DNA methyltransferase 1 (DNMT1). Treatment with RX-3117 leads to a decrease in DNMT1 expression [1].
The drug is incorporated into DNA and RNA, inhibiting their synthesis [1].

A549 and SW1573 cells demonstrate anti-proliferative activity in response to RX-3117 (11.7, 21 μM; 48 hours) [1]. Urinary cytochrome pump 2 (RX-3117; 1–25 μM; 72 h) inhibits the growth of RX-3117 in HCT-116, MDA-MB-231, PANC-1, Caki-1, MCF7, A549, MKN45, and U251 cells. The corresponding values are 0.39, 0.18, 0.62 and 0.84, 0.34, 0.34, 0.50, 0.83 μM, respectively [2]. In S phase and intercellular space, RX-3117 (5, 10 μM; 4 days) causes cell cycle inference [2]. In MDA-MB-231, RX-3117 (1–5 μM; 24 hours) decreases DNMT1 cell mass in a dose-dependent manner [3].

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In A549 and SW1573 non-small cell lung cancer (NSCLC) cell lines, the cytotoxicity of RX-3117 was significantly reduced when cells were transfected with siRNA targeting UCK2, but not UCK1. In A549 cells, 11.7 μM RX-3117 resulted in 63.7% cell growth in control cells, while siUCK2-transfected cells showed 123.6% growth (complete protection, p=0.004). In SW1573 cells, 21 μM RX-3117 resulted in 59% cell growth in controls, while siUCK2-transfected cells showed 83.8% growth (partial protection, p=0.003). 3'-ethynylcytidine (ECyd) was used as a positive control, showing a similar protective effect with UCK2 knockdown [1].
The formation of RX-3117 monophosphate (RX-3117MP), the initial activated metabolite, was measured in a panel of cell lines (U937, A549, CCRF-CEM, SW1573, AG6000, A2780) after exposure to 10 μM [6-³H]-RX-3117 for 120 minutes. The accumulation of RX-3117MP at 60 and 120 minutes showed a strong positive correlation with UCK2 mRNA expression (correlation coefficients of 0.8768 and 0.8891, respectively), but no correlation with UCK1 expression [1].
Exposure of A549 and SW1573 cells to 33.3 μM RX-3117 for 24 hours led to an accumulation of cells in the S-phase of the cell cycle. In A549 cells, the S-phase fraction increased from 20.92% to 38.06%, and in SW1573 cells from 20.91% to 24.87%. This S-phase arrest was completely abolished when the cells were co-incubated with 100 μM uridine or 100 μM cytidine, which are natural substrates of UCK2 [1].
The proliferation of A549 and SW1573 cells was inhibited by treatment with 33.3 μM RX-3117 for 24 hours. This anti-proliferative effect was completely reversed by the addition of 100 μM uridine or 100 μM cytidine [1].
Western blot analysis showed high UCK2 protein expression in the RX-3117-sensitive cell lines A2780 and U937, while UCK1 protein expression was low across the tested cell lines [1].

In mice that were left naked, RX-3117 (2, 10 mg/kg; intraperitoneal injection; three times a week for five weeks) demonstrated anti-tumor activity [3].

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In initial studies, RX-3117 showed potent activity in nine xenograft models, including gemcitabine-resistant xenografts. Overall, it was effective against 17 human colon, renal, and pancreatic cancer xenograft models, with efficacy comparable to or better than other analogs or drugs like paclitaxel [2].
In colorectal and pancreatic xenograft models, the combination of RX-3117 with nab-paclitaxel (Abraxane) resulted in superior tumor growth inhibition compared to RX-3117 alone, even in a model derived from a patient resistant to nab-paclitaxel monotherapy [2].
In the same colorectal and pancreatic xenograft models, the combination of RX-3117 with an anti-PD1 immunotherapy also showed promising activity, leading to higher survival rates compared to anti-PD1 treatment alone [2].

UCK Enzyme Activity Correlation: To determine the correlation between UCK enzyme activity and UCK1/UCK2 expression, UCK activity was measured in lysates from six tumor cell lines and seven xenograft-derived cells. The activity was determined using radioactively labeled substrates: [³H]-RX-3117 and [2-¹⁴C]-uridine. The measured UCK enzyme activity showed a significant (p<0.001) positive linear correlation with UCK2 mRNA expression in both the cell line panel (r = 0.803 for [³H]-RX-3117; r = 0.828 for [2-¹⁴C]-uridine) and the xenograft cell panel (r = 0.915 for [³H]-RX-3117; r = 0.878 for [2-¹⁴C]-uridine). No such correlation was found with UCK1 mRNA expression, which showed negative or no correlation [1].

Cell Viability Assay[1]
Cell Types: A549, SW1573 cells
Tested Concentrations: 11.7 . , 21 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: Displayed anti-proliferative activity in A549 (63.7% cell growth), SW1573 cells (59% cell growth).

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Apoptosis analysis [2]
Cell Types: A549, SW1573 NSCLC cell
Tested Concentrations: 5 µM for A549 cells, 10 µM for SW1573 cells
Incubation Duration: 4 days
Experimental Results: Induced cell cycle arrest in S phase and apoptosis.

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siRNA Transfection and Chemosensitivity: A549 and SW1573 cells were seeded in 96-well plates. After 24 hours, they were transfected with 25 nM of siRNA targeting UCK1, UCK2, or a non-targeting control using a transfection reagent. Twenty-four hours post-transfection, cells were exposed to RX-3117 (11.7 μM for A549, 21 μM for SW1573) or the control drug ECyd for 48 hours. Cell growth was then assessed using the sulforhodamine B (SRB) assay. Cells were fixed with cold trichloroacetic acid, stained with SRB, and optical density was measured at 540 nm [1].
RNA Isolation and qRT-PCR: To confirm gene knockdown, cells were seeded in 6-well plates and transfected as above. Seventy-two hours post-transfection, RNA was isolated using TRIzol reagent. cDNA was synthesized using a cDNA synthesis kit with random hexamer primers. Quantitative RT-PCR was performed using a Lightcycler 2.0 with Taqman primers specific for UCK1, UCK2, and β-actin (as a housekeeping gene) to quantify mRNA expression levels [1].
Western Blotting: To confirm protein knockdown, transfected cells were lysed in a lysis buffer containing a protease inhibitor cocktail. Protein concentration was determined using a standard assay. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with primary antibodies against UCK2 and β-actin. Secondary antibodies conjugated with infrared dyes were used for detection with an infrared imager [1].
Immunocytochemistry: Cells were fixed on microscope slides with 4% paraformaldehyde. Endogenous peroxidase was blocked with H₂O₂, and non-specific binding was blocked with BSA and Triton X-100. Cells were incubated with a primary polyclonal rabbit antibody against UCK2, followed by a biotinylated anti-rabbit antibody and HRP-streptavidin. Staining was visualized with 3,3'-diaminobenzidine (DAB) and H₂O₂ [1].
Immunohistochemistry: Frozen tissue sections from human placenta, normal liver, and liver metastases were fixed. The same staining procedure as for immunocytochemistry was then followed, using the UCK2 primary antibody and DAB for visualization [1].
RX-3117 Nucleotide Accumulation: Intact cells from a panel of lines were exposed to 10 μM [6-³H]-RX-3117 for 120 minutes. The reaction was stopped by acid precipitation, and RX-3117 and its phosphorylated metabolites (mono-, di-, and triphosphate) were separated and analyzed using thin-layer chromatography [1].
Cell Cycle Analysis: A549 and SW1573 cells were seeded in 12-well plates. After 24 hours, they were treated with 33.3 μM RX-3117, alone or in combination with 100 μM uridine or cytidine. After 24 hours of treatment, both adherent and floating cells were collected, centrifuged, and resuspended in a propidium iodide (PI) solution containing sodium citrate, Triton X-100, and ribonuclease A. After incubation on ice, samples were analyzed by flow cytometry to determine cell cycle distribution [1].
Proliferation Analysis: A549 and SW1573 cells were seeded and treated as described for the cell cycle analysis. Cells were counted at the start of treatment and after 24 hours using a Coulter counter. The doubling time was calculated based on these counts [1].

Animal/Disease Models: Nude mice (human colon cancer HCT116 xenograft model) [3]
Doses: 2, 10 mg/kg
Route of Administration: IP; 3 times a week for five weeks
Experimental Results: Doses of 2 and 10 mg/kg It has a significant inhibitory effect on tumor growth.

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Xenograft Efficacy Studies:** The antitumor efficacy of RX-3117 was evaluated in various human tumor xenograft models, including colon, renal, pancreatic, and gemcitabine-resistant tumors. The specific details of the protocol, such as drug formulation, route of administration (likely oral, based on its properties), dosing schedule, and duration, are referenced from previous work (Yang et al., 2014) but not detailed in this review [2].
* **Combination Xenograft Studies:** In colorectal and pancreatic xenograft models, the efficacy of RX-3117 in combination with other agents was tested. Mice bearing xenografts were treated with RX-3117 alone, nab-paclitaxel (Abraxane) alone, or the combination. In a separate study, the combination of RX-3117 with an anti-PD1 antibody was also tested, and its effect on tumor growth and survival was monitored. Specific protocol details are not provided in this review [2].

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Xenograft Efficacy Studies: The antitumor efficacy of RX-3117 was evaluated in various human tumor xenograft models, including colon, renal, pancreatic, and gemcitabine-resistant tumors. The specific details of the protocol, such as drug formulation, route of administration (likely oral, based on its properties), dosing schedule, and duration, are referenced from previous work (Yang et al., 2014) but not detailed in this review [2].
Combination Xenograft Studies: In colorectal and pancreatic xenograft models, the efficacy of RX-3117 in combination with other agents was tested. Mice bearing xenografts were treated with RX-3117 alone, nab-paclitaxel (Abraxane) alone, or the combination. In a separate study, the combination of RX-3117 with an anti-PD1 antibody was also tested, and its effect on tumor growth and survival was monitored. Specific protocol details are not provided in this review [2].

RX-3117 is taken up into cells via human equilibrative nucleoside transporter 1 (hENT1) [1].
RX-3117 is sequentially phosphorylated to its monophosphate, diphosphate, and triphosphate forms to exert its cytotoxic activity [1].
The first, rate-limiting phosphorylation step is catalyzed specifically by uridine-cytidine kinase 2 (UCK2), not UCK1 [1].
Unlike gemcitabine, RX-3117 is not a substrate for deamination by cytidine deaminase (CDA), making it resistant to this common inactivation pathway [1].

The literature suggests that because UCK2 expression is low in normal tissues (with the exception of placenta), RX-3117 is unlikely to be activated in normal tissues, which could potentially reduce the occurrence of toxicity at therapeutic doses [1].
Immunohistochemistry staining showed that UCK2 is highly expressed in human placenta and in liver metastases derived from colon cancer, but is not detectable in normal liver tissue from the same patients [1].

[1]. The Cytidine Analog Fluorocyclopentenylcytosine (RX-3117) Is Activated by Uridine-Cytidine Kinase 2. PLoS One. 2016 Sep 9;11(9):e0162901.

[2]. RX-3117 (fluorocyclopentenyl cytosine): a novel specific antimetabolite for selective cancer treatment. Expert Opin Investig Drugs. 2019 Apr;28(4):311-322.

[3]. DNA methyltransferase inhibitors in cancer: a chemical and therapeutic patent overview and selected clinical studies. Expert Opin Ther Pat. 2012 Dec;22(12):1427-42.

Roducitabine is a triol with the structure (1S,2R)-4-fluoro-3-(hydroxymethyl)cyclopentan-3-en-1,2-diol, substituted at the 5-position with a 4-amino-2-oxopyrimidin-1(2H)- group. It is a cytosine analog with anticancer activity against various cancers, including gemcitabine-resistant tumors. It can function as an antimetabolite, antitumor drug, prodrug, DNA synthesis inhibitor, and apoptosis inducer. It is an organofluorine compound, a primary allyl alcohol, and a triol. Fluorocyclopentenylcytosine is being investigated in the clinical trial NCT03189914 (RX-3117 in combination with Abraxane® for the treatment of metastatic pancreatic cancer patients). Roducitabine is an orally administered small molecule nucleoside antimetabolite with potential antitumor activity. Following administration, roducitabine is taken up by cells via a carrier-mediated transporter, phosphorylated by uridine cytidine kinase (UCK), and further phosphorylated to diphosphate (RX-DP) and triphosphate (RX-TP). The triphosphate form is incorporated into RNA, inhibiting RNA synthesis. RX-DP diphosphate is reduced to dRX-DP by ribonucleotide reductase (RR); its triphosphate form (dRX-TP) is incorporated into DNA. Furthermore, roducitabine also inhibits DNA methyltransferase 1 (DNMT1). This ultimately leads to cell cycle arrest and apoptosis. UCK is the rate-limiting enzyme in the pyrimidine nucleotide rescue pathway.

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RX-3117 (fluorocyclopentenylcytosine) is an orally available cytidine analog that was in Phase I clinical trial at the time of this publication [1].
It has shown promising antitumor activity in various human tumor xenograft models, including tumors resistant to gemcitabine, suggesting a lack of cross-resistance [1].
The lack of cross-resistance with gemcitabine is explained by their different activation pathways: gemcitabine is activated by deoxycytidine kinase (DCK), while RX-3117 is activated by UCK2 [1].
RX-3117 inhibits both DNA and RNA synthesis, with a more pronounced effect on DNA synthesis [1]. It also targets DNA methyltransferase 1 (DNMT1), leading to its downregulation, which suggests it may act as a demethylating agent [1].
The study concludes that UCK2 expression may be used as a predictive biomarker to select patients who are potentially sensitive to RX-3117 treatment, as UCK2 expression correlates with both drug activation and cytotoxic effect [1].

C10H12FN3O4

257.22

257.081

C, 46.69; H, 4.70; F, 7.39; N, 16.34; O, 24.88

865838-26-2

865838-26-2

11242315

White solid powder

1.8±0.1 g/cm3

516.3±60.0 °C at 760 mmHg

266.1±32.9 °C

0.0±3.0 mmHg at 25°C

1.721

-0.72

4

5

2

18

474

3

FC1=C(CO)[C@@H](O)[C@@H](O)[C@@H]1N1C=CC(N)=NC1=O

QLLGKCJUPWYJON-HLTSFMKQSA-N

InChI=1S/C10H12FN3O4/c11-6-4(3-15)8(16)9(17)7(6)14-2-1-5(12)13-10(14)18/h1-2,7-9,15-17H,3H2,(H2,12,13,18)/t7-,8-,9+/m1/s1

4-amino-1-[(1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl]pyrimidin-2-one

RX3117; TV1360; RX3117; TV1360; RX 3117; TV 1360; fluorocyclopentenylcytosine

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: ≥ 50 mg/mL (~194.4 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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