DNA (incorporation induces strand breaks)

The mechanism of action of CNDAC is distinct; upon entering DNA, it causes single-strand breaks (SSBs), which are subsequently transformed into double-strand breaks (DSBs) during the second S phase of cell division [1]. CNDAC (0-100 μM; 3 days) prevents HL-60 and THP-1 cells from swelling [2]. A number of cells, including Rad51D and XRCC3, are susceptible to CNDAC (0–1 μM; 24 h) [1]. After a delayed S phase, CNDAC (6 μM; 48 h) causes cell cycle arrest in HCT116 cells during the G2 phase [3]. -10 μM; 3–6 days) to cause HL-60 and THP-1 cells to become disinfected [2].

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In clonogenic survival assays using Chinese hamster ovary (CHO) cell lines, CNDAC demonstrated potent cytotoxic activity. The IC₅₀ value for CNDAC in wild-type AA8 cells was 0.48 µM. Cells deficient in the homologous recombination (HR) repair pathway were significantly more sensitive to CNDAC. Specifically, Rad51D-deficient cells (51D1) showed an IC₅₀ of 0.006 µM, representing approximately 50-fold sensitization compared to Rad51D-complemented cells (51D1.3, IC₅₀ = 0.32 µM). XRCC3-deficient cells (irs1SF) exhibited even greater sensitivity, with an IC₅₀ of 0.0053 µM, which is about 89-fold more sensitive than wild-type AA8 cells (IC₅₀ = 0.48 µM). This stark contrast highlights that the cytotoxicity of CNDAC is highly dependent on functional HR for repair. In contrast, cells deficient in Rad51D or XRCC3 were not sensitized to cytarabine or gemcitabine (sensitization factor ~1). [1]
The anti-proliferative and cytotoxic effects of CNDAC were evaluated against acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary AML cells from bone marrow (BM) and peripheral blood (PB).
In the Alamar Blue assay, the IC₅₀ values for CNDAC against HL-60 and THP-1 cells were determined (Figure 1 and Table 2). For HL-60 cells plated at high density (0.5 × 10⁶ cells/mL), the IC₅₀ was 5.356 µM on day 3 and decreased to <0.5 µM by day 6. For HL-60 cells plated at low density (0.05 × 10⁶ cells/mL), the IC₅₀ was consistently <0.5 µM from day 3 to day 6. For THP-1 cells (which are less sensitive to Ara-C), the IC₅₀ for CNDAC was 0.929 µM (low density, day 3) and >10 µM (high density, day 3), decreasing to 1.104 µM and 2.095 µM respectively by day 6. [2]
Using trypan blue exclusion assays, CNDAC induced significant cell death in both AML cell lines at concentrations ranging from 0.5 µM to 10 µM. In HL-60 cells (Ara-C sensitive), CNDAC was more effective than Ara-C at equivalent concentrations, especially at lower cell seeding densities. In THP-1 cells (Ara-C resistant), CNDAC showed significantly higher cell death than Ara-C at doses >2 µM. CNDAC demonstrated a delayed effect in THP-1 cells, with more robust cytotoxicity observed on days 4-6 compared to day 3. [2]
Flow cytometry analysis (7-AAD/Annexin V staining) confirmed the induction of apoptosis by CNDAC in both cell lines. The distribution of early and late apoptotic events differed between HL-60 and THP-1 cells. [2]
In primary AML mononuclear cells (MNCs) from 5 patients, CNDAC demonstrated potent cytotoxic activity. Cells were treated for 4 days, and survival was assessed up to 35 days post-treatment. Treatment with 10 µM (medium dose) CNDAC resulted in a significant and sustained reduction in cell survival for both PB and BM MNCs compared to untreated controls on days 4, 7, and 14. Even at a low dose (1 µM), CNDAC treatment led to significantly lower overall survival of PB and BM cells over the 35-day culture period compared to untreated cells and to cells treated with 1 µM Ara-C. Importantly, residual cells after CNDAC treatment did not expand in culture post-drug washout, unlike cells treated with Ara-C. [2]
CNDAC showed potent growth inhibitory activity against murine leukemia L1210 cells with an IC₅₀ of 0.53 μM and against human oral epidermoid carcinoma KB cells with an IC₅₀ of 5.2 μM.
It exhibited a broad spectrum of cytotoxicity against 15 human solid tumor cell lines, including lung adenocarcinoma (PC-8, PC-9), stomach adenocarcinoma (ST-KM, MKN-45), colon adenocarcinoma (Colo-320), breast adenocarcinoma (MCF-7), osteosarcoma (OST, MNNG/HOS), fibrosarcoma (HT-1080), and melanoma (A-375), with IC₅₀ values ranging from 0.53 μM to >360 μM.
CNDAC was more cytotoxic than ara-C against several cell lines refractory to ara-C.
N⁴-Acetyl-CNDAC (6b) was less potent than CNDAC, and other pyrimidine analogues (CNDAT, CNDAU) were inactive up to 100 μg/mL.
The elimination product 10 (2′-C-cyano-2′,3′-didehydro-2′,3′-dideoxycytidine) was much less effective than CNDAC, indicating that the 3′-hydroxyl group is important for activity.
CNDAA (adenine analogue) showed cytotoxicity against L1210 cells but was inactive against human solid tumor cell lines.[4]

In mice with active tumors, CNDAC (20 mg/kg; ip; daily for 10 days) demonstrated resistance [4].

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CNDAC showed strong antitumor activity against M5076 mouse reticulum cell sarcoma when administered orally at 400 mg/kg/day on days 1, 4, 7, 10, 13, and 16, achieving 99% tumor volume inhibition on day 20 (T/C = 133%).
At 200 mg/kg/day under the same schedule, it showed 93% tumor volume inhibition (T/C = 136%).
Long-term survivors were observed in both dose groups.
CNDAC was also highly effective against P388 mouse leukemia with T/C >600% and survival over 60 days in 5 out of 6 mice at 20 mg/kg/day intraperitoneally on days 1–10.
It was effective against HT1080 human fibrosarcoma implanted in chick embryos or athymic mice, which is refractory to ara-C.[4]

Cell viability assay [1]
Cell Types: Rad51D-deficient 51D1, Rad51D-complemented 51D1.3, wild-type AA8 and XRCC3-deficient irs1SF CHO cell
Tested Concentrations: 0-1 μM
Incubation Duration: 24 h
Experimental Results: Inhibition of cell survival. The IC50 against Rad51D-deficient 51D1, Rad51D-complemented 51D1.3, wild-type AA8, and XRCC3-deficient irs1SF cell lines were 0.006, 0.32, 0.48, and 0.0053 μM, respectively.

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Cell proliferation assay [2]
Cell Types: HL-60 and THP-1 Cell
Tested Concentrations: 0-100 μM
Incubation Duration: 3 days
Experimental Results: Inhibitory effect on the proliferation of HL-60 and THP-1 cells, IC50 is 1.5832 μM and 0.84 μM, respectively.

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Apoptosis analysis[2]
Cell Types: HL-60 and THP-1 Cell
Tested Concentrations: 0, 0.5, 1, 2, 3, 4, 5 and 10 μM
Incubation Duration: 3, 4, 5 and 6 days
Experimental Results: Induction Apoptosis in both cells.

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Cell cycle analysis [3]
Cell Types: HCT116
Tested Concentrations: 6 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: 36% and 36% of cells were arrested in late S and G2/M phases respectively.

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Clonogenic survival assays were performed to evaluate the cytotoxicity of CNDAC and its dependence on DNA repair pathways. Exponentially growing CHO cells were seeded in 6-well plates and allowed to attach overnight. The following day, cells were exposed to a range of concentrations of CNDAC for 24 hours. After the treatment period, the drug-containing medium was removed, cells were washed, and fresh drug-free medium was added. Cells were then incubated for an additional 4 to 6 days to allow colony formation. Subsequently, colonies were fixed and stained with a crystal violet solution in ethanol. The stained plates were scanned, and colonies were counted electronically using a colony counting system. Survival curves were plotted, and IC₅₀ values were calculated from these curves. [1]
Alamar Blue Assay for IC₅₀ Determination: HL-60 and THP-1 cells were plated in 96-well flat-bottom plates at 5 × 10³ cells/well and incubated for 24 hours. Cells were then treated with a range of concentrations of CNDAC (0.005 to 100 µM) in triplicate for 72 hours. After treatment, Alamar Blue reagent was added to each well at a final concentration of 10%. Plates were returned to the incubator for 8 hours. Absorbance was measured at 570 nm and 600 nm using a plate reader. The percentage reduction of Alamar Blue was calculated, and from this, the percentage inhibition of cell proliferation was determined. Non-linear regression standard curves were generated to calculate IC₅₀ values. [2]
Cell Viability and Proliferation Assay (Trypan Blue Exclusion): Cell lines (HL-60, THP-1) were seeded in 48-well plates at two densities (0.05 × 10⁶ cells/mL and 0.5 × 10⁶ cells/mL). Cells were treated with CNDAC at concentrations ranging from 0.5 µM to 10 µM for up to 6 days. At designated time points (3, 4, 5, 6 days), cells were collected, mixed with trypan blue dye, and counted using a hemocytometer. The percentage of dead cells was calculated from raw counts. [2]
Primary AML PB MNCs were treated with CNDAC (1, 10, 100 µM) in suspension for 4 days. Primary AML BM MNCs were treated with the same concentrations of CNDAC but in a co-culture system with irradiated M2-10B4 mouse stromal cells for 4 days. After treatment, cells were washed, re-plated onto fresh stromal layers, and cultured for an additional 31 days (total 35-day observation). Cell survival was assessed by trypan blue exclusion at days 4 (end of treatment), 7, 14, and 35. Survival percentage was calculated relative to the initial number of cells plated. [2]
Apoptosis Assay (Flow Cytometry): Cell lines treated with CNDAC were collected, washed with cold PBS, and resuspended in binding buffer. Cells were then stained with Annexin V and 7-AAD according to the manufacturer's instructions, incubated in the dark for 15 minutes at room temperature, washed, and analyzed by flow cytometry within 1 hour. Cells were categorized as live (Annexin V⁻ 7-AAD⁻), early apoptotic (Annexin V⁺ 7-AAD⁻), and late apoptotic/necrotic (Annexin V⁺ 7-AAD⁺). [2]
Tumor cell growth inhibitory activity was assessed using a 72-hour incubation assay. Cells (2 × 10³ per well) were incubated with test compounds, followed by addition of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). After 4 hours, formazan was dissolved in DMSO and absorbance at 540 nm was measured. Percent inhibition was calculated as [1 − (OD of sample well / OD of control well)] × 100. IC₅₀ was defined as the concentration causing 50% inhibition of cell growth.[4]
Cytotoxicity against various human tumor cell lines was tested similarly.[4]

Animal/Disease Models: CDF1 mouse, P388 tumor model [4]
Doses: 20 mg/kg
Route of Administration: intraperitoneal (ip) injection, daily for 10 days
Experimental Results: Greatly improved survival time and survival rate.

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M5076 reticulum cell sarcoma cells (10⁶) were implanted subcutaneously into the axillary region of female BD2F₁ mice. CNDAC was administered orally on days 1, 4, 7, 10, 13, and 16 at doses of 50, 100, 200, 300, and 400 mg/kg/day. Tumor volume was measured as 0.5 × length × width². Tumor growth inhibition and T/C (%) were calculated on day 20. Body weight changes were also recorded.[4]
For P388 leukemia, CNDAC was administered intraperitoneally once daily on days 1–10 at 20 mg/kg/day.[4]

The provided literature [2] does not describe the ADME (absorption, distribution, metabolism, excretion) or pharmacokinetic properties (e.g., half-life, bioavailability) of CNDAC itself. The literature mentions that human pharmacokinetic studies have detected plasma concentrations of up to 0.25 µM after administration of its prodrug, sapaccitabine. [2]

[1]. Sapacitabine, the prodrug of CNDAC, is a nucleoside analog with a unique action mechanism of inducing DNA strand breaks. Chin J Cancer. 2012 Aug;31(8):373-80.

[2]. Bone Marrow and Peripheral Blood AML Cells Are Highly Sensitive to CNDAC, the Active Form of Sapacitabine. Adv Hematol. 2012;2012:727683.

[3]. Antiproliferative effects of sapacitabine (CYC682), a novel 2'-deoxycytidine-derivative, in human cancer cells. Br J Cancer. 2007 Sep 3;97(5):628-36.

[4]. Nucleosides and nucleotides. 122. 2'-C-cyano-2'-deoxy-1-beta-D-arabinofuranosylcytosine and its derivatives. A new class of nucleoside with a broad antitumor spectrum. J Med Chem. 1993 Dec 24;36(26):4183-9.

Lagocitabine hydrochloride is the hydrochloride form of lagocitalbine, which is an analog of the nucleoside deoxycytidine and has potential antitumor activity. After administration, lagocitalbine can be incorporated into DNA and directly inhibit the activity of DNA polymerase, which may lead to DNA replication inhibition and cell cycle arrest in the S and G2/M phases, DNA fragmentation, and tumor cell apoptosis. CNDAC (2'-C-cyano-2'-deoxy-1-β-D-arabinopentaurose cytosine) is the active metabolite of sapaccitabine, a prodrug with high oral bioavailability. Both are undergoing clinical studies in hematologic malignancies and solid tumors. [1] CNDAC has a unique mechanism of action among deoxycytidine analogs. After incorporation into DNA during DNA replication, the cyano sugar moiety induces DNA structural instability. This leads to β-elimination rearrangement, resulting in single-strand breaks (SSBs) and the formation of chain termination residues (CNddCs) at the 3' end. These SSBs can be repaired via the transcription-coupled nucleotide excision repair (TC-NER) pathway. If not repaired, the SSBs will transform into single-end double-strand breaks (DSBs) when the cell enters the second S phase. These lethal DSBs are primarily repaired via the homologous recombination (HR) pathway. Defects in key HR components (e.g., Rad51D, XRCC3) significantly enhance the cell’s sensitivity to CNDAC. [1] This unique mechanism and repair dependence distinguishes CNDAC from other deoxycytidine analogues (e.g., cytarabine and gemcitabine), which do not rely on HR for repair. This suggests that CNDAC/sapacitabine may have different clinical applications and may be effective against HR-deficient tumors or overcome resistance to other nucleoside analogues. [1] Clinical trials of sapacitabine (a prodrug) are underway for various cancers, including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic lymphocytic leukemia (CLL), and non-small cell lung cancer (NSCLC). [1] CNDAC is the active metabolite of the oral prodrug sapacitabine. It is a deoxycytidine analogue, structurally related to cytarabine (Ara-C) and gemcitabine, except that the 2' hydrogen of the glycosyl moiety is replaced by a cyano group. [2] CNDAC has a unique mechanism of action. The incorporation of the cyano group into DNA leads to structural instability, which in turn triggers β-elimination rearrangement. This results in single-strand breaks (SSBs) and a chain-terminating residue lacking a 3'-OH group (CNddC). These SSBs have poor repair capabilities (slow nucleotide excision repair) and are converted into double-strand breaks (DSBs) during subsequent DNA replication, ultimately leading to cell death. [2]
CNDAC is reported to be a weak substrate of cytidine deaminase (CDA), an enzyme that inactivates cytarabine (Ara-C), which may explain its activity in Ara-C resistant models such as the THP-1 cell line. [2]
This study showed thatCNDAC (administered in the form of sapaccitabine) had higher activity than Ara-C in vitro against acute myeloid leukemia (AML) cell lines and primary patient cells (including cells from the matrix-protected bone marrow microenvironment). It maintained its long-lasting efficacy after drug washout and remained active at low doses, highlighting its potential clinical value, especially for elderly patients with acute myeloid leukemia (AML) or those resistant to conventional cytarabine (Ara-C) therapy. [2]
Sapaccitabine (prodrug) is currently undergoing clinical trials in elderly newly diagnosed AML patients (a phase III clinical trial has been mentioned). [2]
CNDAC is a cytosine nucleoside analog designed with an electron-withdrawing cyano group at the 2′-β position. It is speculated that CNDAC can be phosphorylated to 5′-triphosphate and incorporated into DNA, where the cyano group may promote β-elimination, leading to DNA strand breaks or the formation of baseless sites, thereby exerting its antitumor effect. [4]
As a nucleoside, CNDAC is chemically stable, but its activity is enhanced after incorporation into DNA. [4]
Compared to cytarabine, CNDAC has a different antitumor spectrum and is effective against cytarabine-resistant tumors. [4]

C10H12N4O4.HCL

288.68762

288.063

134665-72-8

CNDAC;135598-68-4

3035200

White to off-white solid powder

596.9ºC at 760mmHg

314.8ºC

9.77E-17mmHg at 25°C

4

5

2

19

466

4

C1=CN(C(=O)N=C1N)[C@H]2[C@H]([C@@H]([C@H](O2)CO)O)C#N.Cl

PKGUOXDXRLGZBN-KUAPJGQRSA-N

InChI=1S/C10H12N4O4.ClH/c11-3-5-8(16)6(4-15)18-9(5)14-2-1-7(12)13-10(14)17;/h1-2,5-6,8-9,15-16H,4H2,(H2,12,13,17);1H/t5-,6+,8-,9+;/m0./s1

(2R,3S,4S,5R)-2-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-5-(hydroxymethyl)oxolane-3-carbonitrile;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~125 mg/mL (~432.99 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.)