New 5-Substituted SN38 Derivatives: A Stability Study and Interaction with Model Nicked DNA by NMR and Molecular Modeling Methods.

The new 5-substituted SN-38 derivatives, 5(R)-(N-pyrrolidinyl)methyl-7-ethyl-10-hydroxycamptothecin (1) and its diastereomer 5(S) (2), were investigated using a combination of nuclear magnetic resonance (NMR) spectroscopy and molecular modeling methods. The chemical stability, configuration stability, and propensity to aggregate as a function of concentration were determined using 1H NMR. The calculated self-association constants (Ka) were found to be 6.4 mM-1 and 2.9 mM-1 for 1 and 2, respectively. The NMR experiments were performed to elucidate the interaction of each diastereomer with a nicked decamer duplex, referred to as 3. The calculated binding constants were determined to be 76 mM-1 and 150 mM-1 for the 1-3 and 2-3 complexes, respectively. NMR studies revealed that the interaction between 1 or 2 and the nicked decamer duplex occurred at the site of the DNA strand break. To complement these findings, molecular modeling methods and calculation protocols were employed to establish the interaction mode and binding constants and to generate molecular models of the DNA/ligand complexes.

Investigation of the Uptake and Transport of Two Novel Camptothecin Derivatives in Caco-2 Cell Monolayers.

Irinotecan and Topotecan are two Camptothecin derivatives (CPTs) whose resistance is associated with the high expression of breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). To reverse this resistance, two novel CPTs, FL77-28 (7-(3-Fluoro-4-methylphenyl)-10,11-methylenedioxy-20(S)-CPT) and FL77-29 (7-(4-Fluoro-3-methylphenyl)-10,11-methylenedioxy-20(S)-CPT), were synthesized by our group. In this study, the anti-tumor activities of FL77-28, FL77-29, and their parent, FL118 (10,11-methylenedioxy-20(S)-CPT), were evaluated and the results showed that FL77-28 and FL77-29 had stronger anti-tumor activities than FL118. The transport and uptake of FL118, FL77-28, and FL77-29 were investigated in Caco-2 cells for the preliminary prediction of intestinal absorption. The apparent permeability coefficient from apical to basolateral (Papp AP-BL) values of FL77-28 and FL77-29 were (2.32 ± 0.04) × 10-6 cm/s and (2.48 ± 0.18) × 10-6 cm/s, respectively, suggesting that the compounds had moderate absorption. Since the transport property of FL77-28 was passive diffusion and the efflux ratio (ER) was less than 2, two chemical inhibitors were added to further confirm the involvement of efflux proteins. The results showed that FL77-28 was not a substrate of P-gp or BCRP, but FL77-29 was mediated by P-gp. In conclusion, FL77-28 might be a promising candidate to overcome drug resistance induced by multiple efflux proteins.

Design, Synthesis, and In Vitro/In Vivo Anti-Cancer Activities of Novel (20S)-10,11-Methylenedioxy-Camptothecin Heterocyclic Derivatives.

A novel camptothecin analogue, (20S)-10,11-methylenedioxy-camptothecin (FL118), has been proven to show significant antitumor efficacy for a wide variety of solid tumors. However, the further development of FL118 is severely hindered due to its extremely poor water solubility and adverse side effects. Here, two series of novel 20-substituted (20S)-10,11-methylenedioxy-camptothecin coupled with 5-substituted uracils and other heterocyclic rings through glycine were synthesized. All the derivatives showed superior cytotoxic activities in vitro with IC50 values in the nanomolar range. Among them, 12e displayed higher cytotoxic activities in several cancer cell lines with better water solubility than FL118. Our results further showed that, like FL118, 12e inhibited cell proliferation resulting from cell cycle arrest and apoptosis by blocking the anti-apoptotic gene transcription of survivin, Mcl-1, Bcl-2, and XIAP in both A549 cells and NCI-H446 cells. Furthermore, 12e did not show any inhibitory activity on Topo I, which is involved in hematopoietic toxicity. In vivo, 12e showed similar antitumor efficacy to FL118 but lower toxicity. Our findings indicate that 12e is a promising therapeutic agent for cancer treatment, and the core structure of FL118 represents a promising platform to generate novel FL118-based antitumor drugs.

Synthesis and antitumor activity of biotinylated camptothecin derivatives as potent cytotoxic agents.

A series of biotinylated camptothecin derivatives were designed and synthesized. The key to the synthesis was achieved by employing an esterification reaction and click chemistry. All of the new derivatives were tested for cytotoxicity against five human tumor cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW480 with IC50 values ranging from 0.13 to 21.53 µM. Most of the derivatives exhibited potent cytotoxicity, especially compound 17 (IC50 = 0.13-3.31 µM) and compound 18 (IC50 = 0.23-1.48 µM), which exhibited the highest potencies. The structure-activity relationships (SARs) of the biotinylated camptothecin derivatives were discussed for exploring novel anticancer agents.

Synthesis and antitumor activity of a series of lactone-opened camptothecin derivatives.

A series of E-ring lactone-opened camptothecin (CPT) derivatives bearing with terminal aza-heterocyclic groups were synthesized, and their antitumor activity was evaluated both in vitro and in vivo. Hydroxyl-amide analogues with morpholin-4-yl displayed excellent antitumor activity in vitro and efficient inhibition on tumor xenograph model in nude mice. Ester-amide compounds acted less active in vitro cytotoxicity and lower inhibition activity in vivo. Substitutions at 7- and 10- positions favored the antitumor activity.

Synthesis, insecticidal activity and inhibition on topoisomerase I of 20(S)-t-Boc-amino acid derivatives of camptothecin.

Camptothecin (CPT), a quinolone alkaloid extracted from Camptotheca acuminata Decne, exhibits potential insecticidal activities against various insect species. Our previous studies have showed that CPT induced apoptosis in Spodoptera exigua Hübner cell line and inhibited the relaxation activity of topoisomerase I (Topo I). In this study, total seven 20(S)-t-butoxy carbonyl-amino acid derivatives of CPT were synthesized and evaluated for insecticidal activities, cytotoxicity and Topo I inhibitory activities. Results showed that introduction of t-Boc amino acids to 20-position on CPT improves contact assay and cytotoxicity of most derivatives toward S. exigua but reduces inhibitory effect on relaxation activity of S. exigua Topo I. Furthermore, compounds 1d and 1g demonstrated higher level of contact activities and cytotoxicity than CPT and hydroxyl-camptothecin (HCPT), which are potential to be developed as potential insecticides targeted at more than Topo I.

Low toxic and high soluble camptothecin derivative 2-47 effectively induces apoptosis of tumor cells in vitro.

The cytotoxic activity of camptothecin derivatives is so high that these compounds need to be further modified before their successful application as anti-cancer agents clinically. In this study, we reported the synthesis and biological evaluation of a novel camptothecin derivative called compound 2-47. The changes in structure did not reduce its activity to inhibit DNA topoisomerase I. Compound 2-47 induced apoptosis of many tumor cells including leukemia cells K562, Jurkat, HL-60, breast cancer cell BT-549, colon cancer cell HT-29 and liver cancer cell HepG2 with a half maximal inhibitory concentration (IC50) of 2- to 3-fold lower than HCPT as a control. In particular, 2-47 inhibited the proliferation of Jurkat cells with an IC50 of as low as 40 nM. By making use of Jurkat cell as a model, following treatment of Jurkat cells, compound 2-47 activated caspase-3 and PARP, resulting in a decreased Bcl-2/Bax ratio. These data showed that compound 2-47 induces Jurkat cell death through the mitochondrial apoptotic pathway. In addition, compound 2-47 showed a decreased cytotoxic activity against normal cells and an improved solubility in low-polar solvent. For example, compound 2-47 solutes in CHCl3 130-fold higher than HCPT. Taken together, our data demonstrated that camptothecin derivative 2-47 notably inhibits the tumor cell proliferation through mitochondrial-mediated apoptosis in vitro.

Assessment of the chemotherapeutic potential of a new camptothecin derivative, ZBH-1205.

CPT-11 (irinotecan) is a derivative of camptothecin which is a natural product derived from the Chinese tree Camptotheca acuminta and widely used in antitumor therapy. Here, the in vitro anti-tumor activity and associated mechanisms of a novel derivative of camptothecin, ZBH-1205, were investigated in a panel of 9 human tumor cell lines, as well as in HEK 293 and SK-OV-3/DPP, a multi-drug resistant (MDR) cell line, and compared to CPT-11 and 7-ethyl-10-hydroxy-camptothecin (SN38). Comparisons between the different compounds were made on the basis of IC50 values as determined by the MTT assay, and flow cytometry was used to evaluate cell cycle progression, apoptosis, and the levels of pro- and active caspase-3 among different treatment groups. Interaction between the molecules and topoisomerase-1 (Topo-1)-DNA complexes was detected by a DNA relaxation assay. Our results demonstrated that IC50 values for ZBH-1205 ranged from 0.0009 µmol/L to 2.5671 µmol/L, which were consistently lower than IC50 values of CPT-11 or SN38 in the panel of cell lines, including SK-OV-3/DPP. Furthermore, ZBH-1205 was more effective than CPT-11 or SN38 at stabilizing Topo-1-DNA complexes and inducing tumor cell apoptosis. Therefore, ZBH-1205 is a promising chemotherapeutic agent to be further assessed in large-scale clinical trials.

Drug combinations of camptothecin derivatives promote the antitumor properties.

Camptothecin (CPT) derivatives are widely used as small molecule chemotherapeutic agents and have demonstrated efficacy in the treatment of diverse solid tumors. A variety of derivatives have been developed to resolve the drawbacks of poor water solubility, high toxicity and rapid hydrolysis in vivo. However, the obstacles, such as acquired resistance and toxicity, still exist. The utilization of rational drug combinations has the potential to enhance the efficacy and mitigate the toxicity of CPT derivatives. This paper provides an overview of CPT derivatives in combination with other drugs, with a particular focus on cell cycle inhibitors, DNA synthesis inhibitors, anti-metastatic drugs and immunotherapy agents. Concurrently, the mechanisms of antitumor activity of combinations of different classes of drugs and CPT derivatives are elucidated. While the various combination strategies have yielded more favorable therapeutic outcomes, the efficacy and toxicity of the drug combinations are influenced by the inherent properties of the drugs involved. Moreover, a summary of the drug conjugates of CPT derivatives was provided, accompanied by an analysis of the structural activity relationship (SAR). This paves the way for the subsequent developments in drug combinations and delivery modes.

Rational Identification of Novel Antibody-Drug Conjugate with High Bystander Killing Effect against Heterogeneous Tumors.

Bystander-killing payloads can significantly overcome the tumor heterogeneity issue and enhance the clinical potential of antibody-drug conjugates (ADC), but the rational design and identification of effective bystander warheads constrain the broader implementation of this strategy. Here, graph attention networks (GAT) are constructed for a rational bystander killing scoring model and ADC construction workflow for the first time. To generate efficient bystander-killing payloads, this model is utilized for score-directed exatecan derivatives design. Among them, Ed9, the most potent payload with satisfactory permeability and bioactivity, is further used to construct ADC. Through linker optimization and conjugation, novel ADCs are constructed that perform excellent anti-tumor efficacy and bystander-killing effect in vivo and in vitro. The optimal conjugate T-VEd9 exhibited therapeutic efficacy superior to DS-8201 against heterogeneous tumors. These results demonstrate that the effective scoring approach can pave the way for the discovery of novel ADC with promising bystander payloads to combat tumor heterogeneity.

Design, synthesis, and biological evaluation of novel 7-substituted 10,11-methylenedioxy-camptothecin derivatives against drug-resistant small-cell lung cancer in vitro and in vivo.

A series of novel 7-substituted 10,11-methylenedioxy-camptothecin (FL118) derivatives were designed, synthesized, and biologically evaluated. All the FL118 analogues showed significant cytotoxic activities in vitro with IC50 values in the nanomolar range and were more potent than topotecan. The most active compound 9c exhibited more significant anti-tumor activity against small-cell lung cancer (NCI-H446, H69, drug-resistant H69AR cells, drug-resistant NCI-H446/Irinotecan cells and drug-resistant NCI-H446/EP cells) in vitro. Additionally, 9c could also induce the expression of apoptosis proteins such as caspase-3, caspase-9, and PARP in small-cell lung cancer. Further studies showed that 9c induced apoptosis by inhibiting the expression of Mcl-1, Bcl-2, XIAP and survivin in small-cell lung cancer. In vivo9c also showed better anti-tumor efficacy, with the tumor growth inhibition rates were 40.4% (0.75 mg/kg), 73.7% (1.5 mg/kg), and 95.5% (3 mg/kg). It is noteworthy that 9c also demonstrated potent inhibition of drug-resistant tumor growth in NCI-H446/Irinotecan and NCI-H446/EP xenograft models, the tumor growth inhibition rates were 93.42% and 84.46%, respectively. Taken together, these findings indicated that compound 9c displays outstanding antitumor activity and drug-resistance in small-cell lung cancer both in vivo and in vitro, which could be worth further research as a novel anti-tumor drug against small-cell lung cancer.

Synthesis and Biological Evaluation of 10-Substituted Camptothecin Derivatives with Improved Water Solubility and Activity.

Despite remarkable clinical achievements, camptothecin (CPT) still suffers from poor solubility and severe toxicity. Therefore, it is necessary to redevelop CPT derivatives as supplementary antitumor agents with good water solubility and small side effects. In this work, 27 camptothecin derivatives were synthesized and screened for their cytotoxicity against A549 (lung) and HCT-116 (colon) cancer cell lines. Among them, compound B7, 7-ethyl-10-(2-oxo-2-(4-methylpiperidin-1-yl)ethoxy)camptothecin,was demonstrated in vitro to be a more potent antitumor agent than SN-38 by comparison of their inhibitory activities against cell proliferation and colony formation and interference effect on process of cell cycle and cell apoptosis. Additionally, a molecular docking model revealed that B7 can interact with the topoisomerase I-DNA complex, and that the solubility of B7 reached 5.73 µg/mL in water. Moreover, B7 significantly inhibited tumor growth in an A549 xenograft model at dosages of 0.4 and 2.0 mg/kg, and exhibited minimum lethal doses comparable to those of irinotecan. These results indicated that B7, with improved solubility, enhanced activity and acceptable acute toxicity, can be used as a lead compound for the development of novel anticancer agents.

In-silico Studies and Wet-Lab Validation of Camptothecin Derivatives for Anti-Cancer Activity Against Liver (HepG2) and Lung (A549) Cancer Cell Lines.

BACKGROUND: In the present study, we have explored the utility of QSAR modelling, in silico ADMET, docking, chemical semi-synthesis, and in vitro evaluation studies for the identification of active camptothecin (CPT) derivatives against cancer-targeting human liver (HepG2) and lung (A549) cancer cell lines. METHODS: Two QSAR models were developed as screenings tools using the multiple linear regression (MLR) method followed by ADMET and docking studies. The regression coefficient (r2) and cross-validation regression coefficients (rCV2T) of the QSAR model for the HepG2 cell line was 0.95 and 0.90, respectively, and for the A549 cell line, it was 0.93 and 0.81, respectively. RESULTS: In silico studies show that CPT derivatives (CPT-1 and CPT-6) possess drug-like properties. Docking performed on DNA Topoisomerase-I showed significant binding affinity. Finally, predicted active derivatives were chemically semi synthesized, spectroscopically characterized, and evaluated in-vitro for cytotoxic/anticancer activity against HepG2 and A549 cell lines. CONCLUSION: The experimental results are consistent with the predicted results. These findings may be of immense importance in the anticancer drug development from an inexpensive and widely available natural product, camptothecin.

F10, a new camptothecin derivative, was identified as a new orally-bioavailable, potent antitumor agent.

Topoisomerases are interesting targets for drug discovery. In the present study, we attached saturated carbon atoms to the 10-position of camptothecin and synthesized 10 new camptothecin derivatives from 10-HCPT or SN-38. The activities of new compounds were evaluated both in vitro and in vivo. The most promising compound F10, 7-ethyl-10-(2-oxo-2-(piperidin-1-yl)ethoxy)camptothecin, inhibited cancer cells growth with the IC50 of 0.002, 0.003, 0.011 and 0.081 µM on Raji, HCT116, A549 and Lovo cells, respectively. Meanwhile, oral administration of F10 remarkably suppressed the HCT116-xenograft tumor growth in the nude-mice model at the dosage of 0.5, 2.0 and 8.0 mg/kg in vivo. Intraperitoneal administration of F10 can completely inhibit Raji-xenograft tumor growth in established NPG mouse model at 2.0 and 4.0 mg/kg. In addition, the minimum lethal doses of F10 and SN-38 in mice by intravenous administration were 80 and 40 mg/kg (or 0.155, 0.102 mmol/kg), respectively. The solubility of F10 reached 9.86 µg/mL in a buffer solution of pH 4.5. The oral bioavailability of F10 achieved 22.4% in mice. The molecular docking model revealed that F10 can interact with topoisomerase I-DNA complex. Our findings indicate that F10 is a new orally-oavailable antitumor agent with potent anticancer effect. Furthermore, attaching a ring hydrophobic moiety to the 10-position of camptothecin provides a favorable approach in the optimization of camptothecin.

Two novel camptothecin derivatives inhibit colorectal cancer proliferation via induction of cell cycle arrest and apoptosis in vitro and in vivo.

At present, chemotherapy is still to be the preferred and most significant therapeutic strategy for cancer patients in clinical practice. Although Camptothecin (CPT) has been discovered for over half century, a series of CPT derivatives such as Topotecan (TPT) and irinotecan (CPT-11) have been approved and are still to be the first-line medicines for clinical application. Up to now, the topoisomerase 1 inhibitor continues to be a significant drug development research field. Based on previous study of the structure-activity relationship, we consider that the introduction of lipophilic group at C7 position can prolong the retention time and the hydroxyl esterification at C20 can eliminate the hydrogen bond interaction, stabilize the E-lactone form and promote the anti-cancer effect. In this study, we carried out an optimization at C7 and C20 positions to afford two CPT derivatives 3g and 3j. Firstly, we predicted the possibly binding sites of two compounds with topoisomerase 1 by molecular docking. Then we evaluated the anti-proliferation effect of the two novel derivatives and compared the IC50 with CPT-11. Furthermore, the induction of cell cycle arrest and apoptosis was explored through karyomorphology, flow cytometry (FCM) and Western blot analysis. At last, we evaluated the anti-cancer effect and detected the mechanism in colorectal cancer xenograft model. In brief, all the data showed that the novel CPT derivatives (3g and 3j) could inhibit colorectal cancer proliferation via induction of cell cycle arrest and apoptosis in vitro and in vivo. It suggested that the two agents may be a new potential therapeutic strategy in the future.