ph), 7.02−7.05 (m, 2H, −Ph),
5.94−5.98 (m, 2H, −CHCH2), 5.02−5.10 (m, 4H, −CHCH2),
4.13 (t, J = 6.0 Hz, 2H, −CH2−), 4.07 (s, 2H, −CH2−), 3.99 (t, J =
6.0 Hz, 2H, −CH2−), 3.83 (t, J = 6.0 Hz, 2H, −CH2−), 3.56−3.58
(m, 4H, −CH2−), 3.34 (s, 6H, N-CH3), 3.27 (s, 2H, −CH2−), 3.17−
3.18 (m, 2H, −CH2−), 3.17 (s, 6H, N-CH3), 3.08−3.11 (m, 4H,
−CH2−), 2.18−2.22 (m, 4H, −CH2−), 2.11−2.13 (m, 4H, −CH2−),
1.38−1.41 (m, 4H, −CH2−), 1.26−1.30 (m, 4H, −CH2−), 0.83 (t, J
= 7.2 Hz, 6H, −CH3); 13C NMR (150 MHz, DMSO-d6) δ: 163.4,
163.2, 155.5, 153.8, 138.4, 137.5, 133.0, 130.8, 130.3, 128.8, 128.5,
127.9, 116.0, 113.8, 111.6, 65.9, 64.7, 62.7, 62.5, 51.7, 42.6, 40. 51,
39.1, 38.7, 34.5, 32.2, 31.1, 22.9, 19.9, 14.0; HRMS (ESI)
C40H64Br2N4O4 [M − 2Br]/2+ calcd = 332.2459; found = 332.2472.
4,4′-((3′,5-Diallyl-[1,1′-biphenyl]-2,4′-diyl)bis(oxy))bis(N-(2-(bu-
tylamino)-2-oxoethyl)-N,N-dimethylbutan-1-aminium) Bromide
(5o). 5o was prepared from compound 1C (0.2 mmol, 107.3 mg)
and intermediate 4F (0.6 mmol, 94.9 mg) following a similar
procedure as that for 5i. Data for 5o: Yield: 39%, pale-yellow liquid,
1H NMR (600 MHz, DMSO-d6) δ: 8.60−8.63 (m, 2H, −NH−), 7.32
(dd, J = 8.4, 2.4 Hz, 1H, −Ph), 7.27 (d, J = 2.4 Hz, 1H, −Ph), 7.08
(dd, J = 8.4, 2.4 Hz, 1H, −Ph), 7.06 (d, J = 2.4 Hz, 1H, −Ph), 7.01 (d,
J = 8.4 Hz, 1H, −Ph), 6.98 (d, J = 8.4 Hz, 1H, −Ph), 5.93−6.01 (m,
2H, −CHCH2), 5.03−5.10 (m, 4H, −CHCH2), 4.11 (s, 2H,
−CH2−), 4.03−4.04 (m, 4H, −CH2−), 3.95 (t, J = 6.0 Hz, 2H,
−CH2−), 3.58−3.61 (m, 2H, −CH2−), 3.33−3.34 (m, 4H, −CH2−),
3.22 (s, 6H, N-CH3), 3.12 (s, 6H, N-CH3), 3.09−3.12 (m, 4H,
−CH2−), 1.90−1.92 (m, 2H, −CH2−), 1.77−1.79 (m, 4H, −CH2−),
1.65−1.67 (m, 2H, −CH2−), 1.39−1.43 (m, 4H, −CH2−), 1.27−
1.30 (m, 4H, −CH2−), 0.86 (t, J = 7.2 Hz, 2H, −CH3), 0.85 (t, J =
7.2 Hz, 2H, −CH3); 13C NMR (150 MHz, DMSO-d6) δ: 163.4,
163.3, 155.5, 154.0, 138.5, 137.5, 132.6, 131.0, 130.8, 130.7, 130.1,
128.7, 128.4, 127.7, 116.1, 116.0, 113.5, 111.5, 67.7, 67.2, 64.64, 64.6,
62.3, 62.2, 51.7, 51.5, 40.5, 39.1, 38.7, 34.4, 31.2, 31.1, 26.3, 26.1,
21.4, 19.9, 19.6, 19.4, 14.0; HRMS (ESI) C42H68Br2N4O4 [M − 2Br]/
2+ calcd = 346.2615; found = 346.2621.
−Ph), 7.10 (dd, J = 8.4, 2.4 Hz, 1H, −Ph), 7.05 (d, J = 2.4 Hz, 1H,
−Ph), 7.00 (dd, J = 8.0, 2.0 Hz, 1H, −Ph), 6.95 (d, J = 2.0 Hz, 1H,
−Ph), 6.72 (d, J = 8.4 Hz, 1H, −Ph), 5.91−5.98 (m, 2H, −CH
CH2), 5.02−5.10 (m, 4H, −CHCH2), 4.42 (s, 2H, −CH2−), 3.99
(t, J = 5.2 Hz, 2H, −CH2−), 3.62 (t, J = 8.0 Hz, 2H, −CH2−), 3.31−
3.36 (m, 4H, −CH2−), 3.16−3.26 (m, 2H, −CH2−), 3.12 (s, 6H, N-
CH3), 2.10−2.11 (m, 2H, −CH2−), 1.51−1.57 (m, 2H, −CH2−),
1.33−1.38 (m, 2H, −CH2−), 0.89 (t, J = 7.2 Hz, 3H, −CH3−); 13C
NMR (100 MHz, CDCl3) δ: 162.4, 153.4, 152.2, 137.7, 137.3, 133.6,
131.8, 131.6, 131.4, 128.9, 128.8, 128.1, 125.8, 115.9, 115.5, 112.4,
64.4, 64.3, 63.8, 51.2, 51.0, 39.6, 39.3, 30.8, 23.1, 20.2, 13.6. HRMS
(ESI) C29H41BrN2O3 [M-Br]+ calcd = 465.3112; found = 465.3120.
N-(2-(Butylamino)-2-oxoethyl)-4-((5,5′-diallyl-2′-hydroxy-[1,1′-
biphenyl]-2-yl)oxy)-N,N-dimethylbutan-1-aminium Bromide (6o).
6o was prepared from compound 2C (0.2 mmol, 80.3 mg) and
intermediate 4F (0.6 mmol, 94.9 mg) following a similar procedure as
that for 5i. Data for 6o: Yield: 60%, pale-yellow liquid, 1H NMR (400
MHz, CDCl3) δ: 8.77 (s, 1H, −NH−),7.14 (dd, J = 8.4, 2.0 Hz, 1H,
−Ph), 7.08 (d, J = 2.0 Hz, 1H, −Ph), 6.99−7.05 (m, 2H, −Ph), 6.90
(d, J = 8.4 Hz, 1H, −Ph), 6.68 (s, 1H, −Ph), 5.92−5.97 (m, 2H,
−CHCH2), 5.04−5.11 (m, 4H, −CHCH2), 4.38 (s, 2H,
−CH2−), 4.04 (t, J = 4.8 Hz, 2H, −CH2−), 3.41−3.45 (m, 2H,
−CH2−), 3.35 (d, J = 6.8 Hz, 2H, −CH2−), 3.32 (d, J = 6.8 Hz, 2H,
−CH2−), 3.20−3.25 (m, 2H, −CH2−), 3.10 (s, 6H, N-CH3), 1.71−
1.83 (m, 4H, −CH2−), 1.52−1.56 (m, 2H, −CH2−), 1.32−1.38 (m,
2H, −CH2−), 0.88 (t, J = 7.2 Hz, 3H, −CH3); 13C NMR (100 MHz,
CDCl3) δ: 162.4, 153.5, 152.1, 137.6, 137.3, 133.8, 132.2, 132.0,
131.4, 129.2, 128.9, 127.4, 126.1, 116.1, 115.9, 115.7, 112.8, 67.9,
66.0, 62.8, 51.7, 39.5, 39.37, 39.33, 30.9, 25.7, 20.1, 19.9, 13.6; HRMS
(ESI) C30H43BrN2O3 [M-Br]+ calcd = 479.3268; found = 479.3281.
Determination of MICs. MICs were determined according to the
guidelines of the Clinical and Laboratory Standards Institute
(CLSI).55 To determine the MICs, S. aureus or MRSA-16 was
cultured in 1 mL of Mueller−Hinton broth (MHB) for 3−5 h at 37
°C and 200 rpm and then was diluted to a final concentration of 1 ×
105 CFU/mL. The stock solutions of the tested compounds were set
at a concentration of 256 μg/mL and serially diluted by the 2-fold
dilution method. Next, 100 μL of each dilution and 100 μL of
bacterial suspension were successively added to 96-well plates. The
positive groups were levofloxacin, tigecycline, and vancomycin, and
the negative and blank groups were 200 μL of media and bacterial
solution (1 × 105 CFU/mL). Finally, the plate was incubated at 37 °C
for 16−18 h, and the results were observed by the naked eye. The
MIC was defined as the lowest concentration of tested compounds
that inhibited the visible growth of bacteria completely. The
determination of MICs was repeated at least three times.
Hemolytic Activity. Stock solutions of the tested compounds
were prepared in phosphate-buffered saline (PBS) at a concentration
of 5120 μg/mL and serially diluted by the 2-fold dilution method, and
then 50 μL of each dilution was added to 96-well plates. RBCs were
obtained from the fresh sterile sheep blood and resuspended in PBS
to 5% (v/v). The RBC suspension (150 μL) was added to 96-well
plates, and then the plates were incubated at 37 °C for 1 h. The
negative control was an RBC suspension with PBS only, and the
positive control was PBS containing 0.1% (v/v) Triton X-100. After
incubation, the mixture was centrifuged (3500 rpm for 5 min), and
then the supernatant (100 μL) was pipetted into a fresh 96-well plate.
Finally, hemolysis was calculated by measuring the absorbance at 540
nm.
Time-Kill Kinetics Study. The clinical MRSA-16 isolate was
incubated in 1 mL of Muller-Hinton broth (MHB) broth at 37 °C for
16−18 h, diluted 1000 times with MHB, and cultured for 2.5−5 h.
After that, the bacterial suspension was diluted to 1 × 105 CFU/mL
(early exponential phase) and 1 × 108 CFU/mL (late exponential
phase). Next, the bacterial suspension was challenged with compound
5i (1×, 4×, 6× and 8× MIC) and cultured at 37 °C and 200 rpm for 6
h (early exponential phase) or 8 h (late exponential phase). The
colony count was measured at 0, 0.5, 1, 2, 3, 4, 5, and 6 h for the early
exponential phase and 0, 1, 2, 4, 6, and 8 h for the late exponential
phase. Tigecycline (1×, 8× MIC) was used as a positive control.
N-(2-(Butylamino)-2-oxoethyl)-3-((5,5′-diallyl-2′-hydroxy-[1,1′-
biphenyl]-2-yl)oxy)-N,N-dimethylpropan-1-aminium Bromide (6i).
6i was prepared from compound 2B (0.2 mmol, 77.5 mg) and
intermediate 4F (0.6 mmol) following a similar procedure as that for
1
5i. Data for 6i: Yield: 97%, pale-yellow liquid, H NMR (400 MHz,
CDCl3) δ: 8.67 (t, J = 8.4 Hz, 1H, −NH−), 7.15 (d, J = 8.0 Hz, 1H,
J
J. Med. Chem. XXXX, XXX, XXX−XXX