Research Article Print
Research Article
﻿Antiproliferative activity of a new derivative from the class of N-glycoside of indolo[2,3-a]pyrrolo[3,4-c]carbazoles
Marina P. Kiseleva, Larisa M. Borisova, Galina B. Smirnova, Yulia A. Borisova, Anna V. Lantsova, Ekaterina V. Sanarova, Lyudmila L. Nikolaeva§, Lydia V. Ektova, Marina V. Komarova|
‡ N.N. Blokhin National Medical Research Center of Oncology, Moscow, Russia
§ Sechenov University, Moscow, Russia
| Samara National Research University, Samara, Russia
 Corresponding author: Lyudmila L. Nikolaeva ( alima91@yandex.ru ) Academic editor: Mikhail Korokin © 2022 Marina P. Kiseleva, Larisa M. Borisova, Galina B. Smirnova, Yulia A. Borisova, Anna V. Lantsova, Ekaterina V. Sanarova, Lyudmila L. Nikolaeva, Lydia V. Ektova, Marina V. Komarova.This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Kiseleva MP, Borisova LM, Smirnova GB, Borisova YuB, Lantsova AV, Sanarova EV, Nikolaeva LL, Ektova LV, Komarova MV (2022) ﻿Antiproliferative activity of a new derivative from the class of N-glycoside of indolo[2,3-a]pyrrolo[3,4-c]carbazoles. Research Results in Pharmacology 8(2): 49-57. https://doi.org/10.3897/rrpharmacology.8.79424

# ﻿Abstract

Introduction: The creation of highly effective original anticancer drugs remains an urgent direction of scientific research in tumor therapy. One of the promising groups in this regard is ﻿indolocarbazoles﻿ and their derivatives, which are capable of initiating various pathways of tumor cell death. The aim of the study was to evaluate an antiproliferative activity of a new, Russian derivative of N-glycoside substituted ﻿indolocarbazole﻿ 6-amino-12-(α-L-arabinopyranosyl)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (LCS-1208) on models of transplantable tumors of mice and on human tumors in Balb/c nude mice.

Materials and methods: ﻿Indolocarbazole﻿ sensitivity to LCS-1208 was assessed on transplantable tumors of mice – lymphatic leukemia L-1210, cervical carcinoma (CC-5), and colon adenocarcinoma (CAC) by five-fold intraperitoneal administration (ip) of the LCS-1208 substance in single doses of 50, 75, 100 mg/kg. Investigation into the effectiveness of the LCS-1208 lyo dosage form was performed on subcutaneous xenografts of human colon cancer SW620 by an intravenous administration (iv). The antitumor effect was evaluated by the tumor growth inhibition (TGI) and an increase in life span (ILS) of the treated animals as compared with the control ones. Evaluation of specific antitumor activity on xenografts was performed according to the tumor/control (T/C%) criterion (maximum criterion T/C≤42%).

Results and discussion: According to the results of the study, the most sensitive to the action of the LCS-1208 substance in the case of an ip administration of a total dose of 375 mg/kg were CAC with TGI=97–62%, p≤0.001 up to 16 days after the treatment, and ILS=36% (criteria for TGI≥70% and ILS≥25%). On xenografts of a human colon cancer SW620, the effectiveness of the LCS-1208 lyo drug dosage form within the range of total doses from 50 to 150 mg/kg in case of iv to Balb/c nude mice was set at T/C = 35–2% (criterion T/C<42%).

Conclusion: The presented results suggest possible effectiveness of LCS-1208 in treatment of colon malignant tumors of humans.

# Keywords

﻿indolocarbazoles﻿, transplantable tumors of mice, subcutaneous xenografts, tumor growth inhibition.

# ﻿Introduction

﻿Indolocarbazoles﻿ and their derivatives constitute a wide class of natural and synthetic compounds with various types of biological activity, including antitumor activity, and this allows us to consider these compounds as potential antitumor agents. Drugs from the class of ﻿indolocarbazoles﻿ have attracted the attention of researchers by their ability to interact with several intracellular targets and the ability to induce different cell death paths. Among the representatives of this class, there are the compounds that cause damage to DNA structure by means of intercalation, and that are inhibitors of topoisomerases controlling the processes of DNA replication, transcription and repair, as well as inhibitors of kinases, in particular, CDK-1 kinase and protein kinase C involved in transmission of intracellular proliferative signals (Civenni et al. 2016; Lafayette et al. 2017; Zenkov et al. 2020).

So far, an extensive database on the antiproliferative activity of ﻿indolocarbazoles﻿ with various chemical structures has been compiled, which makes it possible to consider such substances as promising for carrying out further study (Caruso et al. 2019; Zenkov et al. 2021). ﻿Indolocarbazole﻿ derivatives containing glycoside substituents attached to the pharmacophore via nitrogen atoms are of special interest (Sánchez et al. 2006; Wada et al. 2007; Kiseleva et al. 2019). Among the biologically active N-glycosides of ﻿indolocarbazoles﻿, the alkaloid ﻿staurosporine﻿, an effective protein kinase C inhibitor, is well known (Tanramluk et al. 2009). The natural antibiotic ﻿rebeccamycin﻿ and its water-soluble derivative ﻿becatecarin﻿ have the properties of topoisomerase I inhibitors (Issa et al. 2019). The manifestation of high antiproliferative activity in such compounds as ﻿rebeccamycin﻿ and ﻿staurosporine﻿ determined the search for effective antitumor drugs among their synthetic analogues and low molecular weight derivatives with lower toxic properties. The representatives of this class: ﻿midostaurin﻿ (Li et al. 2022), ﻿enzastaurin﻿ (Sadeghi et al. 2021), ﻿lestaurtinib﻿ (Kangussu-Marcolino and Singh 2022), ﻿becatecarin﻿ (Schwandt et al. 2012), and ﻿edotecarin﻿ (Buzun et al. 2020) are undergoing clinical trials. The introduction of new antitumor agents from N-glycosides of ﻿indolocarbazoles﻿ class into clinical practice is also extremely important for overcoming the drug resistance of tumor cells to treatment.

At the N.N. Blokhin National Medical Research Center of Oncology (NMRCO), a method was developed to synthesize indolo[2,3-a]pyrrolo[3-4-c] carbazole derivative (LCS-1208). This substance is almost insoluble in water and in most organic solvents, which was a major problem when developing the dosage form. As a result of the performed investigations, a lyophilized dosage form: “LCS-1208, 9 mg, lyophilizate for preparation of injection solution” (LCS-1208 lyo) was created and protected by the patent of the Russian Federation (Lantsova et al. 2014; Gulyakin et al. 2021). In the course of studying the effectiveness of the dosage form of LCS-1208 lyo usage, a method to treat human colon cancer SW620 was patented and proposed in the experiment.

The aim of the study was to evaluate the antiproliferative activity of a new Russian N-glycoside substituted ﻿indolocarbazole﻿ derivative LCS-1208 on transplanted mice tumor models and on human tumors in Balb/c nude mice.

# ﻿Materials and methods

## ﻿Animals

The work was performed on 24 sexually mature female BDF1-hybrid mice F1 (C57Bl/6j x DBA/2), 24 female CBA/Lac and 26 Balb/c mice strains, 38 male Balb/c nude mice, weighing 20–22 g. Before treatment, the animals were divided into groups. The number of animals in the control groups was 8–10 mice, and in the test groups – 7–10 animals. Ethics Committee Minutes No. 04Р of September 18, 2020.

Mice tumor models include lymphocytic leukemia L-1210, cervical carcinoma (CC-5), and colon adenocarcinoma (CAC). During the experiments, 2–5th in vivo passages were used. Transplantation was performed according to a standard technique (Treshchalina et al. 2012; Treshalina 2017).

L-1210 cells were transplanted into female BDF1 hybrids via the intraperitoneal administration (ip), 106 cells per mouse in 0.3 ml of nutrient medium 199. During the experiment, CC-5 was inoculated to CBA female mice, and CAC – to Balb/c female mice. During the transplantation, inoculation of tumor cells was performed subcutaneously into the right axillary region of each mouse by 50 mg of tumor suspension in nutrient medium 199 at a dilution of 1:10 (5×106 cells). The treatment was started 24 h after transplantation of L-1210 cells and 48 h after in the case of CC-5 and CAC (Sof’ina et al. 1980). The start of treatment corresponded to the intensive reproduction time of tumor cells, which ensures their being in the most chemotherapy-sensitive state (Polin et al. 2011).

The CAC and CC-5 tumor strains of mice were generated at the N.N. Blokhin NMRCO. CAC arose in 1971 from a subcutaneous syngeneic transplant of embryo colon in a Balb/c mouse. CC-5 was induced by methylcholanthrene in the CBA mice’ cervix subcutaneous autotransplant in 1970 (Sof’ina et al. 1980).

During the experiments on immunodeficient mice, a transplantable human colon cancer strain SW620, grown in the form of subcutaneous xenografts, was used (Treshchalina 2012). Each Balb/c nude mouse were injected subcutaneously 0.2 ml of a 20% suspension (40 mg of tumor tissue). Treatment was started 48 h after transplantation.

The test compound – 6-amino-12-(α-L-arabinopyranosyl)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (LCS-1208 substance) was dissolved in ﻿dimethyl sulfoxide﻿ (﻿DMSO﻿) and diluted with saline up to 10% concentration of ﻿DMSO﻿. In the experiments on mice with L-1210, CC-5 and CAC, we used a 5-day regimen with an interval of 24 h of ip administration of the substance LCS-1208 in effective doses of 50, 75 and 100 mg/kg, which were found in the previous experiments (Kiseleva et al. 2015).

The dosage form – LCS-1208 lyo – is an amorphous orange powder containing 98% of the main active ingredient in a vial. Before use, the content of the vial was rehydrated with water for injection to form a homogenous orange solution.

A study of the antitumor activity of LCS-1208 lyo was carried out on SW620 with two-fold intravenous administrations (iv) of 25, 50 and 75 mg/kg doses to Balb/c nude mice with an interval of 96 h.

## ﻿Evaluation of the antitumor effect

The efficacy of treating mice with CC-5, CAC and L-1210 was evaluated according to the standard criteria: tumor growth inhibition (TGI, %) and an increase in life span (ILS, %). The evaluation of specific antitumor activity on xenografts of human tumors was carried out according to the TGI index calculated by the ratio of the average volumes of tumor nodes in the treated and control groups of mice, T/C% (”treatment/control”), taking into account that in the control group T/C=100% and using the maximum criterion T/C≤42% for experiments with developed tumors.

The degree of tumor growth inhibition (TGI and T/C) were calculated by formulas (1, 2):

$TGI%=Vc-VtVc×100%$, (1)

$TC%=VtVc×100%$, (2)

where Vc and Vt are the average volumes of tumors (mm3) in the control and treatment groups, which for each solid tumor was defined as a number obtained by multiplication of sizes of three perpendicular diameters of the tumor node. Tumor volume was measured at different periods of time after treatment.

ILS of the treated animals in comparison with the animals from the control group was calculated by formula (3):

$ILS%=ALSt-ALScALSc×100%$, (3)

where ALSt and ALSc are the average life spans (days) in the treatment and control groups of animals.

Doses causing TGI≥70% lasting at least 7 days after completion of the treatment and ILS≥25% for animals with solid tumors and ILS≥75% for the animals with lymphocytic leukemia were considered to be effective (Treshchalina et al. 2012).

## ﻿Evaluation of treatment tolerance

During the experiments, follow-up of the animals was continued until their death. The tolerability of the LCS-1208 was judged by the state and behavior of the mice. The toxicity of the studied regimens and doses of the LCS-1208 substance and LCS-1208 lyo was evaluated by the time of death of the treated animals in comparison with the death of the animals in the control group, as well as by a decrease in their body weight (≥20%) and spleen mass (indirect signs of general, hematological and immunological toxicity) (Teicher and Andrews 2004; Treshchalina et al. 2012).

Statistical evaluation of the results was performed by using the IBM SPSS Statistics 21 package (license number 20130626-3), followed by comparing the separate groups with one another according to the Tukey’s test, and using Excel software when calculating the Fisher criterion. Differences between the compared groups were considered statistically significant at p <0.05.

# ﻿Results and discussion

## ﻿Study of the effectiveness of the LCS-1208 substance in mice on lymphocytic leukemia L-1210

The study of the effectiveness of LCS-1208 on lymphocytic leukemia L-1210 was performed with five-fold ip administrations of the LCS-1208 substance in single doses of 50 and 75 mg/kg (total doses 250 and 375 mg/kg) (Table 1). The antitumor effect of the LCS-1208 substance is shown in the form of ILS = 43% (p = 0.001 in relation to the control) and ILS = 47% (p < 0.001 in relation to the control), for the studied doses, respectively.

Table 1.

Antitumor Effect of the LCS-1208 Substance When Intraperitoneal Administration to Mice With L-1210

Dose, mg/kg Total dose, mg/kg Increase of life span, % P in relation to the control group
50 250 43 0.001
75 375 47 <0.001

In the test groups, the differences between the total doses of 250 mg/kg and 350 mg/kg were insignificant (p = 0.925), indicating an equal but insufficient antitumor activity of the LCS-1208 substance on lymphocytic leukemia L-1210 (criterion of effectiveness for animals with lymphocytic leukemia is ILS ≥ 75%).

## ﻿Study of the effectiveness of LCS-1208 substance in mice with CC-5

The LCS-1208 substance with five-fold ip administrations in single doses of 75 and 100 mg/kg (total doses of 375 and 500 mg/kg) generated positive, but not high inhibition of tumor growth in CC-5 with respect to the control group: TGI = 97–52% (p ≤ 0.015) and TGI=98–59% (p < 0.001), respectively, within 7 days (Table 2, Fig. 1).

Table 2.

Antitumor Effect of the LCS-1208 Substance When Intraperitoneal Administration to Mice with CC-5

Dose, mg/kg Total dose, mg/kg Tumor growth inhibition (TGI) % Toxicity death, %
Days after treatment
1 7 11 18 24
75 375 97 52 20 17 42 29
100 500 98 59 39 17 10 43

A single dose of 75 mg/kg (total dose of 375 mg/kg) caused the death of mice in 29% of cases on the 20th day after treatment with an average life span of control mice of 43 (39–54) days. Increasing a single dose to 100 mg kg (total dose of 500 mg/kg) led to the death of mice in 43% of cases at the earlier periods of follow-up (on the 15th day after the end of treatment). Signs of toxicity were expressed as a slight decrease in body weight of the animal by 10% of the initial weight. At autopsy, there were no differences in the weight of the spleen in comparison with the control group of animals.

## ﻿Study of the effectiveness of the LCS-1208 substance on CAC of mice

When using the LCS-1208 substance a in single dose of 75 mg/kg (total dose of 375 mg/kg), in comparison with the control group, a reliable prolonged inhibition of primary subcutaneous tumor node growth within 16 days after the end of the treatment (TGI = 97–62%, p≤0.001), and ILS of mice with CAC by 36% was observed (Table 3, Fig. 2).

Table 3.

Antitumor Effect of the LCS-1208 Substance When Intraperitoneal Administration to Mice With CAC

Dose, mg/kg Total dose, mg/kg Tumor growth inhibition (TGI), % Increase in life span (ILS), % Toxicity death, %
Days after treatment
1 7 11 16
75 375 97 97 86 62 36 0
100 500 99 99 94 78 13 50

An increase in a single dose of LCS-1208 to 100 mg/kg (total dose of 500 mg/kg) reliably increased the inhibition of CAC growth by 99–78% (p≤0.001) within 16 days of follow-up in comparison with the control group, but led to death of 50% mice on the 3rd-5th days after the end of treatment. Signs of toxicity were expressed in a decreased motor activity, a decreased body weight of animals by 46% when compared to their initial weight, as well as in significantly decreased (2–3 times) spleen mass at autopsy in comparison with the spleen mass in mice from the control group.

## ﻿Study of the effectiveness of LCS-1208 lyo on subcutaneous xenografts of human colon cancer SW620 in vivo

The antitumor activity of LCS-1208 lyo was studied on human tumors in Balb/c nude mice in the conditions of an optimal application regimen under the control of tolerance.

The results of the study showed that subcutaneous xenografts of colon cancer SW620 without treatment have dynamics of steady growth. Tumor nodes grow quite quickly and show an 8-time increase within 12 days after transplantation, from Vav=328±148 mm3 to Vav=2697±414 mm3. Against this background, all the doses of the LCS-1208 lyo were highly effective and gave a significant, reliable antitumor effect, showing an increase in a level and degree of significance within 12 days after the end of treatment (Table 4). Thus, on the 1st, 6th, 12th days, at a single dose of 25 mg/kg (total dose of 50 mg/kg) – T/C=35%; 13%; 19%, respectively. At a single dose of 50 mg/kg (total dose of 100 mg/kg) – T/C=28%; 6%; 6%, respectively. At a single dose of 75 mg/kg (total dose of 150 mg/kg) – T/C=23%; 2%; 2%, respectively.

Table 4.

Study of the Efficacy of LCS-1208 Lyo on Subcutaneous Xenographs of Human Colon Cancer SW620 in Vivo With Double (after 96 h) Intravenous Administration

No. mice, indicators Average tumor volumes and performance indicators at various times after treatment
1st day 6th day 12th day
TGC Single dose (total dose), mg/kg TGC Single dose (total dose), mg/kg TGC Single dose (total dose), mg/kg
25(50) 50(100) 75(150) 25(50) 50(100) 75(150) 25(50) 50(100) 75(150)
1 333 53 105 78 843 243 53 29 2209 878 148 68
2 359 89 64 145 973 199 50 62 2157 599 98 81
3 401 156 62 29 1926 357 56 25 3012 701 85 death
4 573 121 44 136 1657 157 55 78 3406 545 93 death
5 407 218 72 29 1225 117 95 14 2745 237 250 68
6 295 8 106 41 1398 231 102 23 2840 330 228 59
7 124 59 116 132 1578 249 85 65 2723 348 108 75
8 131 170 181 30 1450 94 101 15 2484 617 215 90
9 166 40 108 59 46 32 715 182 65
10 117 131 19 53 141 27 150 133 21
Vav 328 116 92 75 1381 176 78 34 2697 512 154 66
S 148 64 44 51 358 98 32 23 414 235 61 21
Т/С% 100 35 28 23 100 13 6 2 100 19 6 2
Ttest 0.00000002 0.0002 0.0006 0.000002 0.000001 0.00004 0.00004 0.0004 0.00005

However, using a 75 mg/kg single dose (total dose of 150 mg/kg) on the 12th day of treatment led to the death of 20% of mice from toxicity, which was expressed in a decreased motor activity and decreased body weight of animals by 15% compared to the initial one. In preliminary studies, the LCS-1208 lyo in a dose of 150 mg/kg with a slow stream iv infusion to mice with SW620 caused the death of animals from toxicity one day after the administration.

In the framework of this study, in tolerated doses of 25, 50 and 75 mg/kg (total doses of 50, 100 and 150 mg/kg, respectively), the condition and behavior of the mice during 20 days was satisfactory, with no evidence of any side effects. The comparative dynamics of tumor growth, evaluated visually in these animals on the 12th and 20th days after treatment, showed that the tumor growth in the LCS-1208 lyo group almost stabilized, since the tumor sizes were smaller than the tumor sizes in mice from the control group, measured on 12th day after the end of the treatment. Balb/c nude mice with subcutaneous xenografts of human colon cancer SW620 are shown on the 4th day (Fig. 3) and 16th day (Fig. 4) after the end of treatment in comparison with the control.

# ﻿Conclusion

A necessary condition when developing and studying new drugs is a careful selection of sensitive models in the experiment and special attention to the ratio of risk and effectiveness under the control of treatment tolerance.

As a result of the study of sensitivity of transplanted tumors of mice with L-1210, CC-5 and CAC to the LCS-1208 substance, a high antitumor activity of the LCS-1208 substance with respect to CAC was shown with a 5-fold ip administration of a single dose of 75 mg/kg (total dose of 375 mg/kg). Administration of a total dose of 500 mg/kg resulted in the death of mice.

The antitumor effect of the LCS-1208 substance on CC-5 was noted; however, in the indicated doses and regimen of administration, there was observed the death of animals from toxicity (29–43%).

The LCS-1208 substance in the studied doses with a 5-fold ip administration to mice with lymphocytic leukemia L-1210 did not cause toxicity. The life span of animals was 43–47%, which turned out to be lower than the criterion of effectiveness.

High results of LCS-1208 substance antitumor effect on mice with CAC with indices of TGI=97–62% up to 16 days after treatment and ILS=36% became the basis for a further study of the effectiveness of LCS-1208 lyo dosage form on subcutaneously transplanted xenografts. In our studies performed on xenografts of human colon cancer SW620, the LCS-1208 lyo in the range of total doses from 50 to 150 mg/kg with iv administration to Balb/c nude mice showed effectiveness in inhibiting tumor growth T/C=35–2% (T/C criterion<42%). The obtained results demonstrate the high activity of all the studied doses of the LCS-1208 lyo drug substance with direct dependence of an antitumor effect on a total dose (Fig. 5).

The results of this study are comparable with the data obtained by other authors during investigation of the antiproliferative activity of various representatives of the ﻿indolocarbazole﻿ class. For example, Ciomei M. et al. (2006) studied the antitumor effect of ﻿edotecarin﻿ when used alone or in combination with ﻿5-fluorouracil﻿, ﻿irinotecan﻿, ﻿cisplatin﻿, ﻿oxaliplatin﻿ and the multi-target tyrosine kinase inhibitor SU11248 on the model of human colon cancer xenograft HCT-116. In all the studies, ﻿edotecarin﻿ was active both as monotherapy and in combination with other antitumor agents (Ciomei et al. 2006). Another analogue of ﻿rebeccamycin﻿ – NB-506 – in a dose of 300 mg/m2 inhibited the growth of human colon cancer tumor cells HCT-116 and LS-180, grown as subcutaneous xenografts in immunodeficient mice (Delgado et al. 2018).

﻿Indolocarbazole﻿ from ﻿staurosporine﻿ derivatives CEP-7055 inhibited the growth of subcutaneously transplanted colon cancerxenografts HT-29 and HCT-116 by 50–90% (Ruggeri et al. 2003). CEP-7055 in combination with ﻿irinotecan﻿ and, to a lesser extent, with ﻿oxaliplatin﻿, showed a decrease in primary metastases of colon and liver carcinoma than in case of monotherapy with ﻿irinotecan﻿ or ﻿oxaliplatin﻿ (Jones-Bolin et al. 2006). However, further research was discontinued, as CEP-7055 showed no activity during phase I clinical trials (Williams 2008).

The data presented indicate the need to continue preclinical studies of the LCS-1208 lyo drug, and suggest the effectiveness of its use for treatment of malignant tumors of colon in humans.

# ﻿Conflict of interest

The authors declare no competing interests.

# ﻿References

• Caruso A, Ceramella J, Iacopetta D, Saturnino C, Mauro MV, Bruno R, Aquaro S, Sinicropi MS (2019) Carbazole derivatives as antiviral agents: An overview. Molecules 24(10): e1912. ﻿https://doi.org/10.3390/molecules24101912﻿ [﻿PubMed﻿] [﻿PMC﻿]
• Ciomei M, Croci V, Ciavolella A, Ballinari D, Pesenti E (2006) Antitumor efficacy of edotecarin as a single agent and in combination with chemotherapy agents in a xenograft model. Clinical Cancer Research 12(9): 2856–2861. ﻿https://doi.org/10.1158/1078-0432﻿ [﻿PubMe﻿d]
• Civenni G, Longoni N, Costales P, Dallavalle C, García Inclán C, Albino D, Nuñez LE, Morís F, Carbone GM, Catapano CV (2016) EC-70124, a novel glycosylated indolocarbazole multikinase inhibitor, reverts tumorigenic and stem cell properties in prostate cancer by inhibiting STAT3 and NF-κB. Molecular Cancer Therapeutics 15(5): 806–818. ﻿https://doi.org/10.1158/1535-7163.MCT-15-0791﻿ [﻿PubMed﻿]
• Gulyakin I, Lantsova A, Nikolaeva L, Dmitrieva M, Oborotova N, Orlova O, Zhuravleva N (2021) Features of the development of a lyophilized injectable dosage form of the original anticancer drug LCS-1208. International Journal of Applied Pharmaceutics 13(4): 102–105. ﻿https://doi.org/10.22159/ijap.2021v13i4.41371﻿
• Issa S, Prandina A, Bedel N, Rongved P, Yous S, Le Borgne M, Bouaziz Z (2019) Carbazole scaffolds in cancer therapy: a review from 2012 to 2018. Journal of Enzyme Inhibition and Medicinal Chemistry 34(1): 1321–1346. ﻿https://doi.org/10.1080/14756366.2019.1640692﻿ [﻿PubMed﻿] [﻿PMC﻿]
• Jones-Bolin S, Zhao H, Hunter K, Klein-Szanto A, Ruggeri B (2006) The effects of the oral, pan-VEGF-R kinase inhibitor CEP-7055 and chemotherapy in orthotopic models of glioblastoma and colon carcinoma in mice. Molecular Cancer Therapeutics 5(7): 1744–1753. ﻿https://doi.org/10.1158/1535-7163.MCT-05-0327﻿ [﻿PubMed﻿]
• Kangussu-Marcolino MM, Singh U (2022) Ponatinib, lestaurtinib, and mTOR/PI3K inhibitors are promising repurposing candidates against entamoeba histolytica. Antimicrobial Agents and Chemotherapy 66(2): e01207–01221. ﻿https://doi.org/10.1128/AAC.01207-21﻿ [﻿PubMed﻿]
• Kiseleva MP, Pokrovsky VS, Borisova LM, Golubeva IS, Ektova LV (2019) N-glycosidesindolo[2,3,-a]pyrrolo[3,4,-c]carbazole derivatives chemical structure influence on antitumor activity. Russian Journal of Biotherapy 18(2): 32–39. ﻿https://doi.org/10.17650/1726-9784-2019-18-2-32-39﻿ [in Russian]
• Kiseleva MP, Smirnova ZS, Borisova LM, Kubasova IYu, Ektova LV, Miniker TD, Plikhtiak IL, Medvedeva LA, Eremina VA, Tikhonova NI (2015) Search for new antitumor compounds among n-glycoside indolo[2,3-a]carbazole derivatives. Russian Journal of Oncology 20(1): 33–37. [in Russian]
• Lafayette EA, de Almeida SMV, Cavalcanti Santos RV, de Oliveira JF, Amorim CADC, da Silva RMF, Pitta MGDR, Pitta IDR, de Moura RO, de Carvalho Júnior LB, de Melo Rêgo MJB, de Lima MDCA (2017) Synthesis of novel indole derivatives as promising DNA-binding agents and evaluation of antitumor and antitopoisomerase I activities. European Journal of Medical Chemistry 136: 511–522. ﻿https://doi.org/10.1016/j.ejmech.2017.05.012﻿ [﻿PubMed﻿]
• Lantsova AV, Sanarova EV, Oborotova NA, Poloskova AP, Orlova OL, Shprakh ZS, Kiseleva MP, Gulyakin ID, Smirnova ZS (2014) Development of technology for injectable dosage form based on the national substange from the class of indolocarbazoles – LHS-1208. Russian Journal of Biotherapy [Rossijskij Bioterapevticheskij Zhurnal] 13(3): 25–32. [in Russian]
• Li Z, Wang J, Ge S-S, Qiu Q-C, Du J-H, Shan S-S, Shen X-D, Wan C-L, Wang B-R, Wu D-P, Qiu H-Y, Xue S-L (2022) Combination of venetoclax and midostaurin efficiently suppressed relapsed t(8;21) acute myeloid leukemia with mutant KIT after failure of venetoclax plus azacitidine treatment. Frontiers in Oncology 12: e841276. ﻿https://doi.org/10.3389/fonc.2022.841276﻿ [﻿PubMed﻿] [﻿PMC﻿]
• Polin L, Corbett TH, Roberts BJ, Lawson AJ, Leopol III WR, White K, Kushner J, Hazeldine S, Moore R, Rake J, Horwitz J (2011) Transplantable syngeneic rodent tumors: solid tumors in mice. In: Tumor Models in Cancer Research. Cancer Drug Discovery and Development. Humana Press, Totowa, New Jersey, 43–78. ﻿https://doi.org/10.1007/978-1-60761-968-0_3﻿
• Ruggeri B, Singh J, Gingrich D, Angeles T, Albom MS, Yang SX, Chang H, Robinson C, Hunter K, Dobrzanski P, Jones-Bolin S, Pritchard S, Aimone IL, Klein-Szanto A, Herbert J, Bono F, Schaeffer P, Cassellas P, Bourie B, Pili R, Isaacs J, Ator M, Hudkins R, Vaught J, Mallamo J, Dionne C (2003) CEP-7055: A novel, orally active pan inhibitor of vascular endothelial growth factor receptor tyrosine kinases with potent antiangiogenic activity and antitumor efficacy in preclinical models. Cancer Research 63(18): 5978–5991. [﻿PubMed﻿]
• Sadeghi MM, Salama MF, Hannun YA (2021) Protein kinase c as a therapeutic target in non-small cell lung cancer. International Journal of Molecular Sciences 22(11): e5527. ﻿https://doi.org/10.3390/ijms22115527﻿ [﻿PubMed﻿] [﻿PMC﻿]
• Sánchez C, Méndez C, Salas JA (2006) Indolocarbazole natural products: occurrence, biosynthesis, and biological activity. Natural Product Reports 23(6): 1007–1045. ﻿https://doi.org/10.1039/b601930g﻿ [﻿PubMed﻿]
• Schwandt A, Mekhail T, Halmos B, O’Brien T, Ma PC, Fu P, Ivy P, Dowlati A (2012) Phase-II trial of rebeccamycin analog, a dual topoisomerase-I and -II inhibitor, in relapsed “sensitive” small cell lung cancer. Journal of Thoracic Oncology 7(4): 751–754. ﻿https://doi.org/10.1097/JTO.0b013e31824abca2﻿ [﻿PubMed﻿] [﻿PMC﻿]
• Sofina ZP, Syrkin AB, Goldin A, Klein A (1980) Experimental Evaluation of Antitumor Drugs in the USSR and the USA. Medicine Press, Moscow, 296 pp. [in Russian]
• Tanramluk D, Schreyer A, Pitt WR, Blundell TL (2009) On the origins of enzyme inhibitor selectivity and promiscuity: a case study of protein kinase binding to staurosporine. Chemical Biology & Drug Design 74(1): 16–24. ﻿https://doi.org/10.1111/j.1747-0285.2009.00832.x﻿ [﻿PubMed﻿] [﻿PMC﻿]
• Teicher BA, Andrews PA (Eds) (2004) Anticancer drug development guide. preclinical screening, clinical trials, and approval. 2nd Edn. Humana Press, Totowa, New Jersey, 450 pp. ﻿https://doi.org/10.1007/978-1-59259-739-0﻿
• Treshalina EM (2017) Immunodeficient mice balb/c nude and modeling of various types of tumor growth for preclinical studies. Russian Journal of Biotherapy [Rossijskij Bioterapevticheskij Zhurnal] 16(3): 6–13. ﻿https://doi.org/10.17650/1726-9784-2017-16-3-6-13﻿ [in Russian]
• Treshchalina EM, Zhukova OS, Gerasimova GK, Andronova NV, Garin AM (2012) Guidelines for preclinical study of antitumor activity of drugs. In: Mironov AN (Ed.) Preclinical Drug Research Guide, part 1. Grif & K, Moscow, 642–657. [in Russian]
• Wada Y, Nagasaki H, Tokuda M, Orito K (2007) Synthesis of N-protected staurosporinones. The Journal of Organic Chemistry 72(6): 2008–2014. ﻿https://doi.org/10.1021/jo062184r﻿ [﻿PubMed﻿]
• Zenkov RG, Ektova LV, Vlasova OА, Belitskiy GA, Yakubovskaya MG, Kirsanov KI (2020) Indolo[2,3-a]carbazoles: diversity, biological properties, application in antitumor therapy. Chemistry of Heterocyclic Compounds 56: e644658. ﻿https://doi.org/10.1007/s10593-020-02714-4﻿
• Zenkov RG, Vlasova OA, Maksimova VP, Fetisov TI, Karpechenko NY, Ektova LV, Eremina VA, Popova VG, Usalka OG, Lesovaya EA, Belitsky GA, Yakubovskaya MG, Kirsanov KI (2021) Molecular mechanisms of anticancer activity of N-Glycosides of indolocarbazoles LCS-1208 and LCS-1269. Molecules 26(23): e7329. ﻿https://doi.org/10.3390/molecules26237329﻿ [﻿PubMed﻿] [﻿PMC﻿]

# ﻿Author Contribution

Marina P. Kiseleva, PhD in Biological sciences, Senior Researcher of the Laboratory of Experimental Chemotherapy, Research Institute of Experimental Diagnostic and Therapy of Tumors, e-mail: ﻿marina-kiselyova@mail.ru﻿, ORCID ID ﻿http://orcid.org/0000-0002-4309-6722﻿. Writing the article, the development of the research design, and the administration of the drugs to the animals.

Larisa M. Borisova, PhD in Biological sciences, Head Laboratory of Experimental Chemotherapy, Research Institute of Experimental Diagnostic and Therapy of Tumors, e-mail: ﻿larib@inbox.ru﻿, ORCID ID ﻿http://orcid.org/0000-0001-6554-1949﻿. Writing the article, the development of the research design, and the administration of the drugs to the animals.

Galina B. Smirnova, PhD in Biological sciences, Senior Researcher of the Laboratory of Laboratory of Combined Therapy, Research Institute of Experimental Diagnostic and Therapy of Tumors, e-mail: ﻿gsmir53@yandex.ru﻿. Writing the article, the development of the research design, and the administration of the drugs to the animals.

Yulia A. Borisova, Junior Researcher of the Laboratory of Laboratory of Combined Therapy, Research Institute of Experimental Diagnostic and Therapy of Tumors, e-mail: ﻿ulkabor@yandex.ru﻿. Writing the article, the development of the research design, and the administration of the drugs to the animals.

Anna V. Lantsova, PhD in Pharmacy, Leading researcher of the Laboratory for the Development of Dosage Forms, Research Institute of Experimental Diagnostic and Therapy of Tumors, e-mail: ﻿lantsova1979@mail.ru﻿, ORCID ID ﻿https://orcid.org/0000-0002-0650-2023﻿. Writing the article, analyzing the literature, and interpreting the data.

Ekaterina V. Sanarova, PhD in Pharmacy, Senior researcher of the Laboratory for the Development of Dosage Forms, Research Institute of Experimental Diagnostic and Therapy of Tumors, e-mail: ﻿sanarova8686@mail.ru﻿, ORCID ID ﻿https://orcid.org/0000-0002-5592-5137﻿. Writing the article, analyzing the literature, and interpreting the data.

Lyudmila L. Nikolaeva, PhD in Pharmacy, Researcher of the Laboratory for the Development of Dosage Forms, Research Institute of Experimental Diagnostic and Therapy of Tumors, N. N. Blokhin National Medical Research Center of Oncology, Senior Lecturer, Department of Pharmaceutical Technology and Pharmacology, Sechenov University. e-mail: ﻿alima91@yandex.ru﻿, ORCID ID ﻿https://orcid.org/0000-0001-8003-8241﻿. Writing the article, analyzing the literature, and interpreting the data.

Lydia V. Ektova, PhD in Chemistry, Consultant of the Laboratore Chemical Synthesis, Research Institute of Experimental Diagnostic and Therapy of Tumors, e-mail: ﻿ektova@ronc.ru﻿, ORCID ID ﻿https://orcid.org/0000-0002-3987-6072﻿. Synthesis of the substance.

Marina V. Komarova, PhD in Biological sciences, Associate professor, Department of laser and biotechnical systems, e-mail: ﻿marinakom@yandex.ru﻿, ORCID ID ﻿https://orcid.org/0000-0001-6545-0035﻿. Interpreting the data.