Spliceosome

FR901464 is a potent inhibitor of spliceosomes. It has shown remarkable anticancer activity against multiple human cancer cell lines. [2]
Researchers selected FR901464 as a candidate compound and investigated cell cycle transition, chromatin status and endogenous gene expression in FR901464-treated tumor cells having elevated transcriptional activity. FR901464 induced characteristic G1 and G2/M phase arrest in the cell cycle and internucleosomal degradation of genomic DNA with the same kinetics as activation of SV40 promoter-dependent cellular transcription in M-8 tumor cells. In contrast to the potent activation of the viral promoter, FR901464 suppressed the transcription of some inducible endogenous genes but not house keeping genes in M-8 cells. These results suggest that FR901464 may induce a dynamic change of chromatin structure, giving rise to strong antitumor activity, and therefore may represent a new type of drug for cancer chemotherapy.[1]

FR901463, FR901464 and FR901465, novel antitumor substances, were isolated from the fermentation broth of Pseudomonas sp. No. 2663. Their antitumor activities were examined in three mouse tumor systems and one human tumor system. The three FR compounds prolonged the life of mice bearing murine ascitic tumor P388 leukemia (T/C values were 160%, 145% and 127% for FR901463, FR901464 and FR901465, respectively), and inhibited the growth of a human solid tumor, A549 lung adenocarcinoma, with different effective dose ranges. FR901464 exhibited most prominent effects on these tumor systems among the three FR compounds. FR901464 also inhibited the growth of murine solid tumors, Colon 38 carcinoma and Meth A fibrosarcoma. [1]

In Vitro Splicing Reactions[2]
Pre-mRNA substrate was derived from the adenovirus major late transcript. A 32P-UTP body-labeled G(5′)ppp(5′)G-capped substrate was generated by T7 runoff transcription followed by gel purification. Nuclear extract was prepared from HeLa cells grown in DMEM/F12 1:1 and 5% (v/v) newborn calf serum. For splicing reactions, 10 nM pre-mRNA substrate was incubated with 60 mM potassium glutamate, 2 mM magnesium acetate, 2 mM ATP, 5 mM creatine phosphate, 0.05 mg mL–1 tRNA, and 50% (v/v) HeLa nuclear extract at 30 °C.

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Denaturing Gel Analysis[2]
RNA was extracted from in vitro splicing reaction and separated on a 15% (v/v) denaturing polyacrylamide gel. 32P-labeled RNA species were visualized by phosphorimaging and quantified with ImageQuant software. Splicing efficiency is the amount of mRNA relative to total RNA and normalized to a DMSO control reaction. IC50 values for inhibitors are the concentration of inhibitor that causes 50% decrease of splicing efficiency, which were derived from averaged plots of splicing efficiency vs compound concentration.

Native Gel Analysis[2]
Splicing reactions were set up as described above and incubated at 30 °C for 4–30 min. Time point samples were kept on ice until all samples were ready for analysis. Amounts of 10 μL of splicing reactions were mixed with 10 μL of native gel loading buffer (20 mM Trizma base, 20 mM glycine, 25% (v/v) glycerol, 0.1% (w/v) cyan blue, 0.1% (w/v) bromophenol blue, 1 mg mL–1 of heparin sulfate) and incubated at room temperature for 5 min before loading onto a 2.1% (w/v) low-melting temperature agarose gel. Gels were run at 72 V for 3.5 h, dried onto Whatman paper, and exposed to phosphorimaging screens, which were digitized with a Typhoon Scanner.

FR901463, FR901464 and FR901465, novel antitumor substances, were isolated from the fermentation broth of Pseudomonas sp. No. 2663. Their antitumor activities were examined in three mouse tumor systems and one human tumor system. The three FR compounds prolonged the life of mice bearing murine ascitic tumor P388 leukemia (T/C values were 160%, 145% and 127% for FR901463, FR901464 and FR901465, respectively), and inhibited the growth of a human solid tumor, A549 lung adenocarcinoma, with different effective dose ranges. FR901464 exhibited most prominent effects on these tumor systems among the three FR compounds. FR901464 also inhibited the growth of murine solid tumors, Colon 38 carcinoma and Meth A fibrosarcoma. To address the involvement of transcriptional activation ability of the three FR compounds in the antitumor effect, we selected FR901464 as a candidate compound and investigated cell cycle transition, chromatin status and endogenous gene expression in FR901464-treated tumor cells having elevated transcriptional activity. FR901464 induced characteristic G1 and G2/M phase arrest in the cell cycle and internucleosomal degradation of genomic DNA with the same kinetics as activation of SV40 promoter-dependent cellular transcription in M-8 tumor cells. In contrast to the potent activation of the viral promoter, FR901464 suppressed the transcription of some inducible endogenous genes but not house keeping genes in M-8 cells. These results suggest that FR901464 may induce a dynamic change of chromatin structure, giving rise to strong antitumor activity, and therefore may represent a new type of drug for cancer chemotherapy.[1]

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Drug Formulation:** For in vivo studies, FR901464 was dissolved and diluted in a 10% polyoxyethylated (60 mol) hydrogenated castor oil in saline (HCO60 solution). All drugs were administered to mice at a volume of 10 ml/kg body weight. [1]
* **Murine Ascitic P388 Leukemia Model:** P388 cells (1 × 10⁶) were inoculated intraperitoneally (ip) into BDF₁ mice on Day 0. FR901464 was administered ip once daily for 4 days (Days 1-4). Antitumor efficacy was assessed by the percentage of median survival time of the treated group (T) to that of the control group (C), calculated as T/C (%). [1]
* **Murine Solid Tumor Models (Colon 38 and Meth A):** For Colon 38, tumor fragments (2×2×2 mm) were implanted subcutaneously (sc) in BDF₁ mice on Day 0. FR901464 was administered intravenously (iv) once a day on Days 1, 4, 7, and 10. For Meth A, cells (1 × 10⁵) were inoculated intradermally (id) in BALB/c mice on Day 0. FR901464 was administered iv once a day on Days 8, 11, and 14. Tumor sizes were measured with calipers, and tumor weight was calculated. Efficacy was expressed as growth inhibition (1 - T/C) %. [1]
* **Human A549 Lung Adenocarcinoma Xenograft Model (Subrenal Capsule Assay):** A 1 mm³ tumor fragment was implanted under the kidney capsule of BDF₁ mice on Day 0. The immunosuppressive agent FK-506 (32 mg/kg) was injected subcutaneously on Days 1, 2, 5, 7, 9, and 12 to prevent xenograft rejection. FR901464 was administered ip on Days 1, 5, and 9 (every 4 days). On Day 14, mice were sacrificed, and the final tumor size was measured. Efficacy was calculated as growth inhibition (1 - T/C) %. [1]

[1]. New antitumor substances, FR901463, FR901464 and FR901465. II. Activities against experimental tumors in mice and mechanism of action. J Antibiot (Tokyo). 1996 Dec;49(12):1204-11.

[2]. Enantioselective total syntheses of FR901464 and spliceostatin A and evaluation of splicing activity of key derivatives. J Org Chem. 2014 Jun 20;79(12):5697-709.

FR901464 is a spirocyclic oxide with potent anticancer activity, capable of reducing the mRNA levels of oncogenes and tumor suppressor genes. It was isolated from Pseudomonas strain 2663. It can be used as an antibacterial agent, antitumor agent, and bacterial metabolite. It is an acetate, cyclic hemiketal, pyran compound, monocarboxylic acid amide, and spirocyclic oxide.
(2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-4,7-dihydroxy-7-methyl-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpentan-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate has been reported in Burkholderia thailandensis, and relevant data are available.

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FR901464 (1) and splice repressin A (2) are potent inhibitors of the spliceosome. These compounds have shown significant anticancer activity against a variety of human cancer cell lines. This paper describes the efficient enantioselective synthesis of FR901464, splice repressor A, and six corresponding diastereomers, and evaluates their splicing activities. The synthesis of splice repressor A and FR901464 followed the longest linear sequences, involving 9 and 10 steps, respectively. To construct the highly functionalized tetrahydropyran A ring, we employed CBS reduction, Achmatowicz rearrangement, Michael addition, and reductive amination as key steps. The Michael addition reaction exhibited significant diastereoselectivity, which we validated for different substrates and reaction conditions. Side chain B was prepared from an optically active alcohol, followed by acetylation and Lindlar-catalyzed hydrogenation. Another highly functionalized tetrahydropyran C ring was derived from readily available (R)-isopropylglyceraldehyde, and its synthetic route involved 1,2-addition, cyclic ketalization, and regioselective epoxidation. These fragments were later coupled together via amidation and cross-metathesis reactions in a convergent synthesis. Six key diastereomers were then synthesized to investigate the importance of specific stereochemical features of FR901464 and splicing repressor A to their in vitro splicing activity. [2]

C27H41NO8

507.624

507.283

C, 63.89; H, 8.14; N, 2.76; O, 25.21

146478-72-0

10553647

White to off-white solid powder

1.21g/cm3

702.7ºC at 760mmHg

378.8ºC

7.94E-23mmHg at 25°C

1.553

3.152

3

8

9

36

900

9

O1C([H])([H])C21C([H])([H])[C@@](C([H])([H])[H])(O[H])O[C@]([H])(/C(/[H])=C(\[H])/C(/C([H])([H])[H])=C(\[H])/C([H])([H])[C@@]1([H])[C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([C@@]([H])(C([H])([H])[H])O1)N([H])C(/C(/[H])=C(\[H])/[C@]([H])(C([H])([H])[H])OC(C([H])([H])[H])=O)=O)[C@@]2([H])O[H]

PJKVJJDQXZARCA-QHYZBLTGSA-N

InChI=1S/C27H41NO8/c1-16(8-11-23-25(31)27(15-33-27)14-26(6,32)36-23)7-10-22-17(2)13-21(19(4)35-22)28-24(30)12-9-18(3)34-20(5)29/h7-9,11-12,17-19,21-23,25,31-32H,10,13-15H2,1-6H3,(H,28,30)/b11-8+,12-9-,16-7+/t17-,18-,19+,21+,22-,23+,25+,26-,27+/m0/s1

[(Z,2S)-5-[[(2R,3R,5S,6S)-6-[(2E,4E)-5-[(3R,4R,5R,7S)-4,7-dihydroxy-7-methyl-1,6-dioxaspiro[2.5]octan-5-yl]-3-methylpenta-2,4-dienyl]-2,5-dimethyloxan-3-yl]amino]-5-oxopent-3-en-2-yl] acetate

FR 901464; FR901464; FR901464; 146478-72-0; (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-4,7-dihydroxy-7-methyl-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate; CHEBI:65915; [(Z,2S)-5-[[(2R,3R,5S,6S)-6-[(2E,4E)-5-[(3R,4R,5R,7S)-4,7-Dihydroxy-7-methyl-1,6-dioxaspiro[2.5]octan-5-yl]-3-methylpenta-2,4-dienyl]-2,5-dimethyloxan-3-yl]amino]-5-oxopent-3-en-2-yl] acetate; FR-901464; [(E,2S)-4-[[(2R,3R,5S,6S)-6-[(2E,4E)-5-[(3R,4R,5R,7S)-4,7-dihydroxy-7-methyl-1,6-dioxaspiro[2.5]octan-5-yl]-3-methylpenta-2,4-dienyl]-2,5-dimethyloxan-3-yl]carbamoyl]but-3-en-2-yl] acetate; CHEMBL494107; FR-901464

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~197.00 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.92 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.92 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)