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4.1.2. N-(3,6-Dioxocyclohexa-1,4-dienyl)-2-
(methyl(phenyl)amino)acetamide (2)
1H NMR (DMSO-d6,400 MHz) d 9.75 (s, 1H), 7.34 (d, J = 2.4 Hz,
1H), 7.17 (t, J = 8.0 Hz, 2H), 6.92 (d, J = 10.0 Hz, 1H), 6.79 (dd,
J = 2.8, 10.4 Hz, 1H), 6.68–6.64 (m, 3H), 4.33 (s, 2H), 3.01 (s, 3H).
13C NMR (DMSO-d6, 400 MHz) d 187.85 (C), 182.33 (C), 170.84
(C), 149.00 (C), 138.04 (CH), 137.79 (C), 133.25 (CH), 129.53 (CH),
119.72 (CH), 115.28 (CH), 113.90 (CH), 59.88 (CH2), 40.41 (CH3).
HRMS (ESI) for [MH]+ calculated: 271.10772, observed: 271.10771.
4.1.3. N-(2,5-Dimethoxyphenyl)-2-(methyl(phenyl)amino)
acetamide (3)
1H NMR (CDCl3, 400 MHz) d 8.96 (br s, 1H), 8.13 (dd, J = 3.2 Hz,
1H), 7.32 (d, J = 7.6 Hz, 2H), 6.89–6.84 (m, 3H), 6.76 (d, J = 9.2 Hz,
1H), 6.59 (dd, J = 3.2,8.8 Hz, 1H),4.02 (s, 2H), 3.82 (s, 3H), 3.63 (s,
3H), 3.12 (s, 3H). 13C NMR (CDCl3, 400 MHz) d 169.85 (C), 153.77
(C), 149.19 (C), 143.06 (C), 128.90 (CH), 127.30 (C), 118.16 (CH),
112.93 (CH), 111.31 (CH), 108.35 (CH), 106.55 (CH), 58.20 (CH2),
55.64 (CH3), 54.67 (CH3), 38.82 (CH3). HRMS (ESI) for [MH] + calcu-
lated: 301.15467, observed: 301.15468.
Figure 7. Compound 14 lowers ROS and leads to electrophilic stress. (A) Addition of
20 lM 14 to AML cells leads to a time dependent loss in cellular ROS levels as
measured by a DCF fluorescence assay. (B) Electrophilic molecules, like 14, are
detected by a loss in concentration of the sensor protein KEAP1 (p < 0.05 in all
cases).
4.1.4. N-(2-Hydroxyphenyl)-2-(methyl(phenyl)amino)
acetamide (4)
1H NMR (CDCl3, 400 MHz) d 9.08 (s, 1H), 8.70 (s, 1H), 7.36–7.32
(m, 2H), 7.17–7.13 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.95 (t, J = 7.6 Hz,
1H), 6.87–6.81 (m, 4H), 4.04 (s, 2H), 3.12 (s, 3H).
from each other) with the endpoint having an intensity of
5.02 0.08. Thus, 14 enters AML cells and lowers total ROS as it
is activated. Finally, we wanted to assess if 14 was a reactive
molecule within a cell (Fig. 7B). To accomplish this western blot
against KEAP1, normalized to beta-actin was performed. KEAP1 is
a signaling molecule that inhibits NRF2,25 the transcription factor
that regulates anti-oxidant concentrations in cells. Loss of KEAP1
is dependent on electrophilic and reactive stressors since it
possesses several easy to modify thiols. Addition of 14 lead to a rel-
ative concentration of 0.65 0.09 (p < 0.04) after 24 h.
13C NMR (CDCl3, 400 MHz) d 170.65 (C), 149.15 (C), 148.82 (C),
129.62 (CH), 127.44 (CH), 124.71 (C), 122.24 (CH), 122.15 (CH),
120.39 (CH), 119.87 (CH), 113.89 (CH), 59.44 (CH2), 40.18 (CH3).
HRMS (ESI) for [MH]+ calculated: 257.12845, observed: 257.12845.
4.1.5. N-(3-Hydroxyphenyl)-2-(methyl(phenyl)amino)
acetamide (5)
1H NMR (CD3OD, 400 MHz) d 7.24–7.19 (m, 2H), 7.15–7.07 (m,
2H), 6.92 (d, J = 8.0 Hz, 1H), 6.78–6.71 (m, 3H), 6.54 (m, 1H), 4.05 (s,
2H), 3.08 (s, 3H). 13C NMR (CD3OD, 400 MHz) d 170.36 (C), 157.51
(C), 149.52 (C), 138.86 (C), 129.10 (CH), 128.71 (CH), 117.37 (CH),
112.52 (CH), 111.24 (CH), 111.08 (CH), 107.30 (CH), 57.24 (CH2),
38.85 (CH3). HRMS (ESI) for [MH]+ calculated: 257.12845,
observed: 257.12845.
3. Conclusion
In conclusion, we have synthesized a series of ROS-activatable
compounds. The best compounds are cytotoxic to AML cancer cells
but much less so to normal cells. The ROS-activatable portion is a
simple hydroquinone. When oxidized, this module reacts with
the pendant amine to form an unusual bicyclic ring. Interestingly
14 displays unique selectivity compared to its peers. We propose
that this complex chemistry is integral to selectivity. Current
efforts in our laboratory are focusing on optimization of this design
and making this design suitable for in vivo administration via tun-
ing of pharmacokinetic properties.
4.1.6. N-(2,5-Dihydroxyphenyl)acetamide (6)
1H NMR (CD3OD, 400 MHz) d 7.20 (t, J = 1.6 Hz, 1H), 6.71 (dd,
J = 1.6 Hz, 8.8 Hz, 1H), 6.49–6.46 (m, 1H), 2.16 (s, 3H). 13C NMR
(CD3OD, 400 MHz) d 170.70 (C), 149.86 (C), 140.85 (C), 126.21
(C), 116.36 (CH), 111.68 (CH), 109.10 (CH), 22.14 (CH3). HRMS
+
(ESI) for [MH] calculated: 168.06552, observed: 168.06551.
4. Material and methods for the synthesis
4.1. Synthesis
4.1.7. 2-Aminocyclohexa-2,5-diene-1,4-dione (7)
1H NMR (DMSO-d6, 400 MHz) d 9.22 (s, 1H), 8.96 (s, 1H), 6.67 (d,
J = 8.4 Hz, 1H), 6.40 (dd, J = 2.8, 8.4 Hz, 1H), 6.33 (d, J = 2.8 Hz, 1H).
13C NMR (CD3OD, 400 MHz) d 150.67 (C), 142.39 (C), 126.73 (C),
116.74 (CH), 112.04 (CH), 106.38 (CH). HRMS (ESI) for [MH]+ calcu-
lated: 124.03930, observed: 124.03929.
All compounds were made by divergent routes. Final com-
pounds were prepared with more than 98% purity and character-
ized by 1H NMR, 13C NMR and HRMS. Structural characterizations
for the final compounds are given below.
4.1.8. N-(2,5-Dihydroxyphenyl)-2-hydroxyacetamide (8)
1H NMR (CD3OD, 400 MHz) d 7.72 (d, J = 2.4, 1H), 6.71 (dd,
J = 1.2, 8.8 Hz, 1H), 6.46–6.43 (m, 1H), 4.13 (s, 2H).13C NMR (MeOD,
400 MHz) d 171.49 (C), 149.76 (C), 139.66 (C), 125.91 (C), 115.06
(CH), 110.57 (CH), 107.37 (CH), 61.66 (CH2). HRMS (ESI) for [MH]
calculated: 184.06043, observed: 184.06043.
4.1.1. N-(2,5-Dihydroxyphenyl)-2-(methyl(phenyl)amino)
acetamide (1)
1H NMR (CD3OD, 400 MHz) d 7.64 (d, J = 2.91 Hz, 1H), 7.25 (dd,
J = 7.12, 8.69 Hz, 2H), 6.81 (d, J = 8.30 Hz, 2H), 6.65 (d, J = 8.56, 1H),
6.41 (dd, J = 2.86, 8.61, 1H), 4.03 (s, 2H), 3.12 (s, 3H). 13C NMR (CD3-
OD, 400 MHz) d 169.13 (C), 149.82 (C), 148.59 (C), 139.37 (C),
129.03 (CH), 128.98 (CH), 128.91 (CH), 125.96 (C), 119.41 (CH),
115.13 (CH), 113.83 (CH), 110.74 (CH), 107.70 (CH), 58.59 (CH2),
39.54 (CH3).HRMS (ESI) for [MH]+ calculated: 273.12337, observed:
273.12338.
+
4.1.9. N-(2,5-Dihydroxyphenyl)-2-(phenylamino)acetamide (9)
1H NMR (CD3OD, 400 MHz) d 7.68 (s, 1H), 7.15 (t, J = 7.6 Hz, 2H),
6.73–6.60 (m, 4 H), 6.38 (d, J = 8.8 Hz, 1H), 3.85 (s, 2H). 13C NMR
(CD3OD, 400 MHz) d 170.79 (C), 149.81 (C), 147.92 (C), 139.66