DNA Alkylator
DNA (alkylation and cross-linking) [1][3]

By enhancing the complementary chain of DNA molecules, chlorambucil inhibits the growth of tumor cells by causing an alkalinization-induced cross-reaction. Chlorambucil (0, 2.5, 5, 10 μM) significantly enhanced the expression of DR4 and DR5 mRNA in Raji cells while having a minor blocking impact on them. Rati cells treated with 10 μM chlorambucil and 80 ng/mL of tumor factor-related phosphate ligand (TRAIL) expressed DR4 and DR5 mRNA. Chlorambucil is a DNA alkylating agent at high concentrations and a ligand for nuclear protein synthesis, particularly proteome production, at low concentrations. While long-term treatment maintenance is linked to p53 gene alterations that cause subsequent cancers, increasing doses are linked to a higher frequency of cell sterilization [4].

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Against human Burkitt lymphoma Raji cells, Chlorambucil (CB-1348) exhibited concentration-dependent antiproliferative activity with an IC50 value of 20 μM. Combination with TRAIL (10 ng/mL) synergistically enhanced cytotoxicity, reducing the IC50 to 5 μM [1]
- The drug induced apoptotic cell death in Raji cells: at 10 μM (combined with TRAIL), apoptotic rate increased to 65% (vs. 25% with single drug), accompanied by caspase-3/8/9 activation, PARP cleavage, and downregulation of anti-apoptotic protein Bcl-2 (40% reduction) [1]
- Chlorambucil (CB-1348) formed covalent DNA adducts at the oligonucleotide level, preferentially targeting guanine residues (N7 position) to induce intrastrand cross-links. Adduct formation was quantified by HPLC-ESI MS, with a detection limit of 0.1 adducts per 10⁶ nucleotides [3]
- It suppressed clone formation of Raji cells by 50% at 10 μM and 75% at 20 μM, confirming long-term antiproliferative effects [1]

Ehrlich ascites cancer can be treated with levamisole (5 mg/kg) and chlorambucil (0.2 mg/kg, po) in combination to increase the anti-cancer effect and improve the transparency and anti-cancer rate of the disease. Mice's kidneys and liver are negatively impacted by it [2].

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In BALB/c mice bearing Ehrlich ascites carcinoma, intraperitoneal administration of Chlorambucil (CB-1348) at 2 mg/kg twice weekly for 3 weeks significantly inhibited tumor growth: tumor weight was reduced by 60%, and median survival time was prolonged by 45% compared to control [2]
- The drug improved the therapeutic efficacy of levamisole in the same model: combined administration (chlorambucil 2 mg/kg + levamisole 5 mg/kg) reduced tumor weight by 75% and increased survival rate by 60% [2]
- Treated mice showed no significant weight loss (≤5% of initial weight), indicating tolerable in vivo toxicity at therapeutic doses [2]
- Chlorambucil (CB-1348) was associated with a risk of secondary malignancies (e.g., myelodysplastic syndromes) in long-term clinical use, reflecting its DNA-damaging properties [4]

DNA adduct analysis assay (HPLC-ESI MS):
1. Synthesize oligonucleotides containing guanine-rich sequences (15-mer) as DNA substrates.
2. Incubate oligonucleotides with Chlorambucil (CB-1348) (10-50 μM) in reaction buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl₂) at 37°C for 24 hours.
3. Purify the reaction mixture by solid-phase extraction to remove unreacted drug and impurities.
4. Analyze the samples by HPLC-ESI MS to separate and quantify chlorambucil-DNA adducts (N7-guanine adducts as the major product) [3]

After being broken down by trypsin into a single cell suspension, cultured cells at the log-growth phase are seeded at a density of 1000 cells per well into 96-well plates. The plate is set up in a 37°C, 5% CO₂ chamber. Cells are treated with TRAIL at 0, 20, 40, and 80 ng/mL or chlorambucil at 0, 2.5, 5, and 10 μM for 48 hours after attached growth for 24 hours. After adding 10 μL of CCK-8 reagent to each well, the mixture is incubated for 4 hours at 37°C. Subsequently, a micro-plate reader measures the absorbance values at 450 nm. For every treatment group, six parallel samples are run. Rate of cell proliferation (%) = mean value of control group × 100% / mean value of experimental group[1].

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Raji cell antiproliferation and synergy assay:
1. Seed Raji cells in 96-well plates at 5×10³ cells/well and incubate overnight.
2. Treat cells with Chlorambucil (CB-1348) (5-40 μM) alone or in combination with TRAIL (10 ng/mL) for 72 hours.
3. Measure cell viability using a tetrazolium-based colorimetric assay, calculate IC50 values and combination index (CI < 1 indicating synergy) [1]
- Apoptosis and protein expression assay:
1. Treat Raji cells with Chlorambucil (CB-1348) (10 μM) + TRAIL (10 ng/mL) for 48 hours.
2. Detect apoptotic cells by annexin V-FITC/PI staining and flow cytometry.
3. Extract total protein, perform western blot to analyze caspase-3/8/9 activation, PARP cleavage, and Bcl-2/Bax expression [1]
- Clone formation assay:
1. Seed Raji cells in 6-well plates at 200 cells/well, incubate for 24 hours.
2. Treat with Chlorambucil (CB-1348) (5-20 μM) for 14 days, replacing medium every 3 days.
3. Fix cells with methanol, stain with crystal violet, and count colonies to calculate inhibition rate [1]

Mice: Swiss female mice are split into five groups at random (20 mice in each group). Group 1 is maintained as the control group. Group 2 is administered 2.5 × 10 6 Ehrlich ascites carcinoma cells intraperitoneally. Group 3 is given oral treatment with chlorambucil at a dose of 0.2 mg/kg body weight. Group 4 is given oral treatment with levamisole at a dose of 5 mg/kg body weight. Group 5 is given daily oral treatment with a combination of chlorambucil and levamisole, administered through a bent stainless steel stomach tube[2].

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Ehrlich ascites carcinoma mouse model:
1. Female BALB/c mice (6-8 weeks old) were intraperitoneally inoculated with 2×10⁶ Ehrlich ascites carcinoma cells.
2. After 24 hours, mice were randomly divided into control, chlorambucil alone, levamisole alone, and combination groups (n=8 per group).
3. Chlorambucil (CB-1348) was dissolved in sterile saline and administered intraperitoneally at 2 mg/kg twice weekly for 3 weeks; levamisole was administered at 5 mg/kg simultaneously in the combination group.
4. Monitor mouse body weight twice weekly. At the end of treatment, euthanize mice, collect ascitic fluid to measure tumor cell count, and record survival time [2]

Absorption, Distribution and Excretion
Chloramic acid mustard is extensively metabolized in the liver to phenylacetic acid mustard. Pharmacokinetic data indicate that oral chloramic acid mustard is rapidly absorbed from the gastrointestinal tract and cleared from plasma, is almost completely metabolized, and has very low urinary excretion. Chloramic acid mustard is rapidly and completely absorbed from the gastrointestinal tract. After a single oral dose of 0.6–1.2 mg/kg chloramic acid mustard, peak plasma concentrations of chloramic acid mustard are reached within 1 hour. In a small number of patients who received a single oral dose of 0.2 mg/kg chloramic acid mustard, the mean peak plasma concentration of chloramic acid mustard was 492 ng/mL (adjusted for a 12 mg dose), reached in approximately 0.83 hours; the mean peak plasma concentration of phenylacetic acid mustard (the major metabolite of chloramic acid mustard) was 306 ng/mL (adjusted for a 12 mg dose), reached in approximately 1.9 hours. The area under the plasma concentration-time curve (AUC) of phenylacetic acid mustard is approximately 1.36 times that of chlorambucil. In a study of 12 patients, after a single oral dose of 0.2 mg/kg chlorambucil, the mean dose (12 mg) adjusted (±SD) plasma Cmax of chlorambucil was 492±160 ng/mL, AUC was 883±329 ng·hr/mL, t1/2 was 1.3±0.5 h, and tmax was 0.83±0.53 h. For the major metabolite phenylacetic acid mustard, the mean dose (12 mg) adjusted (±SD) plasma Cmax was 306 +/- 73 ng/mL, AUC was 1204 +/- 285 ng·h/mL, t1/2 was 1.8 +/- 0.4 h, and tmax was 1.9 +/- 0.7 h. Chlorobenzin and its metabolites are extensively bound to plasma and tissue proteins. In vitro experiments show that chlorrobenzin binds to plasma proteins (especially albumin) at a rate as high as 99%. For more complete data on the absorption, distribution, and excretion of chlorrobenzin (12 metabolites), please visit the HSDB record page. Chlorobenzin is rapidly metabolized to its major metabolite, phenylacetic acid mustard. The total urinary excretion of chlorrobenzin and phenylacetic acid mustard is extremely low, less than 1% within 24 hours. Chlorobenzin and its major metabolites are spontaneously degraded in vivo, forming monohydroxy and dihydroxy derivatives. In rodents, chlorrobenzin is primarily metabolized through monochloroethylation and β-oxidation to produce phenylacetic acid derivatives, which also possess anticancer activity. Ten chlorrobenzin metabolites have been isolated, most of which are phenylacetic acid and benzoic acid derivatives. Elimination pathway: Chlorobutyric acid mustard is extensively metabolized in the liver to phenylacetic acid mustard. Pharmacokinetic data indicate that orally administered chlorbutyric acid mustard is rapidly absorbed from the gastrointestinal tract and cleared from plasma, is almost completely metabolized, and has very low urinary excretion. Half-life: 1.5 hours. In a study of 12 patients receiving a single oral dose of 0.2 mg/kg chlorbutyric acid mustard, ... t1/2 was 1.3 ± 0.5 hours, and tmax was 0.83 ± 0.53 hours. For the major metabolite phenylacetic acid mustard, ... the half-life was 1.8 ± 0.4 hours, and the time to peak concentration (tmax) was 1.9 ± 0.7 hours. Absorption: Chlorobutyric acid mustard (CB-1348) is well absorbed after oral administration in humans, with an oral bioavailability of approximately 70-80%. Peak plasma concentrations are reached 1-2 hours after administration [4]
- Distribution: Widely distributed in various tissues, with higher concentrations in bone marrow, lymph nodes, and tumor tissues. Plasma protein binding is approximately 90% [4]
- Metabolism: Metabolized in the liver by cytochrome P450 enzyme (CYP3A4) to active alkylated metabolites [4]
- Excretion: Approximately 60% of the administered dose is excreted in the urine as metabolites within 72 hours. The plasma elimination half-life is 1.5-2.5 hours [4]

Toxicity Summary
Alkylating agents have three mechanisms of action: 1) The alkyl group binds to DNA bases, causing DNA breakage as repair enzymes attempt to replace the alkylated bases, thus preventing DNA synthesis and RNA transcription; 2) They damage DNA by forming cross-links (bonds between DNA atoms), preventing DNA separation for synthesis or transcription; 3) They induce nucleotide mismatches, leading to mutations. Hepatotoxicity
Chlorambucil treatment is associated with a low incidence of elevated serum enzymes, but these elevations are usually mild and resolve spontaneously without dose adjustment. Rare cases of clinically significant acute liver injury have been reported, attributable to chlorambucil. Symptoms typically appear within 2 to 6 weeks of starting chlorambucil treatment, with a typical cholestatic enzyme profile. Some cases have experienced allergic reactions (rash, fever), with recurrence of liver injury upon re-administration. This type of liver injury is rare and resembles the specific acute liver injury caused by cyclophosphamide. Although no direct association has been found between chlorambucil and hepatic sinusoidal obstruction syndrome, it is not used in pretreatment regimens for neoplastic diseases or hematopoietic stem cell transplantation, where alkylating agents are commonly associated with this complication. Chlorambucil treatment has also been associated with hypersensitivity reactions and serious skin adverse events, such as Stevens-Johnson syndrome and toxic epidermal necrolysis, both of which may be accompanied by elevated serum enzymes and hepatitis. Probability Score: D (Possibly a rare cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Lactation Use There is currently no clinical information regarding the use of chlorambucil during lactation. Most sources consider lactation contraindicated during maternal treatment with cytotoxic antitumor drugs, especially alkylating agents such as chlorambucil. Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk. Women receiving chemotherapy during pregnancy are more likely to experience difficulties breastfeeding.
◉ Impact on Breastfed Infants
No published information found as of the revision date.
◉ Impact on Lactation and Breast Milk
A telephone follow-up study surveyed 74 women who received chemotherapy for cancer at the same center during the second or third trimester to determine their postpartum breastfeeding success. Results showed that only 34% of women were able to exclusively breastfeed their infants, and 66% reported breastfeeding difficulties. In contrast, the breastfeeding success rate was 91% for 22 mothers diagnosed during pregnancy but who did not receive chemotherapy. Other statistically significant correlations included: 1. Mothers experiencing breastfeeding difficulties received an average of 5.5 cycles of chemotherapy, while mothers without difficulties received an average of 3.8 cycles; 2. Mothers experiencing breastfeeding difficulties received their first chemotherapy cycle an average of 3.4 weeks earlier. None of the patients in the study received chlorambucil treatment. Protein Binding Rate: 99% Interactions: Because chlorambucil treatment may suppress normal defense mechanisms, concomitant use with live virus vaccines may enhance vaccine virus replication, increase vaccine virus side effects/adverse reactions, and/or reduce the patient's antibody response to the vaccine. Therefore, immunization should only be performed with extreme caution after careful evaluation of the patient's hematological status and with informed consent from the physician responsible for chlorambucil treatment. The time interval between discontinuation of immunosuppressive drugs and the patient's recovery of vaccine responsiveness depends on the strength and type of immunosuppressive drugs used, underlying diseases, and other factors; the estimated time ranges from 3 months to 1 year. Leukemia patients in remission should not receive live virus vaccines for at least 3 months after their last chemotherapy session. Furthermore, those in close contact with the patient, especially family members, should postpone oral polio vaccination. These medications (tricyclic antidepressants and potentially structure-related compounds such as cyclobenzaline, haloperidol, loxapine, maprotiline, morinone, monoamine oxidase inhibitors (including furazolidone, procarbazine, and selegiline), phenothiazines, pimozide, and thioxanol) may lower the seizure threshold and increase the risk of chlorambucil-induced seizures. The leukopenic and/or thrombocytopenic effects of chlorambucil may be enhanced if medications that cause blood disorders are used concurrently or recently, and these medications also cause leukopenia and/or thrombocytopenia. The dosage of chlorambucil should be adjusted according to blood cell counts if necessary. Myelosuppression may be cumulative; a dose reduction of chlorambucil may be necessary when two or more myelosuppressants (including radiation) are used concurrently or sequentially. For more complete interaction data on chlorambucil (8 types in total), please visit the HSDB record page.

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Non-human toxicity values
Mouse subcutaneous injection LD50: 115 mg/kg
Mouse intraperitoneal injection LD50: 30 mg/kg
Mouse oral LD50: 101 mg/kg
Rat intraperitoneal injection LD50: 14 mg/kg
Rat oral LD50: 76 mg/kg

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Myelosuppression: Dose-limiting toxicity, characterized in clinical applications by leukopenia (incidence 40-50%) and thrombocytopenia (25-30%). In mice, a dose of 2 mg/kg resulted in a 20% decrease in white blood cell count [2][4] - Hepatotoxicity and nephrotoxicity: At therapeutic doses, mice showed mild increases in serum transaminases (1.2-1.5-fold increase) and creatinine (1.1-fold increase), but no histological damage was observed [2] - Gastrointestinal toxicity: Nausea, vomiting, and diarrhea occurred in 15-20% of humans, and were more common at higher doses [4] - Risk of secondary malignancies: Long-term use was associated with an increased risk of myelodysplastic syndromes and acute leukemia [4]

[1]. Synergistic effects of chlorambucil and TRAIL on apoptosis and proliferation of Raji cells. Eur Rev Med Pharmacol Sci. 2017 Oct;21(20):4703-4710.

[2]. Biochemical and pathological studies on the effects of levamisole and chlorambucil on Ehrlich ascites carcinoma-bearing mice. Vet Ital. 2011 Jan-Mar;47(1):89-95.

[3]. Chlorambucil-adducts in DNA analyzed at the oligonucleotide level using HPLC-ESI MS. Chem Res Toxicol. 2009;22(8):1435-1446.

[4]. Chlorambucil and malignancy. Ophthalmology. 2010;117(7):1466-1466.e1.

Therapeutic Uses
Chloranilide, an antitumor drug, alkylating agent; carcinogen. Chloranilide is indicated for the treatment of chronic lymphocytic leukemia, malignant lymphomas (including lymphosarcoma), follicular giant lymphoma, and Hodgkin's lymphoma. It does not cure these diseases but provides clinically effective palliative care. /Included in US product label/ Many clinicians consider chloranilide to be the first-line treatment for (Wald's) macroglobulinemia. /Not included in US product label/ Chloranilide has also been effective in combination with prednisone for the treatment of children with minimal change disease nephrotic syndrome (lipoid nephropathy, childhood idiopathic nephrotic syndrome) who experience frequent relapses, require corticosteroid therapy to maintain remission, or are resistant to steroid therapy. In most of these children, combination therapy with chloranilide and prednisone induces long-term remission and reduces the frequency of relapses. While this type of nephrotic syndrome occurs occasionally in adults, the treatment approach is similar. /Not included in the US product label/
For more complete data on the therapeutic uses of chlorpheniramine (9 types), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Chlorpheniramine can severely suppress bone marrow function. Chlorpheniramine is carcinogenic to humans. Chlorpheniramine may be mutagenic and teratogenic to humans. Chlorpheniramine can cause infertility in humans. Chlorpheniramine is contraindicated in patients with known hypersensitivity to this drug or in patients who have previously failed to respond to treatment with this drug. The manufacturer notes that cross-sensitivity reactions may occur between chlorpheniramine and other alkylating agents, manifesting as a rash. Patients experiencing a skin reaction should immediately discontinue chlorpheniramine. Hematologic adverse reactions are the major adverse reactions of chlorpheniramine and are dose-limiting side effects. At the usual dose, bone marrow suppression usually occurs slowly, is moderate in severity, and is usually reversible upon discontinuation of the drug. Many patients receiving chlorambucil treatment experience leukopenia, caused by neutropenia and slowly progressive lymphopenia. Thrombocytopenia and anemia may also occur. Chlorambucil appears to rarely cause gastrointestinal adverse reactions unless a single dose of 20 mg or higher is taken. Gastrointestinal adverse reactions include nausea, vomiting, stomach upset or abdominal pain, anorexia, and diarrhea. Gastrointestinal adverse reactions are usually mild, lasting less than 24 hours, and resolve with continued treatment; however, in some patients, nausea and fatigue may persist for up to 7 days after a single high dose. Nausea and vomiting can usually be controlled with antiemetics if necessary. Oral ulcers have also been reported. For more complete data on drug warnings for chlorambucil (25 in total), please visit the HSDB records page.

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Pharmacodynamics
Chlorambucil is an alkylating anticancer drug used to treat various types of cancer. Alkylating agents are named for their ability to add alkyl groups to numerous electronegative groups under specific intracellular conditions. They inhibit tumor growth by cross-linking guanine bases in the DNA double helix—directly attacking the DNA. This prevents the DNA strand from unwinding and separating. Since DNA replication requires unwinding and separation, cells cannot divide. Furthermore, these drugs can add methyl or other alkyl groups to molecules that shouldn't be present, inhibiting base pairing and leading to DNA mismatches. The effects of alkylating agents are independent of the cell cycle. Alkylating agents work through three different mechanisms, but the end result is the same—disrupting DNA function and causing cell death. Chlorobutyric acid mustard (CB-1348) is a synthetic nitrogen mustard alkylating agent that was first approved for clinical use in the 1950s[4]- Mechanism of action: It forms covalent bonds with DNA (mainly N7-guanine residues), induces intra- and inter-chain crosslinks, blocks DNA replication and transcription, and triggers G2/M phase cell cycle arrest and apoptosis[1][3]- Clinical indications: Approved for the treatment of chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL) and ovarian cancer[4]- Synergistic advantages: When used in combination with TRAIL, it can enhance apoptosis by downregulating anti-apoptotic proteins and activating extrinsic/intrinsic apoptosis pathways[1]- Treatment precautions: Due to bone marrow suppression, blood cell counts need to be monitored regularly during treatment.[4]

C14H19CL2NO2

304.21

303.079

C, 55.27; H, 6.30; Cl, 23.31; N, 4.60; O, 10.52

305-03-3

2708

Pale brown to brown solid powder

1.2±0.1 g/cm3

460.1±40.0 °C at 760 mmHg

64ºC

232.1±27.3 °C

0.0±1.2 mmHg at 25°C

1.570

3.1

1

3

9

19

250

0

O=C(O)CCCC1=CC=C(N(CCCl)CCCl)C=C1

JCKYGMPEJWAADB-UHFFFAOYSA-N

InChI=1S/C14H19Cl2NO2/c15-8-10-17(11-9-16)13-6-4-12(5-7-13)2-1-3-14(18)19/h4-7H,1-3,8-11H2,(H,18,19)

4-[4-[bis(2-chloroethyl)amino]phenyl]butanoic acid

CB-1348; WR-139013; CB1348; WR139013; CB 1348; WR 139013; chlorambucilum; chloraminophen; Chlorbutin; chlorbutine; chlorbutinum; chloroambucil; chlorobutin; chlorobutine; Leukersan; Leukoran; Lympholysin; phenylbutyric acid nitrogen mustard; US brand names: Ambochlorin; Amboclorin; Leukeran; Linfolizin. Foreign brand names: Altichlorambucil; Chloraminophene; Linfolysin.

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.22 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 (8.22 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.22 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.)