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Article

Design, Synthesis, and Acute Toxicity Assays for Novel Thymoquinone Derivative TQFL12 in Mice and the Mechanism of Resistance to Toxicity

by
Ting Li
1,2,
Qi Tan
1,2,
Chunli Wei
1,
Hui Zou
1,3,
**aoyan Liu
1,
Zhiqiang Mei
1,
Pengfei Zhang
4,
**gliang Cheng
1 and
Junjiang Fu
1,*
1
Key Laboratory of Epigenetics and Oncology, The Research Center for Preclinical Medicine, Southwest Medical University, Luzhou 646000, China
2
Basic Medical School, Southwest Medical University, Luzhou 646000, China
3
Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, School of Medicine, Hunan Normal University, Changsha 410013, China
4
NHC Key Laboratory of Cancer Proteomics, Department of Oncology, Central South University, Changsha 410008, China
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(13), 5149; https://doi.org/10.3390/molecules28135149
Submission received: 1 June 2023 / Revised: 26 June 2023 / Accepted: 27 June 2023 / Published: 30 June 2023
(This article belongs to the Special Issue Discovery of Small Molecules for Cancer Therapy: Current Challenges, Recent Trends, and Future Perspectives)
Browse Figures
Versions Notes

Abstract

:
TQFL12 is a novel derivative designed and synthesized on the basis of Thymoquinone (TQ) which is extracted from Nigella sativa seeds. We have demonstrated that TQFL12 was more effective in the treatment of TNBC than TQ. In order to directly reflect the acute toxicity of TQFL12 in vivo, in this study, we designed, synthesized, and compared it with TQ. The mice were administered drugs with different concentration gradients intraperitoneally, and death was observed within one week. The 24 h median lethal dose (LD50) of TQ was calculated to be 33.758 mg/kg, while that of TQFL12 on the 7th day was 81.405 mg/kg, and the toxicity was significantly lower than that of TQ. The liver and kidney tissues of the dead mice were observed by H&E staining. The kidneys of the TQ group had more severe renal damage, while the degree of the changes in the TQFL12 group was obviously less than that in the TQ group. Western blotting results showed that the expressions of phosphorylated levels of adenylate-activated protein kinase AMPKα were significantly up-regulated in the kidneys of the TQFL12 group. Therefore, it can be concluded that the acute toxicity of TQFL12 in vivo is significantly lower than that of TQ, and its anti-toxicity mechanism may be carried out through the AMPK signaling pathway, which has a good prospect for drug development.

1. Introduction

Malignant tumors are a worldwide disease that seriously threaten human health with tumor metastasis and recurrence. The incidence and mortality of tumors compared to chronic diseases are increasing year by year. The latest cancer data shows that breast cancer (BC) has become the number one killer of females due to malignant tumors in the world today [1,2,3,4]. Both the incidence and mortality of breast cancer are the top among female malignant tumors in many countries [5]. Triple-negative breast cancer (TNBC), as a result of lacking estrogen receptor (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), leads to high aggressiveness, strong metastasis, and drug resistance to traditional treatments, and has always attracted much attention in the fields of tumor research [6,7]. Poor diagnostic targets and a lack of effective therapeutic agents are the most two difficult problems. Among the different tumor treatment options including radiotherapy, chemotherapy, biologically targeted therapy, immunotherapy, and endocrine therapy, small-molecule compound therapy is considered to be highly effective and promising [8]. The need to find more highly effective with low toxicity therapeutic drugs targeting breast cancer, especially triple-negative breast cancer, has become urgent.
Natural products or traditional herbal medicine have long been the source of active ingredients for clinical therapeutics. Artemisinin (Qinghaosu) is a source of traditional Chinese herbal medicine with antimalarial effects found by Youyou Tu, for example [9]. Degalactotigonin is a Natural Compound from Solanum nigrum L., which has been demonstrated to inhibit the growth and metastasis of osteosarcoma [10]. ** a higher-effect and lower-toxicity small-molecule compound drug for tumor patients.
Small-molecule compounds from natural products are considered to be an effective therapeutic strategy for TNBC. The goal of researchers is to develop promising small-molecule compound drugs. TQ has been demonstrated to be effective for TNBC, but the high toxicity limits the performance of its effects. We successfully found that TQFL12 was more effective and lower toxicity than TQ in TNBC cells. Hence, TQFL12 could be a candidate compound for the therapy of TNBC and has potential clinical values.

5. Conclusions

In summary, we designed, synthesized, and compared TQFL12 with TQ, and conducted acute toxicity assays on TQFL12. The acute toxicity of TQFL12 in vivo is significantly lower than that of TQ, and its anti-toxicity mechanism may be through the AMPKα signaling pathway. Thus, given the higher anti-tumor effect on breast cancer and lower toxicity, TQFL12 is expected to be a good prospect for drug development in TNBC patients.

Author Contributions

Methodology, T.L. and H.Z.; Investigation, T.L., Q.T., C.W., X.L., Z.M. and J.C.; Resources, H.Z. and P.Z.; Writing—original draft, T.L. and J.F.; Writing—review & editing, J.F.; Supervision, J.F.; Project administration, J.F.; Funding acquisition, J.F. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (81672887, 82073263), the Foundation of Science and Technology Department of Sichuan Province (grant no. 2022NSFSC0737), the Sichuan Science and Technology Program (Nos. 2022YFS0623-C3, 2022YFS0623-C4), the Research Foundation of Luzhou City (grant no. 2021-SYF-37), and the Primary Research & Development Plan of Hunan Province (2020SK2071).

Institutional Review Board Statement

The animal experiments followed the international, national, and institutional guidelines for the care and use of animal subjects. The study has been approved by the Ethical Committee of Southwest Medical University (No.: 20210930-007, date: 30 September 2021).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank people for their help from the Research Center for Preclinical Medicine, Southwest Medical University.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Not available.

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Molecules 28 05149 g001 550
Figure 1. The design and synthesis of the novel compound TQFL12. The NH2 group using NaN3 and acetic acid and 4-chlorobenzaldehyd in EtOH with HCl were added on the TQ position 6. (A) The original structure of TQ. (B) The structure of the novel derivative TQFL12 [(E)-3-((4-chlorobenzylidene) amino)-5-isopropyl-2-methylcyclohexa-2,5-diene-1,4-dione]. (C) HR-ESI-MS spectrum of TQFL12. TQFL12 yields four different mass-to-charge ratios, with maximum abundance at 302.0932, and the relative molecular mass of TQFL12 is correctly verified to m/z 302.0932 [M + H]+ (calcd. for C17H17ClNO2+: 302.0932) after hydrogenation by the mass spectrometer.
Figure 1. The design and synthesis of the novel compound TQFL12. The NH2 group using NaN3 and acetic acid and 4-chlorobenzaldehyd in EtOH with HCl were added on the TQ position 6. (A) The original structure of TQ. (B) The structure of the novel derivative TQFL12 [(E)-3-((4-chlorobenzylidene) amino)-5-isopropyl-2-methylcyclohexa-2,5-diene-1,4-dione]. (C) HR-ESI-MS spectrum of TQFL12. TQFL12 yields four different mass-to-charge ratios, with maximum abundance at 302.0932, and the relative molecular mass of TQFL12 is correctly verified to m/z 302.0932 [M + H]+ (calcd. for C17H17ClNO2+: 302.0932) after hydrogenation by the mass spectrometer.
Molecules 28 05149 g001
Molecules 28 05149 g002 550
Figure 2. The Bioavailability Radar plot and BOILED-Egg of TQFL12 obtained by using Swiss ADME. (A) The 2D chemical structure of TQFL12 (left side) and the Bioavailability Radar plot show the drug-likeness of TQFL12 by five parameters. The pink area represents the optimal range for each property. TQFL12 was predicted to have the potential to become an oral drug. (B) The BOILED-Egg was used for evaluation of passive gastrointestinal absorption (HIA) and brain penetration (BBB). The white zone is for the high probability of passive absorption by the gastrointestinal tract, and the yellow zone (yolk) is for the high probability of brain penetration. Yolk and white areas are not mutually exclusive. In addition, the points are colored in blue if predicted as active by P-gp (PGP+) and in red if predicted as non-substrate of P-gp (PGP−). TQFL12 was predicated to a non-substrate of P-gp and was at the position of the double yellow and white areas, which was to be an oral drug. These things considered, the PAINS group which was predicted as the active group in the TQFL12 structure by Swiss ADME was the active quinone group in the anti-cancer effect.
Figure 2. The Bioavailability Radar plot and BOILED-Egg of TQFL12 obtained by using Swiss ADME. (A) The 2D chemical structure of TQFL12 (left side) and the Bioavailability Radar plot show the drug-likeness of TQFL12 by five parameters. The pink area represents the optimal range for each property. TQFL12 was predicted to have the potential to become an oral drug. (B) The BOILED-Egg was used for evaluation of passive gastrointestinal absorption (HIA) and brain penetration (BBB). The white zone is for the high probability of passive absorption by the gastrointestinal tract, and the yellow zone (yolk) is for the high probability of brain penetration. Yolk and white areas are not mutually exclusive. In addition, the points are colored in blue if predicted as active by P-gp (PGP+) and in red if predicted as non-substrate of P-gp (PGP−). TQFL12 was predicated to a non-substrate of P-gp and was at the position of the double yellow and white areas, which was to be an oral drug. These things considered, the PAINS group which was predicted as the active group in the TQFL12 structure by Swiss ADME was the active quinone group in the anti-cancer effect.
Molecules 28 05149 g002
Molecules 28 05149 g003 550
Figure 3. The representative hematoxylin and eosin (H&E) staining images for liver tissues of mice with the treatments of high dose concentration of the indicated drugs. (A,B) Normal control liver tissues without drug treatments; (C,D) TQFL12-treated group with unobvious damages in the liver; (E,F) TQ-treated group. A little focal necrosis and inflammatory cell infiltration occurred in the liver tissues, as the arrow points to. (B,D) are the enlarged images from (A,C), respectively. The fresh tissues (about 100 mg were obtained from mice, three representative samples were selected for each group) were removed from mice, washed in PBS three times, and fixed in 4% paraformaldehyde (>24 h) after dehydration by a gradient of alcohol (70%, 80%, 90%, 95%, 95%, 100%, and 100%), cleared in xylene three times, in the paraffin for 1 h three times, and then the tissues were quickly clamped out and laid flat in an embedded box with melted paraffin, cooled, and placed overnight. On the second day, the wax blocks were fixed on a paraffin microtome at 4 μm thickness and dried for 2 h for H&E staining.
Figure 3. The representative hematoxylin and eosin (H&E) staining images for liver tissues of mice with the treatments of high dose concentration of the indicated drugs. (A,B) Normal control liver tissues without drug treatments; (C,D) TQFL12-treated group with unobvious damages in the liver; (E,F) TQ-treated group. A little focal necrosis and inflammatory cell infiltration occurred in the liver tissues, as the arrow points to. (B,D) are the enlarged images from (A,C), respectively. The fresh tissues (about 100 mg were obtained from mice, three representative samples were selected for each group) were removed from mice, washed in PBS three times, and fixed in 4% paraformaldehyde (>24 h) after dehydration by a gradient of alcohol (70%, 80%, 90%, 95%, 95%, 100%, and 100%), cleared in xylene three times, in the paraffin for 1 h three times, and then the tissues were quickly clamped out and laid flat in an embedded box with melted paraffin, cooled, and placed overnight. On the second day, the wax blocks were fixed on a paraffin microtome at 4 μm thickness and dried for 2 h for H&E staining.
Molecules 28 05149 g003
Molecules 28 05149 g004 550
Figure 4. The representative H&E staining images for kidney tissues of mice with the treatments of high dose concentration of the indicated drugs. (A,B) Normal control kidney tissues without drug treatments; (C,D) TQFL12-treated group with unobvious damages in the kidney; (E,F) TQ-treated group with strong renal failure. (E) Renal tubular epithelial thinning and interstitial swelling; (F) Obvious vascular endothelial swelling, vascular congestion, hemorrhage, and lumen narrowing. (B,D) are the enlarged images from (A,C), respectively. The sampling and H&E staining are the same as in Figure 3.
Figure 4. The representative H&E staining images for kidney tissues of mice with the treatments of high dose concentration of the indicated drugs. (A,B) Normal control kidney tissues without drug treatments; (C,D) TQFL12-treated group with unobvious damages in the kidney; (E,F) TQ-treated group with strong renal failure. (E) Renal tubular epithelial thinning and interstitial swelling; (F) Obvious vascular endothelial swelling, vascular congestion, hemorrhage, and lumen narrowing. (B,D) are the enlarged images from (A,C), respectively. The sampling and H&E staining are the same as in Figure 3.
Molecules 28 05149 g004
Molecules 28 05149 g005 550
Figure 5. TQFL12 toxicity resistance through the AMPKα signaling pathway. (A,B) TQFL12-treated group shows up-regulated expression of both total and phosphorylated levels of AMPKα in the kidney tissues and the increase was more than that of the TQ group. (C,D) TQFL12-treated group shows up-regulated expression of phosphorylated levels of mTOR and the total expression of that in the liver tissues and the increase in the TQ group was more than that of TQFL12. (B,D) The quantitative data from (A,C), respectively. * p < 0.05, ** p < 0.01. The fresh tissues (about 50 mg were obtained from mice, three representative samples were selected from the TQFL12 group and two from the TQ group) from each group were lysed by EBC buffer for 30 min at 4 °C, taken as homogenate, and high-speed centrifugated at 12,000 rpm for 30 min at 4 °C; then, the liquid supernatant proteins samples were extracted and 2× SDS buffer were added and boiled at 100 °C for 5 min. The protein samples were separated in 8% and 10% sodium dodecyl sulfate–PAGE gel, transferred to the polyvinylidene fluoride with 300 mA for 2 h, and the membranes with the methanol were rinsed by TBST. After blocking in 5% nonfat milk, the membranes were incubated with indicated antibodies for Western blotting.
Figure 5. TQFL12 toxicity resistance through the AMPKα signaling pathway. (A,B) TQFL12-treated group shows up-regulated expression of both total and phosphorylated levels of AMPKα in the kidney tissues and the increase was more than that of the TQ group. (C,D) TQFL12-treated group shows up-regulated expression of phosphorylated levels of mTOR and the total expression of that in the liver tissues and the increase in the TQ group was more than that of TQFL12. (B,D) The quantitative data from (A,C), respectively. * p < 0.05, ** p < 0.01. The fresh tissues (about 50 mg were obtained from mice, three representative samples were selected from the TQFL12 group and two from the TQ group) from each group were lysed by EBC buffer for 30 min at 4 °C, taken as homogenate, and high-speed centrifugated at 12,000 rpm for 30 min at 4 °C; then, the liquid supernatant proteins samples were extracted and 2× SDS buffer were added and boiled at 100 °C for 5 min. The protein samples were separated in 8% and 10% sodium dodecyl sulfate–PAGE gel, transferred to the polyvinylidene fluoride with 300 mA for 2 h, and the membranes with the methanol were rinsed by TBST. After blocking in 5% nonfat milk, the membranes were incubated with indicated antibodies for Western blotting.
Molecules 28 05149 g005
Table
Table 1. The pharmacokinetics, drug-likeness, and medicinal chemistry friendliness of TQFL12.
Table 1. The pharmacokinetics, drug-likeness, and medicinal chemistry friendliness of TQFL12.
PharmacokineticsDrug-LikenessMedicinal Chemistry
GI absorptionHighLipinskiYesPAINS1
BBB permeantYesGhoseYesBrenk2
P-gp substrateNoVeberYesLead-likeness1
CYP1A2 inhibitorYesEganYesSynthetic Accessibility3.29
CYP2C19 inhibitorYesMueggeYes
CYP2C9 inhibitorYesBioavailability Score0.55
CYP2D6 inhibitorNo
CYP3A4 inhibitorYes
Table
Table 2. Results of the lethal doses for TQ and TQFL12 treatments for mice.
Table 2. Results of the lethal doses for TQ and TQFL12 treatments for mice.
Time (h)24487296120144168
TQ12088/////
(mg/kg)8078/////
5078/////
2528/////
LD5033.75
95%IC17.5–46.5
TQFL122501677899
(mg/kg)2002788888
1001345666
500122233
LD50 81.41
95%IC 31.56–123.40
Note: each control group contains 8 mice and each experimental group contains 10 mice. IC—interval confidence.
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Li, T.; Tan, Q.; Wei, C.; Zou, H.; Liu, X.; Mei, Z.; Zhang, P.; Cheng, J.; Fu, J. Design, Synthesis, and Acute Toxicity Assays for Novel Thymoquinone Derivative TQFL12 in Mice and the Mechanism of Resistance to Toxicity. Molecules 2023, 28, 5149. https://doi.org/10.3390/molecules28135149

AMA Style

Li T, Tan Q, Wei C, Zou H, Liu X, Mei Z, Zhang P, Cheng J, Fu J. Design, Synthesis, and Acute Toxicity Assays for Novel Thymoquinone Derivative TQFL12 in Mice and the Mechanism of Resistance to Toxicity. Molecules. 2023; 28(13):5149. https://doi.org/10.3390/molecules28135149

Chicago/Turabian Style

Li, Ting, Qi Tan, Chunli Wei, Hui Zou, **aoyan Liu, Zhiqiang Mei, Pengfei Zhang, **gliang Cheng, and Junjiang Fu. 2023. "Design, Synthesis, and Acute Toxicity Assays for Novel Thymoquinone Derivative TQFL12 in Mice and the Mechanism of Resistance to Toxicity" Molecules 28, no. 13: 5149. https://doi.org/10.3390/molecules28135149

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