Novel Synthesized N-Ethyl-Piperazinyl-Amides of C2-Substituted Oleanonic and Ursonic Acids Exhibit Cytotoxic Effects through Apoptotic Cell Death Regulation
Abstract
:1. Introduction
2. Results and Discussion
2.1. Chemistry
2.2. Biological Activity
2.2.1. NCI-60 Anticancer Drug Screening
2.2.2. Compounds 4 and 6 Induce Apoptosis-Related Nucleus Changes
2.2.3. Compounds 4 and 6 Induce Changes in Cell Morphology and Confluence
2.2.4. rtPCR Assay Highlights Biological Activity at Gene Level
2.3. Compounds 4 and 6 Exhibit In Silico Bcl-XL Inhibition
2.4. Effect of Compounds 4 and 6 on Normal and Tumor Angiogenesis Process by CAM Assay and Irritation Potential Determination Using the HET-CAM Assay
3. Materials and Methods
3.1. Chemistry
3.1.1. General
3.1.2. Synthesis of Compounds 3, 4
3.1.3. Synthesis of Compounds 5–10
3.2. NCI-60 Screening
3.3. Cell Culture
3.4. Compound Stability in Cell Culture Media
3.5. DAPI Assay
3.6. The Chorioallantoic Membrane Assay
3.7. HET-CAM Assay
3.8. Molecular Docking
3.9. Gene Expression Analysis by qRT-PCR
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Compound (NSC) | 60 Cell Lines Assay in 1 Dose 10 µM Concentration | |||||
---|---|---|---|---|---|---|
Mean Growth, % | Range of Growth, % | Most Sensitive Cell Lines | Growth % of the Most Sensitive Cell Lines | Positive Cytostatic Effect a | Positive Cytotoxic Effect b | |
3 (D-811947/1) | 46.01 | −35.82 to 80.36 | HL-60(TB) (Leukemia) | −35.82 | 5/59 | 7/59 |
4 (D-811946/1) | −10.47 | −85.67 to −41.54 | MALME-3M (Melanoma) LOXINVI (Melanoma) | −85.67 −85.17 | 60/60 | 60/60 |
5 (D-818127/1) | 86.73 | 49.25 to 115.49 | CAKI-1 (Renal cancer) | 49.25 | 0/59 | 0/59 |
6 (D-818136/1) | 23.36 | −91.03 to 83.75 | SK-MEL-5 | −91.03 | 30/60 | 4/60 |
U251 (CNS cancer) | −88.15 | |||||
7 (D-818138/1) | 78.38 | 16.53 to 109.58 | HL-60(TB) (Leukemia) | 16.53 | 1/59 | 1/59 |
8 (D-8181948/1) | 92.36 | 74.76 to 107.55 | LOXIMVI (Melanoma) | 74.76 | 0/59 | 0/59 |
9 (D-811949/1) | 25.33 | −38.20 to 65.71 | HL-60 (Luekemia) | −38.20 | 33/59 | 5/59 |
10 (D-811951/1) | 68.42 | 31.17 to 106.80 | SR (Leukemia) | 31.17 | 2/59 | 2/59 |
PC-3 (CNS Cancer) | 32.08 |
Panel/Cell Line | Compound 4 | Compound 6 | Compound 9 | DRB | 5-FU | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
GI50 a, µM | TGI b, µM | LC50 c, µM | GI50 a, µM | TGI b, µM | LC50 c, µM | GI50 a, µM | TGI b, µM | LC50 c, µM | GI50 a, µM | GI50 a, µM | |
Leukemia | |||||||||||
CCRF-CEM | |||||||||||
HL-60(TB) | 0.976 | 2.74 | >50.0 | 2.10 | 9.02 | >100 | 2.78 | 8.97 | >100 | 0.08 | 9.97 |
K-562 | 0.980 | 2.18 | 4.84 | 0.443 | 3.34 | >100 | 2.08 | 5.00 | >100 | 0.19 | 2.30 |
MOLT-4 | 0.994 | 2.90 | >50.0 | 1.43 | >100 | >100 | 2.63 | 7.44 | >100 | NT | 3.58 |
RPMI-8226 | 0.809 | 1.99 | 4.90 | 1.03 | 5.20 | >100 | 2.45 | 7.38 | >100 | 0.03 | 0.35 |
SR | - | - | - | 0.959 | 5.13 | >100 | 2.18 | 6.35 | >100 | 0.08 | 0.04 |
MG_MID d | - | - | - | 0.706 | 5.10 | >100 | 2.38 | 7.09 | >100 | 0.03 | NT |
NSC lung cancer | |||||||||||
A549/ATCC | 1.47 | 5.98 | 21.8 | 2.62 | 13.4 | >100 | 3.15 | 16.8 | >100 | 0.06 | 0.18 |
EKVX | 0.916 | 2.42 | 8.95 | 1.90 | 6.48 | 43.5 | 2.17 | 7.32 | 61.7 | 0.41 | NT |
HOP-62 | 1.38 | 3.09 | >50.0 | 1.88 | 3.75 | 7.47 | 2.43 | 5.86 | >100 | 0.07 | 0.39 |
HOP-92 | 0.713 | 2.06 | 8.22 | 1.06 | 3.26 | 10.1 | 1.57 | 5.73 | 38.8 | 0.10 | 77.9 |
NCI-H226 | 1.78 | 7.01 | 28.7 | 1.76 | 3.80 | - | 1.89 | 4.07 | 8.78 | 0.05 | 54.7 |
NCI-H23 | 1.08 | 2.90 | 11.6 | 2.10 | 5.85 | 27.8 | 2.13 | 5.91 | 31.8 | 0.15 | 0.33 |
NCI-H322M | 1.57 | 5.43 | 16.8 | 2.87 | 9.25 | 33.9 | 2.28 | 8.27 | 28.8 | NT | NT |
NCI-H460 | 0.913 | 1.75 | 3.27 | 1.39 | 2.93 | 6.20 | 1.78 | 3.45 | 6.69 | 0.02 | 0.05 |
NCI-H522 | 0.831 | 1.75 | 3.69 | 1.61 | 3.75 | 8.73 | 1.66 | 3.86 | - | 0.03 | 7.27 |
MG_MID | 1.18 | 3.60 | 17.00 | 1.91 | 5.83 | 29.71 | 2.12 | 6.81 | 47.07 | 0.11 | 20.12 |
Colon Cancer | |||||||||||
COLO 205 | 1.02 | 2.37 | 14.5 | 1.89 | 4.74 | >100 | 2.19 | 5.12 | >100 | 0.18 | 0.15 |
HCC-2998 | 0.922 | 1.82 | 3.60 | 1.58 | 3.12 | 6.17 | 1.55 | 3.00 | 5.79 | 0.26 | 0.05 |
HCT-116 | 0.798 | 1.57 | 3.09 | 1.01 | 2.22 | 4.89 | 1.69 | 3.55 | 7.43 | 0.08 | 0.22 |
HCT-15 | 0.977 | 2.39 | 7.41 | 1.08 | 2.71 | 6.79 | 1.66 | 3.26 | 6.41 | 6.46 | 0.11 |
HT29 | 0.913 | 2.05 | 4.59 | 1.71 | 4.67 | 44.5 | 1.93 | 5.36 | >100 | 0.12 | 0.17 |
KM12 | 1.03 | 2.22 | 4.79 | 1.58 | 3.26 | 6.75 | 1.75 | 3.56 | 7.23 | 0.27 | 0.21 |
SW-620 | 0.902 | 1.76 | 3.44 | 1.78 | 3.88 | 8.46 | 1.90 | 4.14 | 9.05 | 0.09 | 0.92 |
MG_MID | 0.94 | 2.03 | 5.92 | 1.52 | 3.51 | 25.37 | 1.81 | 4.00 | 33.70 | 1.06 | 0.26 |
CNS cancer | |||||||||||
SF-268 | 1.77 | 6.49 | 24.6 | 1.59 | 4.79 | 85.7 | 1.77 | 4.02 | 9.15 | 0.10 | 1.62 |
SF-295 | 0.820 | 1.59 | 3.08 | 1.66 | 3.15 | 5.97 | 1.63 | 3.05 | 5.70 | 0.10 | NT |
SF-539 | 0.843 | 1.54 | 2.80 | 1.62 | 3.00 | 5.55 | 1.66 | 3.05 | 5.58 | 0.12 | 0.06 |
SNB-19 | 0.849 | 1.72 | 3.47 | 1.68 | 3.46 | 7.12 | 1.79 | 3.74 | 7.82 | 0.04 | 3.81 |
SNB-75 | 1.03 | 3.82 | 17.2 | - | - | - | 1.57 | 8.91 | 39.9 | 0.07 | 78.7 |
U251 | 0.854 | 1.85 | 4.00 | 1.58 | 3.11 | 6.13 | 1.82 | 3.67 | - | 0.04 | 0.92 |
MG_MID | 1.028 | 2.84 | 8.69 | 1.63 | 3.50 | 22.09 | 1.71 | 4.41 | 13.63 | 0.08 | 17.02 |
Melanoma | |||||||||||
LOX IMVI | 0.821 | 1.52 | 2.80 | 1.36 | 2.73 | 5.45 | 1.58 | 3.06 | 5.91 | 0.07 | 0.24 |
MALME-3M | 0.807 | 1.51 | 8.84 | 1.53 | 2.91 | 5.54 | 1.40 | 2.75 | 5.39 | 0.12 | 0.05 |
M14 | 0.794 | 1.61 | 3.28 | 1.60 | 3.15 | 6.20 | - | - | - | 0.18 | 0.98 |
MDA-MB-435 | 1.05 | 2.30 | 5.22 | 1.48 | 3.11 | 6.52 | 1.70 | 3.49 | 7.18 | 0.25 | 0.07 |
SK-MEL-2 | 1.29 | 3.33 | 16.2 | 1.93 | 3.96 | 8.16 | 2.12 | 4.76 | 65.5 | 0.17 | 56.7 |
SK-MEL-28 | 0.889 | 1.70 | 3.27 | 1.48 | 2.83 | 5.41 | 1.74 | 3.15 | 5.69 | 0.21 | 1.03 |
SK-MEL-5 | 0.888 | 1.60 | 2.89 | 1.63 | 3.00 | 5.54 | 1.75 | 3.14 | 5.63 | 0.08 | 0.46 |
UACC-257 | 0.985 | 2.19 | 4.88 | 1.61 | 4.29 | 33.8 | 2.21 | 5.95 | >100 | 0.14 | 3.55 |
UACC-62 | 0.835 | 1.61 | 3.09 | 1.28 | 2.83 | 6.23 | 1.51 | 2.96 | 5.80 | 0.12 | 0.52 |
MG_MID | 0.93 | 1.93 | 4.94 | 1.54 | 3.21 | 9.20 | 1.56 | 3.66 | 25.14 | 0.15 | 7.07 |
Ovarian cancer | |||||||||||
IGROV1 | 1.04 | 4.85 | 19.1 | 1.91 | 4.16 | 9.10 | 1.89 | 3.87 | 7.94 | 0.17 | 1.22 |
OVCAR-3 | 1.10 | 2.44 | 6.52 | 1.51 | 2.95 | 5.73 | 1.65 | 3.19 | 6.17 | 0.39 | 0.01 |
OVCAR-4 | 1.26 | 4.74 | 18.0 | 2.17 | 7.85 | 47.8 | 2.57 | 7.36 | 36.6 | 0.37 | 4.43 |
OVCAR-5 | 0.907 | 1.87 | 3.87 | 1.56 | 3.35 | 7.19 | 1.54 | 2.93 | 5.57 | 0.41 | 10.9 |
OVCAR-8 | 1.10 | 2.89 | 30.1 | 2.14 | 5.54 | >100 | 2.32 | 6.29 | >100 | 0.10 | 1.74 |
NCI/ADR-RES | 1.08 | 3.24 | 18.3 | 1.57 | 3.46 | 7.60 | 1.59 | 3.31 | 6.91 | 7.16 | 0.31 |
SK-OV-3 | 1.15 | 4.83 | 17.2 | 4.19 | 1.68 | 76.1 | 2.70 | 9.02 | 90.1 | 0.22 | 21.8 |
MG_MID | 1.19 | 3.55 | 16.16 | 2.15 | 4.14 | 36.22 | 2.04 | 5.16 | 36.18 | 1.26 | 5.77 |
Ranal cancer | |||||||||||
786-0 | 0.814 | 1.70 | 3.56 | 1.60 | 3.08 | 5.91 | 1.98 | 4.37 | - | 0.13 | 0.72 |
A498 | 0.927 | 2.42 | 7.92 | 1.66 | 3.53 | 7.48 | 1.90 | 4.05 | 8.65 | 0.10 | 0.35 |
ACHN | 1.33 | 4.38 | 14.8 | 2.60 | 10.5 | 32.4 | 2.46 | 6.45 | 22.6 | 0.08 | 0.27 |
CAKI-1 | 0.946 | 2.96 | 11.2 | 2.08 | 11.8 | 45.3 | 2.12 | 11.8 | 41.9 | 0.95 | 0.07 |
RXF 393 | 0.822 | 1.74 | 3.69 | 1.42 | 2.77 | 5.41 | 1.45 | 2.83 | 5.53 | 0.10 | 2.61 |
SN12C | 0.776 | 1.48 | 2.81 | 1.22 | 2.54 | 5.30 | 1.61 | 3.01 | 5.66 | 0.07 | 0.49 |
TK-10 | 1.27 | 4.14 | 20.3 | 3.86 | 15.4 | 54.1 | 2.87 | 9.40 | 59.3 | NT | 1.12 |
UO-31 | 0.726 | 1.42 | 2.79 | 1.35 | 2.67 | 5.32 | 1.36 | 2.65 | 5.19 | 0.49 | 1.42 |
MG_MID | 0.95 | 2.53 | 8.38 | 1.97 | 6.54 | 20.15 | 1.97 | 5.57 | 21.26 | 0.27 | 0.88 |
Prostate cancer | |||||||||||
PC-3 | 0.875 | 3.17 | 18.3 | 1.00 | 2.79 | 7.78 | 1.49 | 3.85 | 9.92 | 0.32 | 2.36 |
DU-145 | 1.67 | 5.62 | 18.6 | 1.83 | 3.88 | 8.23 | 2.08 | 4.21 | 8.53 | 0.11 | 0.36 |
MG_MID | 1.27 | 4.40 | 18.45 | 1.42 | 3.34 | 8.01 | 1.79 | 5.96 | 9.23 | 0.21 | 1.36 |
Breast cancer | |||||||||||
MCF7 | 0.892 | 2.03 | 4.63 | 1.40 | 3.07 | 6.71 | 1.64 | 3.42 | 7.14 | 0.03 | 0.07 |
MDA-MB-31/ATCC | 0.779 | 1.50 | 2.88 | 1.40 | 2.84 | 5.78 | 1.75 | 3.59 | 7.39 | 0.51 | 6.60 |
HS 578T | 1.07 | 2.68 | 26.5 | 1.83 | 5.37 | >100 | 1.91 | 4.77 | >100 | 0.33 | 9.77 |
BT-549 | 0.833 | 1.58 | 3.00 | 1.47 | 2.88 | 5.66 | 1.69 | 3.59 | - | 0.23 | 10.6 |
T-47D | 1.33 | 3.89 | >50.0 | 2.61 | 13.7 | >100 | 2.79 | 13.4 | >100 | 0.06 | 8.12 |
MDA-MB-468 | 0.931 | 2.32 | 9.27 | 1.28 | 3.94 | 19.7 | 1.65 | 3.43 | 7.1 | 0.05 | NT |
MG_MID | 0.97 | 2.33 | 16.05 | 1.67 | 5.30 | 39.64 | 1.91 | 5.37 | 36.94 | 0.19 | 7.03 |
MG_MID60 e | 1.03 | 2.74 | 12.31 | 1.53 | 6.29 | 32.37 | 1.93 | 5.29 | 37.95 | 0.38 | 6.97 |
Panel | Compound 4 | Compound 6 | Compound 9 | DRB | 5-FU | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
SI (GI50) | SI (TGI) | SI (LC50) | SI (GI50) | SI (TGI) | SI (LC50) | SI (GI50) | SI (TGI) | SI (LC50) | SI (GI50) | SI (GI50) | |
Leukemia | 1.10 | 1.12 | 0.45 | 1.38 | 0.30 | 0.32 | 0.80 | 0.75 | 0.38 | 4.75 | 2.14 |
NSCL cancer | 0.87 | 0.76 | 0.72 | 0.80 | 1.08 | 1.09 | 0.91 | 0.78 | 0.81 | 3.45 | 0.35 |
Colon Cancer | 1.10 | 1.35 | 2.08 | 1.01 | 1.99 | 1.28 | 1.07 | 1.32 | 1.13 | 0.36 | 26.81 |
CNS cancer | 1.00 | 0.96 | 1.41 | 0.94 | 1.80 | 1.46 | 1.13 | 1.20 | 2.78 | 4.75 | 0.41 |
Melanoma | 1.11 | 1.42 | 2.49 | 0.99 | 1.96 | 3.52 | 0.60 | 1.45 | 1.51 | 2.53 | 0.99 |
Ovarian Cancer | 0.86 | 0.77 | 0.76 | 0.71 | 1.52 | 0.89 | 0.95 | 1.02 | 1.05 | 0.30 | 1.21 |
Renal Cancer | 1.08 | 1.08 | 1.47 | 0.77 | 0.96 | 1.61 | 0.98 | 0.95 | 1.78 | 1.41 | 7.92 |
Prostate cancer | 0.81 | 0.62 | 0.67 | 1.08 | 1.89 | 4.04 | 1.08 | 0.89 | 2.11 | 1.81 | 5.13 |
Breast cancer | 1.06 | 1.17 | 0.77 | 0.92 | 1.17 | 0.82 | 1.01 | 0.98 | 1.03 | 2.00 | 0.99 |
Sample | Bcl-XL | Bcl-2 | BAK | BAX | ||||
---|---|---|---|---|---|---|---|---|
A375 | ||||||||
Mean | Std.Dev | Mean | Std.Dev | Mean | Std.Dev | Mean | Std.Dev | |
DMSO 1 | 4.550 | (2.694) | 2.216 | (0.485) | 5.200 | (0.085) | 4.063 | (0.644) |
Compound 4 | 0.500 * | (0.292) | 0.7366 ** | (0.133) | 0.706 *** | (0.255) | 0.270 *** | (0.069) |
Compound 6 | 0.300 | (0.265) | 1.166 | (0.683) | 0.516 *** | (0.231) | 0.590 *** | (0.157) |
RPMI | ||||||||
DMSO 1 | 18.563 | (6.786) | 4.063 | (0.374) | 4.163 | (0.710) | 3.040 | (0.723) |
Compound 4 | ND | ND | 1.483 * | (1.165) | 156.243 *** | (23.330) | 0.460 ** | (0.324) |
Compound 6 | 1.050 * | (0.900) | 0.236 *** | (0.185) | 11.853 ** | (1.789) | 0.640 ** | (0.310) |
SK-Mel-28 | ||||||||
DMSO 1 | 4.730 | (0.353) | 2.573 | (1.015) | 1.383 | (0.249) | 2.086 | (0.322) |
Compound 4 | 0.086 *** | (0.066) | 0.083 * | (0.056) | 101.820 ** | (37.304) | 0.023 *** | (0.011) |
Compound 6 | 0.003 *** | (0.005) | 0.083 ** | (0.032) | 91.283 *** | (14.461) | 0.140 *** | (0.117) |
Target PDB ID | Binding Free Energy ∆G (kcal/mol) | ||
---|---|---|---|
Co-Crystalized Ligand | Compound 4 | Compound 6 | |
2W3L | −10.3 | −6.5 | −6.7 |
2YXJ | −10.7 | −8.5 | −9.2 |
3EQG | −8.8 | −6.7 | 1.8 |
4FA6 | −9.3 | 8.7 | 24.2 |
4JT5 | −8.5 | 3.1 | 24.7 |
4OQ5 | −12.2 | −2.8 | 37.3 |
Test Compound and Controls | Irritation Score | Type of Effect |
---|---|---|
Distillate water | 0 | Non-irritant |
SLS 0.5% | 17.03 | Strong irritant |
DMSO 0.5% | 0.68 | Non-irritant |
Compound 4 | 0 | Non-irritant |
Compound 6 | 0 | Non-irritant |
PDB ID | Protein | Grid Box Size | Grid Box Center Coordinates | Conformers |
---|---|---|---|---|
2YXJ | Apoptosis regulator Bcl-X (Bcl-XL) | size_x = 18.8213065002 size_y = 30.6345174449 size_z = 10.2997599705 | center_x = −10.2573467499 center_y = −18.1808412775 center_z = 9.46376920765 | 8 |
3EQG | Dual specificity mitogen-activated protein kinase kinase 1 (MEK1) | size_x = 14.6058170561 size_y = 16.2060321551 size_z = 8.70490192182 | center_x = −4.16588471722 center_y = 59.3845284049 center_z = 34.6388482191 | 8 |
2W3L | Apoptosis regulator Bcl-2 (Bcl-2) | size_x = 15.4649240353 size_y = 13.2408048336 size_z = 13.3102000528 | center_x = 37.1927426166 center_y = 26.8056607992 center_z = −12.8141402336 | 8 |
4OQ5 | Induced myeloid leukemia cell differentiation protein (Mcl-1) | size_x = 15.5072934727 size_y = 13.9772309154 size_z = 10.4111215841 | center_x = 11.6262580805 center_y = 3.65908070556 center_z = 7.90001930158 | 8 |
4JT5 | Mammalian target of rapamycin-target of rapamycin complex subunit LST8 (mTOR-LST8) | size_x = 16.0737519631 size_y = 11.2048088799 size_z = 10.5072207821 | center_x = 51.5677297505 center_y = −1.9363339261 center_z = −48.4627944976 | 8 |
4FA6 | Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PI3Kγ) | size_x = 14.3022779071 size_y = 10.3234871012 size_z = 8.72203337452 | center_x = 44.8020362414 center_y = 13.746321532 center_z = 30.3837942752 | 8 |
Forward | Reverse | |
---|---|---|
BAK | 5′ ATGGTCACCTTACCTCTGCAA 3′ | 5′TCATAGCGTCGGTTGATGTCG 3′ |
BCL-XL | 5′ GATCCCCATGGCAGCAGTAAAGCAAG 3′ | 5′CCCCATCCCGGAAGAGTTCATTCACT 3′ |
Bax | 5′ GCCGGGTTGTCGCCCTTTT 3′ | 5′CCGCTCCCGGAGGAAGTCCA 3′ |
Bcl-2 | 5′-CGGGAGATGTCGCCCCTGGT-3′ | 5′-GCATGCTGGGGCCGTACAGT-3′ |
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Kazakova, O.; Mioc, A.; Smirnova, I.; Baikova, I.; Voicu, A.; Vlaia, L.; Macașoi, I.; Mioc, M.; Drăghici, G.; Avram, Ş.; et al. Novel Synthesized N-Ethyl-Piperazinyl-Amides of C2-Substituted Oleanonic and Ursonic Acids Exhibit Cytotoxic Effects through Apoptotic Cell Death Regulation. Int. J. Mol. Sci. 2021, 22, 10967. https://doi.org/10.3390/ijms222010967
Kazakova O, Mioc A, Smirnova I, Baikova I, Voicu A, Vlaia L, Macașoi I, Mioc M, Drăghici G, Avram Ş, et al. Novel Synthesized N-Ethyl-Piperazinyl-Amides of C2-Substituted Oleanonic and Ursonic Acids Exhibit Cytotoxic Effects through Apoptotic Cell Death Regulation. International Journal of Molecular Sciences. 2021; 22(20):10967. https://doi.org/10.3390/ijms222010967
Chicago/Turabian StyleKazakova, Oxana, Alexandra Mioc, Irina Smirnova, Irina Baikova, Adrian Voicu, Lavinia Vlaia, Ioana Macașoi, Marius Mioc, George Drăghici, Ştefana Avram, and et al. 2021. "Novel Synthesized N-Ethyl-Piperazinyl-Amides of C2-Substituted Oleanonic and Ursonic Acids Exhibit Cytotoxic Effects through Apoptotic Cell Death Regulation" International Journal of Molecular Sciences 22, no. 20: 10967. https://doi.org/10.3390/ijms222010967