A Comprehensive Monograph on 6-O-benzylguanine (DB11919): From Promising Chemosensitizer to Indispensable Research Tool

Executive Summary

6-O-benzylguanine, also known as O6-BG, is a synthetic small molecule analogue of guanine, rationally designed as a potent, mechanism-based inactivator of the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (MGMT).[1] Its development was predicated on a clear and compelling therapeutic rationale: to function as a chemosensitizing agent, thereby overcoming a primary mechanism of tumor resistance to a cornerstone class of anticancer drugs, the O⁶-alkylating agents. These agents, which include temozolomide (TMZ) and carmustine (BCNU), are critical in the treatment of various malignancies, particularly glioblastoma. Tumor resistance to these drugs is frequently mediated by high cellular levels of MGMT, which efficiently repairs the cytotoxic DNA lesions they induce.[3]

The molecular mechanism of 6-O-benzylguanine is one of elegant and potent "suicide" inhibition. It acts as a pseudosubstrate for MGMT, binding to the enzyme's active site and irreversibly transferring its benzyl group to a critical cysteine residue. This covalent modification permanently inactivates the MGMT protein, which is subsequently targeted for degradation, thereby preventing the repair of drug-induced DNA damage in cancer cells.[5] This blockade allows the cytotoxic lesions to persist, leading to futile DNA repair cycles, the formation of lethal double-strand breaks, and ultimately, apoptotic cell death.

This well-defined mechanism translated into remarkable success in preclinical studies. Both in vitro experiments on a wide array of cancer cell lines and in vivo studies using human tumor xenograft models demonstrated that 6-O-benzylguanine could dramatically potentiate the antitumor effects of alkylating agents, often reversing resistance and inducing significant tumor regressions.[7] These highly promising results provided a strong impetus for its advancement into clinical trials.

However, the clinical development of 6-O-benzylguanine was met with a formidable and ultimately insurmountable challenge. The very potency and lack of tissue specificity that made it an effective MGMT inhibitor in preclinical models proved to be its critical limitation in human subjects. The drug was unable to uncouple the potent sensitization of tumors from a severe, dose-limiting sensitization of healthy host tissues, most notably the hematopoietic stem cells of the bone marrow. The resulting profound myelosuppression when combined with chemotherapy necessitated significant reductions in the dose of the alkylating agent, which in turn negated the potential therapeutic gains. Pivotal clinical trials, particularly in patients with glioblastoma, failed to demonstrate a significant clinical benefit, leading to the cessation of its development as a therapeutic agent.[2]

Despite its failure to gain regulatory approval, the story of 6-O-benzylguanine did not end. Its exquisite specificity and well-characterized mechanism of action have rendered it an indispensable tool in the field of molecular biology and cancer research. It is now widely used as the gold-standard chemical inhibitor to probe the function of MGMT and the intricacies of DNA repair pathways. Furthermore, the fundamental chemistry of its interaction with MGMT has served as the direct inspiration and foundation for the development of powerful protein labeling technologies, such as the SNAP-tag system, which are now staples in laboratories worldwide.[2] Thus, 6-O-benzylguanine represents a paradigmatic case of a compound whose journey transformed it from a failed clinical candidate into a triumphant and enduring scientific tool.

Identification and Physicochemical Properties

Establishing the precise identity and fundamental characteristics of a chemical entity is the cornerstone of any rigorous scientific monograph. 6-O-benzylguanine is a well-characterized small molecule with a comprehensive set of identifiers across various chemical and pharmacological databases.

Nomenclature and Database Identifiers

The compound is most commonly referred to by its generic name, 6-O-benzylguanine, and the widely used abbreviation O6-BG.[2] It is also known by several synonyms that reflect its chemical structure, including

O(6)-Benzylguanine, 2-Amino-6-(benzyloxy)purine, and the trade name Alkylade.[11]

Its unique identity is cataloged across major international databases. In the DrugBank database, it is assigned the accession number DB11919.[11] The Chemical Abstracts Service has registered it with the CAS Number

19916-73-5.[1] As an investigational agent studied extensively by the National Cancer Institute (NCI), it holds several specific identifiers in the NCI Drug Dictionary: the abbreviation

BG, an Investigational New Drug (IND) number of 45789, and an NSC (National Service Center) code of 637037.[6] Additional key identifiers include a PubChem Compound ID (CID) of

4578, a UNII (Unique Ingredient Identifier) of 01KC87F8FE, and a ChemSpider ID of 4417.[1]

Chemical Structure and Formula

The molecular composition of 6-O-benzylguanine is defined by the chemical formula C12​H11​N5​O.[2] Its formal IUPAC (International Union of Pure and Applied Chemistry) name is

6-(Benzyloxy)-7H-purin-2-amine, with the alternative 6-phenylmethoxy-7H-purin-2-amine also being used.[1] This structure, a purine core with a benzyl group attached via an ether linkage at the 6-position, is unambiguously represented by computational identifiers:

• Canonical SMILES (Simplified Molecular-Input Line-Entry System): C1=CC=C(C=C1)COC2=NC(=NC3=C2NC=N3)N [1]
• InChI (International Chemical Identifier) Key: KRWMERLEINMZFT-UHFFFAOYSA-N [1]

Physicochemical Characteristics

6-O-benzylguanine is a solid substance, typically appearing as a white to light yellow powder or crystalline material.[15] It has a precisely determined molecular weight, with an average mass of approximately 241.25 g/mol and a monoisotopic mass of 241.09635999 Da.[1] The compound exhibits a melting point of approximately 205 °C.[15]

Its solubility profile is a critical characteristic that has influenced its formulation for both research and clinical applications. It is generally considered insoluble in water, with experimental values reported as 87.1 mg/L at 20 °C and slightly soluble at 0.2 mg/mL.[15] This poor aqueous solubility necessitated the development of specialized vehicles for intravenous administration in clinical trials.[4] In contrast, it demonstrates good solubility in several organic solvents, including methanol (20 mg/mL), dimethyl sulfoxide (DMSO) (

≥10 mg/mL), and acetonitrile, as well as in acidic aqueous solutions such as 0.1 M HCl.[16]

For laboratory use, 6-O-benzylguanine is stable as a powder for several years when stored appropriately, either at -20 °C or at room temperature in a cool, dark, and inert environment.[14] When prepared as a solution, for instance in methanol, it is recommended to store it at -20 °C and to use it promptly to avoid degradation.[13]

Table 1: Summary of Physicochemical and Identification Properties of 6-O-benzylguanine

Property	Value	Source(s)
Generic Name	6-O-benzylguanine	11
DrugBank ID	DB11919	11
CAS Number	19916-73-5	1
Molecular Formula	C12​H11​N5​O	11
IUPAC Name	6-(Benzyloxy)-7H-purin-2-amine	2
Average Molecular Weight	241.25 g/mol	1
Physical Appearance	White to light yellow powder/crystal	15
Melting Point	~205 °C	15
Solubility in Water	Insoluble (87.1 mg/L at 20 °C)	15
Solubility in Methanol	20 mg/mL	16
Solubility in DMSO	≥10 mg/mL	16

Molecular Mechanism of Action and Pharmacodynamics

The therapeutic concept behind 6-O-benzylguanine is rooted in a deep understanding of a specific DNA repair pathway that confers resistance to a major class of chemotherapeutic drugs. Its mechanism is a classic example of rational drug design, targeting a single, well-defined molecular entity to modulate a cellular phenotype.

The Central Role of MGMT in Chemoresistance

The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT), also referred to as O⁶-alkylguanine-DNA alkyltransferase (AGT or ATase), plays a pivotal role in maintaining genomic integrity.[17] Its primary function is to protect cells from the mutagenic and cytotoxic effects of alkylating agents, which can be environmental carcinogens or therapeutic drugs.[3] These agents damage DNA by adding alkyl groups to various positions on the DNA bases. One of the most critical lesions is the formation of an adduct at the O⁶-position of guanine.[3]

MGMT functions as a unique "suicide" enzyme. It identifies the O⁶-alkylguanine lesion in DNA and directly reverses the damage in a single-step reaction. The enzyme transfers the alkyl group from the guanine base to one of its own cysteine residues, Cys145, located within its active site.[19] This transfer reaction is stoichiometric and irreversible; one molecule of MGMT is consumed for each lesion it repairs, and the alkylated protein is subsequently ubiquitinated and targeted for proteasomal degradation.[3] The cell's capacity to repair this type of damage is therefore limited by its available pool of active MGMT protein and its rate of new protein synthesis.[3]

This protective function becomes a major clinical obstacle in oncology. The efficacy of O⁶-alkylating chemotherapies, such as the nitrosourea carmustine (BCNU) and the triazene temozolomide (TMZ), depends on their ability to inflict lethal DNA damage. High levels of MGMT expression in tumor cells constitute a primary mechanism of chemoresistance, as the enzyme efficiently removes the drug-induced lesions, allowing the cancer cells to survive and proliferate.[4] Conversely, in tumors with low or absent MGMT expression, often due to epigenetic silencing of the

MGMT gene promoter via methylation, these chemotherapeutic agents are significantly more effective. This correlation is so strong that the methylation status of the MGMT promoter has become a key predictive biomarker for response to temozolomide in patients with glioblastoma.[3]

Mechanism of O6-BG: A Pseudosubstrate Suicide Inactivator

Recognizing MGMT as the central mediator of resistance, 6-O-benzylguanine was rationally designed to neutralize this defense mechanism. It functions as a pseudosubstrate, a molecule that mimics the natural substrate of an enzyme but, upon binding, leads to the enzyme's inactivation.[5] The design was based on the principle of the bimolecular displacement reaction at the MGMT active site. The benzyl group was specifically chosen over a simple alkyl group because its chemical properties allow it to enter this reaction more readily, making for a more potent inhibitor.[4]

When 6-O-benzylguanine enters a cell, it binds to the active site of the MGMT protein. In a reaction analogous to the repair of damaged DNA, the enzyme covalently transfers the benzyl group from 6-O-benzylguanine to its active-site Cys145 residue, forming an S-benzylcysteine adduct.[1] This process is irreversible and constitutes a "suicide" inactivation event. The benzylated MGMT protein is rendered permanently non-functional and is rapidly targeted for degradation by the cellular proteasome system.[4]

The potency and speed of this inactivation are remarkable. In cell culture experiments, micromolar concentrations of 6-O-benzylguanine can lead to a greater than 90% depletion of cellular MGMT activity within minutes.[8] This is far more efficient than inactivation by the natural substrate, O⁶-methylguanine, which requires much higher concentrations and longer incubation times to achieve a lesser effect.[8] In cell-free enzyme assays, the concentration of 6-O-benzylguanine required for 50% inhibition (IC₅₀) is in the low nanomolar range, for example, 39 nM in extracts from HL-60 leukemia cells.[24]

Pharmacodynamic Consequence: Chemosensitization

The direct pharmacodynamic consequence of MGMT depletion by 6-O-benzylguanine is the sensitization of cells to O⁶-alkylating agents. By removing the cell's primary defense, 6-O-benzylguanine allows the drug-induced DNA lesions to persist, triggering cell death pathways.

For methylating agents like temozolomide, the key cytotoxic lesion is O⁶-methylguanine (O⁶-meG). In an MGMT-depleted cell, this unrepaired lesion persists through DNA replication, where it incorrectly pairs with thymine instead of cytosine. This O⁶-meG:T mismatch is recognized by the DNA mismatch repair (MMR) system. The MMR machinery attempts to correct the error by excising the newly incorporated thymine. However, because the original O⁶-meG lesion on the template strand remains, the polymerase will again insert a thymine opposite it. This leads to repeated, "futile" cycles of attempted repair, which ultimately result in the accumulation of persistent single- and double-strand DNA breaks, the collapse of the replication fork, cell cycle arrest, and the initiation of apoptosis.[3]

For chloroethylating agents like carmustine (BCNU), the initial lesion is an O⁶-chloroethylguanine adduct. In a normal cell, MGMT would remove this adduct. In an MGMT-depleted cell, however, this initial adduct is able to undergo a spontaneous intramolecular rearrangement to form a reactive intermediate, which then attacks the opposing DNA strand to form a highly toxic N1-guanine-N3-cytosine interstrand cross-link. These cross-links physically prevent the separation of the DNA strands, making DNA replication and transcription impossible and leading to cell death.[4]

The very mechanism that defines the power of 6-O-benzylguanine also contains the seeds of its clinical downfall. The drug's design achieved exceptional potency and efficiency in inactivating MGMT, a fundamental protein responsible for protecting the genome. However, this action is not confined to tumor cells. MGMT is ubiquitously expressed in healthy tissues, where it plays the same vital protective role. The most vulnerable of these tissues are those with high rates of cell division, such as the hematopoietic progenitor cells in the bone marrow, which are already highly sensitive to the toxic effects of alkylating chemotherapy.[4]

When 6-O-benzylguanine is administered systemically, it depletes MGMT not only in the tumor but also in the bone marrow. This systemic inactivation of the protective enzyme renders the hematopoietic system exquisitely sensitive to the co-administered alkylating agent. The consequence is a dramatic amplification of the chemotherapy's inherent myelosuppressive toxicity. This on-target, systemic effect means that the sensitization of the tumor cannot be uncoupled from the sensitization of the host. This dynamic was borne out in clinical trials, where the combination of 6-O-benzylguanine and chemotherapy led to severe, dose-limiting bone marrow toxicity, forcing clinicians to reduce the dose of the chemotherapeutic agent to a level that potentially negated any benefit gained from tumor sensitization.[9] In essence, the therapeutic window, which relies on a differential effect between tumor and normal tissue, was narrowed or closed entirely. The drug's greatest strength—its potent and irreversible inhibition of a key defense mechanism—proved to be its greatest and unavoidable weakness in a clinical context.

Preclinical Development: Establishing the Therapeutic Rationale

Before its evaluation in humans, 6-O-benzylguanine was subjected to extensive preclinical testing that built a powerful and compelling case for its potential as a chemosensitizing agent. The data from both in vitro cell culture systems and in vivo animal models were remarkably consistent and positive, providing the strong scientific foundation necessary to justify clinical investigation.

In Vitro Evidence of Chemosensitization

Laboratory studies using cultured human cancer cells were the first to demonstrate the potent sensitizing effects of 6-O-benzylguanine. These experiments established several key principles of its action.

First, its activity was shown to be broadly applicable across a diverse range of cancer types. Effective MGMT inactivation and subsequent sensitization to alkylating agents were demonstrated in cell lines derived from glioblastoma (e.g., T98G), colon carcinoma (e.g., HT-29), promyelocytic leukemia (e.g., HL-60), and, importantly, various pediatric brain tumors.[7] This suggested that the strategy could be relevant for multiple clinical indications where alkylating agents are used.

Second, the degree of potentiation was quantified and found to be substantial. In a comprehensive study of pediatric brain tumor cell lines, pretreatment with 6-O-benzylguanine produced a dramatic increase in the cytotoxicity of both BCNU and temozolomide. On average, in MGMT-expressing cell lines, it reduced the LD₁₀ (the lethal dose required to kill 90% of the cells) for BCNU by a factor of 2.6 and for temozolomide by an impressive factor of 26. Even more critically, it lowered the threshold dose required to initiate cell killing by 3.3-fold for BCNU and by a remarkable 138-fold for temozolomide. This effect was significant because it brought the effective concentrations of these drugs down from levels that were often higher than what could be safely achieved in patients to levels well within the clinically achievable plasma concentration range.[7]

Third, these studies confirmed that the effect of 6-O-benzylguanine was directly linked to its intended target. A strong correlation was observed between the baseline level of MGMT activity in a given cell line and the degree of chemosensitization produced by 6-O-benzylguanine. Cell lines with high endogenous MGMT expression exhibited the most dramatic increase in sensitivity, whereas cell lines that were already MGMT-deficient showed little to no benefit from the addition of the inhibitor.[4] This provided clear evidence of its on-target mechanism of action.

Finally, experiments confirmed the downstream molecular consequences of MGMT inhibition. Treatment with 6-O-benzylguanine in combination with alkylating agents was shown to increase the levels of biomarkers associated with DNA damage and apoptosis, such as the phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks, and the cleavage of caspase-3, a key executioner of the apoptotic cascade.[5] This provided a mechanistic link between the inhibition of DNA repair and the ultimate outcome of cell death.

In Vivo Evidence from Animal Models

The promising results from cell culture were subsequently validated in more complex in vivo systems, primarily using immunodeficient mice bearing human tumor xenografts. These animal models provided the crucial proof-of-concept that 6-O-benzylguanine could enhance the therapeutic efficacy of alkylating agents in a living organism.

In numerous studies, the combination of 6-O-benzylguanine administered prior to BCNU or temozolomide resulted in significantly greater antitumor activity than could be achieved with the chemotherapy alone. In models of glioblastoma (e.g., D-456 MG), medulloblastoma, and colon cancer, the combination therapy led to profound tumor growth delays and, in some cases, complete and sustained tumor regressions.[4] This effect was particularly striking in tumor models known to be resistant to alkylating agents due to high MGMT expression, demonstrating that 6-O-benzylguanine could effectively reverse this resistance phenotype

in vivo.[4]

However, these preclinical animal studies also provided the first clear signals of the potential for enhanced host toxicity. While the primary focus was on antitumor efficacy, careful observation revealed that the addition of 6-O-benzylguanine also potentiated the toxicity of the co-administered chemotherapy. Specifically, studies in mice demonstrated that the combination led to more severe bone marrow suppression and an increased frequency of clastogenic events (the formation of micronuclei, indicative of chromosome damage) in bone marrow cells compared to chemotherapy alone.[26] This finding was a critical, albeit perhaps underappreciated, foreshadowing of the dose-limiting toxicities that would ultimately define the drug's clinical profile.

The stark contrast between the overwhelming success of 6-O-benzylguanine in preclinical models and its ultimate failure in pivotal human trials for glioblastoma exemplifies a classic challenge in oncology drug development, often referred to as the "preclinical-to-clinical translation gap." The preclinical xenograft models, while invaluable for demonstrating on-target biological activity, were not sufficiently predictive of the delicate balance between efficacy and toxicity in humans.

Several factors contribute to this gap. The xenograft models typically involve transplanting human tumor cells subcutaneously into immunodeficient mice. This artificial context differs from a human patient in critical ways. The mouse hematopoietic system may possess different sensitivities or recovery kinetics compared to the human system. Furthermore, the primary endpoints in these preclinical studies are typically tumor growth delay or regression, which are direct measures of antitumor efficacy. While toxicity is monitored (e.g., by weight loss or survival), these models are not designed to precisely replicate the complex, multi-cycle, cumulative toxicities, particularly hematological toxicity, that define the maximum tolerated dose in human clinical trials. Although some preclinical reports did note the enhanced bone marrow toxicity [26], the overall assessment from the body of animal work was that the enhancement of antitumor activity was greater than the enhancement of toxicity, suggesting a net positive therapeutic index.[4] This conclusion did not hold true in the human clinical setting, where the amplification of toxicity proved to be the dominant and dose-limiting factor, effectively closing the therapeutic window that had appeared so wide in the preclinical data.[2]

Clinical Development and Efficacy

The journey of 6-O-benzylguanine through human clinical trials was a systematic process that began with establishing its biological activity and safety, proceeded to define its use in combination with chemotherapy, and culminated in efficacy studies that ultimately failed to meet their primary objectives, particularly in its most anticipated indication, glioblastoma.

Phase I Trials: Determining the Biologically Active Dose

The initial Phase I clinical trials were not designed to measure antitumor response but to answer a more fundamental question: could 6-O-benzylguanine effectively and safely achieve its intended biological effect in human patients? The primary goal was to determine the dose and administration schedule required to produce profound and sustained depletion of MGMT activity in both accessible tissues, like peripheral blood mononuclear cells (PBMCs), and, most importantly, within the tumor tissue itself.[31]

A key study of this type was a presurgical or "window-of-opportunity" trial in patients with malignant glioma. In this design, patients received 6-O-benzylguanine at various dose levels prior to their scheduled surgery for tumor resection. The resected tumor tissue was then analyzed to directly measure the level of MGMT activity.[25] This approach provided invaluable pharmacodynamic data and established a critical proof-of-concept. The trial determined that a single intravenous dose of 100 mg/m² was sufficient to reduce tumor MGMT activity to undetectable levels (<10 fmol/mg protein) and to maintain this depletion for at least 18 hours.[25] These studies also confirmed that when administered as a single agent, 6-O-benzylguanine was well-tolerated and did not produce any significant toxicity on its own.[33]

Phase I Combination Trials: Establishing Maximum Tolerated Dose (MTD)

With the biologically active dose of 6-O-benzylguanine established, the next logical step was to combine it with alkylating agents and determine the maximum tolerated dose (MTD) of the chemotherapy in this new context. These Phase I combination trials were designed to find the highest dose of drugs like BCNU or temozolomide that could be safely administered alongside the MGMT inhibitor.[9]

The results of these trials were sobering and highlighted the central challenge of this therapeutic strategy. The addition of 6-O-benzylguanine dramatically increased the hematological toxicity of the chemotherapy, necessitating substantial reductions in the standard doses of the alkylating agents to maintain patient safety. For instance, a Phase I trial established the MTD of a single dose of temozolomide, when given with a 48-hour infusion of 6-O-benzylguanine, to be 472 mg/m².[9] In a different trial exploring a 5-day dosing schedule of temozolomide, the addition of 6-O-benzylguanine forced a reduction of the daily temozolomide dose to just 75 mg/m², significantly lower than standard regimens.[36] This finding was a major warning sign, as it raised the critical question of whether the benefits of sensitizing the tumor could outweigh the necessity of lowering the dose of the cytotoxic agent.

Phase II Trials in Malignant Glioma: The Efficacy Verdict

The definitive test of the 6-O-benzylguanine strategy came in Phase II trials designed to evaluate its antitumor efficacy. A pivotal, multi-center Phase II trial was conducted in patients with recurrent malignant glioma that had become resistant to prior temozolomide therapy. Patients were stratified into two groups based on their tumor histology: glioblastoma multiforme (GBM), the most aggressive type, and anaplastic glioma (a slightly less aggressive form).[9]

The results from this trial were mixed and ultimately disappointing. In the cohort of patients with anaplastic glioma, the combination of 6-O-benzylguanine and temozolomide did show a modest level of activity, suggesting it could restore sensitivity to temozolomide in a subset of these patients. The objective response rate (ORR) was 16% (5 out of 32 evaluable patients), and the 6-month progression-free survival (PFS) rate was 25%.[9]

However, in the primary target population of patients with GBM, the combination therapy was largely ineffective. The ORR was a mere 3% (only 1 of 34 patients), and the 6-month PFS rate was just 9%.[9] Given the strong preclinical data and the clear biological rationale, this lack of significant clinical activity in GBM was a major setback. The trial's conclusion was that the addition of 6-O-benzylguanine to temozolomide did not meaningfully restore chemosensitivity in patients with temozolomide-resistant glioblastoma.[2] This result, combined with the significant toxicity, effectively halted the further development of this combination for this indication.

Investigations in Other Cancers and Settings

The therapeutic potential of 6-O-benzylguanine was explored in a wide range of clinical contexts beyond recurrent glioblastoma. Clinical trials have investigated its use in combination with various chemotherapies for the treatment of newly diagnosed glioblastoma and gliosarcoma, other solid tumors, Hodgkin and non-Hodgkin lymphoma, and pediatric brain tumors.[7] Innovative trial designs have also explored its use in conjunction with other agents like ifosfamide [37] or as part of complex protocols involving gene therapy and autologous stem cell transplantation to protect the bone marrow.[12] Despite this broad and sustained investigational effort spanning decades, 6-O-benzylguanine has not demonstrated a sufficiently favorable risk-benefit profile in any setting to achieve regulatory approval for therapeutic use.

Table 3: Overview of Major Clinical Trials Investigating 6-O-benzylguanine

Trial Identifier (NCT#)	Phase	Condition(s) Treated	Intervention	Primary Objective / Endpoint	Summary of Key Findings / Outcome	Source(s)
NCT00002971	I	Malignant Glioma (presurgical)	O6-BG monotherapy	Determine dose for tumor MGMT depletion	A dose of 100 mg/m² IV achieved complete tumor MGMT depletion for ≥18 hours. O6-BG alone was non-toxic.	25
Not Specified	I	Recurrent Malignant Glioma	O6-BG + Temozolomide (single dose)	MTD of Temozolomide	Established the MTD of a single dose of TMZ at 472 mg/m² when combined with O6-BG.	9
Not Specified	II	Recurrent, TMZ-Resistant Malignant Glioma (GBM and Anaplastic Glioma)	O6-BG + Temozolomide	Objective Response Rate (ORR)	Modest activity in anaplastic glioma (16% ORR), but no significant activity in GBM (3% ORR).	9
PBTC-016 (NCT00105822)	II	Recurrent Pediatric High-Grade and Brainstem Glioma	O6-BG + Temozolomide (5-day schedule)	Sustained Objective Response Rate	Did not meet the target response rate for activity. Primary toxicities were myelosuppression and GI symptoms.	36
NCT00086970	I	Unresectable, Metastatic Solid Tumors	Ifosfamide +/- O6-BG	MTD and Dose-Limiting Toxicities	Study was terminated; explored the combination with a different class of alkylating agent.	37
NCT00669669	I/II	Newly Diagnosed Glioblastoma or Gliosarcoma	O6-BG + TMZ/BCNU + Radiation + Gene-Modified (P140K MGMT) Stem Cell Transplant	Safety and feasibility of transduced stem cell infusion	Investigated a gene therapy approach to protect bone marrow from O6-BG-enhanced toxicity.	38
NCT00003766	II	Surgically Resectable Solid Tumors	O6-BG monotherapy (presurgical)	Determine minimal dose for tumor MGMT depletion	A dose-escalation study to define the optimal biologic dose across different solid tumor types.	39

Pharmacokinetics and Metabolism in Humans

The study of pharmacokinetics—how the body absorbs, distributes, metabolizes, and excretes a drug—is crucial for understanding its activity and safety profile. For 6-O-benzylguanine, the pharmacokinetic story is particularly important, as it is dominated by the rapid conversion of the parent drug into a more persistent and equally active metabolite.

Administration and Distribution

Due to its poor solubility in water, 6-O-benzylguanine is not suitable for oral administration and has been administered intravenously in all clinical trials.[4] Upon entering the bloodstream, it is distributed throughout the body, where it can act on MGMT in various tissues.

Rapid Metabolism to an Active Metabolite

A defining characteristic of 6-O-benzylguanine's behavior in humans is its extremely rapid clearance from the plasma. Pharmacokinetic studies in adult cancer patients have shown that the parent drug has a very short terminal half-life (t1/2​β), estimated to be around 26 minutes.[40] A similar rapid elimination was observed in pediatric patients, with a reported mean half-life of approximately 85 minutes.[21]

This rapid disappearance is not due to excretion but rather to extensive and swift metabolism into its major metabolite, O⁶-benzyl-8-oxoguanine (8-oxo-O6BG).[34] This oxidative reaction is primarily carried out by the cytochrome P450 enzyme system in the liver, with studies identifying CYP1A2 as the main enzyme responsible, and CYP3A4 playing a lesser role.[4] Critically, this metabolite is not an inactive breakdown product. Laboratory studies have confirmed that 8-oxo-O6BG is an equally potent inhibitor of the MGMT enzyme as the parent compound, 6-O-benzylguanine.[40]

Pharmacokinetics of the Active Metabolite (8-oxo-O6BG)

In stark contrast to the parent drug, the active metabolite 8-oxo-O6BG exhibits much more favorable pharmacokinetics for sustained biological activity. It has a significantly longer elimination half-life, which has been shown to increase with the dose of the parent drug, ranging from 2.8 hours at lower doses to 9.2 hours at higher doses in adults.[34] In pediatric patients, the mean terminal half-life of the metabolite was found to be approximately 6 hours (360 minutes), about four times longer than that of the parent drug.[21]

Consequently, both the peak plasma concentration (Cmax​) and the total systemic exposure (Area Under the Curve, or AUC) of 8-oxo-O6BG are substantially greater than those of 6-O-benzylguanine. Studies have reported that the Cmax​ of the metabolite is over twice as high, and the AUC can be anywhere from 12- to 29-fold greater than that of the parent compound.[34] The clearance of 8-oxo-O6BG also exhibits nonlinear kinetics; as the dose of 6-O-benzylguanine increases, the clearance of the metabolite decreases, leading to a disproportionate increase in its concentration and a prolongation of its elimination from the body.[34]

Pharmacodynamic Implications

This pharmacokinetic profile has profound implications for understanding the drug's pharmacodynamic effects. While 6-O-benzylguanine itself is only present in the plasma for a very short time, it effectively serves as a pro-drug for the more stable and persistent active agent, 8-oxo-O6BG. The prolonged and effective depletion of MGMT activity observed in patients for many hours after administration is therefore likely attributable primarily to the sustained exposure to high concentrations of the 8-oxo-O6BG metabolite, rather than the transient presence of the parent drug.[34]

Excretion

The elimination of 6-O-benzylguanine and its metabolites from the body does not occur significantly through the kidneys. Studies have shown that the urinary excretion of both the parent drug and 8-oxo-O6BG is minimal.[40]

Table 2: Summary of Key Pharmacokinetic Parameters for 6-O-benzylguanine and its Metabolite in Humans

Compound	Parameter	Population	Value (Mean ± SD or Range)	Source(s)
6-O-benzylguanine (O6-BG)	Terminal Half-life (t1/2​β)	Adult	26 ± 15 min	40
	Terminal Half-life (t1/2​)	Pediatric	85 ± 140 min	21
	Plasma Clearance	Adult	513 ± 148 mL/min/m²	40
	Plasma Clearance	Pediatric	760 ± 400 mL/min/m²	21
8-oxo-O6-benzylguanine (8-oxo-O6BG)	Terminal Half-life (t1/2​)	Adult	2.8 - 9.2 hours (dose-dependent)	34
	Terminal Half-life (t1/2​)	Pediatric	360 ± 220 min (~6 hours)	21
	Plasma Clearance	Pediatric	30 ± 15 mL/min/m²	21
	Relative Exposure (AUC)	Adult	12- to 29-fold > O6-BG	34
	Relative Peak Conc. (Cmax​)	Adult	2.2-fold > O6-BG	34

Safety, Toxicity, and Drug Interactions

The safety profile of 6-O-benzylguanine is defined by a critical dichotomy: it is relatively benign when administered alone but becomes a potent amplifier of toxicity when used in its intended therapeutic context, in combination with alkylating chemotherapy. This profile, along with its metabolism via the cytochrome P450 system, gives rise to a complex landscape of potential toxicities and drug-drug interactions.

Toxicity Profile

When administered as a monotherapy in Phase I clinical trials, 6-O-benzylguanine was found to be inherently non-toxic and was well-tolerated even at doses that achieved complete biological inhibition of MGMT.[27]

The severe toxicities associated with the drug emerge exclusively during combination therapy. As established by its mechanism of action, 6-O-benzylguanine systemically depletes the MGMT protein, which is essential for protecting rapidly dividing cells from the damaging effects of alkylating agents. Consequently, it dramatically potentiates the myelosuppressive effects of these drugs.[26] The primary and consistently observed dose-limiting toxicity in clinical trials combining 6-O-benzylguanine with temozolomide or BCNU is severe, Grade 4 hematologic toxicity, specifically

neutropenia (a dangerous drop in neutrophils) and thrombocytopenia (a dangerous drop in platelets).[9] This enhanced bone marrow toxicity was first predicted by preclinical animal studies [26] and was ultimately confirmed to be the principal obstacle to its successful clinical application.

Other clinically significant adverse events reported in combination therapy trials include febrile neutropenia (fever during a period of low neutrophil count, indicating a high risk of infection), infections, seizures, and thromboembolic events (blood clots).[9]

Hazard and Handling Information (for Research Use)

For laboratory and research applications, 6-O-benzylguanine is classified as a hazardous chemical substance according to the Globally Harmonized System (GHS). It is typically accompanied by a "Warning" signal word and is associated with the following hazard statements:

• H315: Causes skin irritation [1]
• H319: Causes serious eye irritation [1]
• H335: May cause respiratory irritation [1]

Some safety data sheets also include the hazard statement H302: Harmful if swallowed.[15] Due to these properties, standard safety precautions are required when handling the compound in a non-clinical setting. These include using the substance only in a well-ventilated area, avoiding the creation of dust, and wearing appropriate personal protective equipment (PPE), such as protective gloves and safety glasses or goggles.[43]

Drug-Drug Interactions

The metabolism of 6-O-benzylguanine via the cytochrome P450 system, particularly CYP1A2 and CYP3A4, makes it susceptible to a wide range of pharmacokinetic drug-drug interactions.[4] Co-administration with drugs that inhibit or induce these enzymes can alter the plasma concentrations of 6-O-benzylguanine and its active metabolite, potentially affecting both its efficacy and toxicity.

• Pharmacokinetic Interactions:
• CYP Inhibitors: Drugs that inhibit CYP1A2 or CYP3A4 can decrease the metabolism and clearance of 6-O-benzylguanine, leading to increased serum concentrations. Examples include abametapir, abiraterone, and allopurinol.[11]
• CYP Inducers: Conversely, drugs that induce these enzymes can increase the metabolism of 6-O-benzylguanine, leading to lower serum concentrations and potentially reduced efficacy. Examples include adalimumab, amobarbital, and apalutamide.[11]
• Metabolic Interactions: A large number of drugs can influence the rate of metabolism of 6-O-benzylguanine. Some may decrease its metabolism (e.g., acetaminophen, acenocoumarol, acyclovir, amiodarone), while others may increase it (e.g., abatacept, albendazole, anakinra).[11]
• Pharmacodynamic Interactions: Beyond metabolic effects, 6-O-benzylguanine can interact with other drugs at the level of their biological effect.
• It may decrease the therapeutic efficacy of certain medications, such as 1,2-benzodiazepines and adenosine.[11]
• It can increase the risk or severity of adverse effects when combined with other drugs. For example, co-administration with salbutamol (albuterol) can increase the risk of hypokalemia (low potassium levels), and combination with agents like acebutolol or amphetamine can increase the general risk of adverse effects.[11]

Table 4: Selected Drug-Drug Interactions with 6-O-benzylguanine

Interacting Drug	Type of Interaction	Description of Effect	Source
Abametapir	Pharmacokinetic	The serum concentration of 6-O-benzylguanine can be increased when it is combined with Abametapir (a CYP inhibitor).	11
Apalutamide	Pharmacokinetic	The serum concentration of 6-O-benzylguanine can be decreased when it is combined with Apalutamide (a CYP inducer).	11
Acetaminophen	Metabolic	The metabolism of 6-O-benzylguanine can be decreased when combined with Acetaminophen.	11
Abatacept	Metabolic	The metabolism of 6-O-benzylguanine can be increased when combined with Abatacept.	11
Adenosine	Pharmacodynamic	The therapeutic efficacy of Adenosine can be decreased when used in combination with 6-O-benzylguanine.	11
Alprazolam	Pharmacodynamic	The therapeutic efficacy of Alprazolam can be decreased when used in combination with 6-O-benzylguanine.	11
Acebutolol	Pharmacodynamic	The risk or severity of adverse effects can be increased when Acebutolol is combined with 6-O-benzylguanine.	11
Salbutamol (Albuterol)	Pharmacodynamic	The risk or severity of hypokalemia can be increased when 6-O-benzylguanine is combined with Salbutamol.	11
Ambroxol	Pharmacodynamic	The risk or severity of methemoglobinemia can be increased when 6-O-benzylguanine is combined with Ambroxol.	11

Application as a Research Tool in Molecular Biology

While 6-O-benzylguanine failed to achieve its primary goal as a therapeutic agent, its unique biochemical properties have secured it an enduring and prominent role as a research tool. The very characteristics that limited its clinical utility—its exquisite specificity and high potency for a single molecular target—are precisely what make it an ideal chemical probe for laboratory investigations. This has given the compound a remarkable "second life" that has had a lasting impact on basic and translational science.

Probing the Function of MGMT and DNA Repair

In molecular and cell biology, 6-O-benzylguanine is widely used as the gold-standard laboratory chemical to selectively and completely inhibit MGMT activity.[2] By treating cells or organisms with 6-O-benzylguanine, researchers can create a state of functional MGMT "knockout," allowing them to study the downstream consequences of the loss of this key repair protein. This application has been instrumental in elucidating the intricate details of DNA repair pathways. It has enabled scientists to investigate the cellular response to specific types of DNA damage, to map the interplay between the MGMT pathway and other repair systems like mismatch repair (MMR), and to explore the fundamental mechanisms of alkylation-induced mutagenesis and carcinogenesis.[5]

Development of Novel MGMT Assays and Imaging Agents

The specific, high-affinity, and covalent interaction between 6-O-benzylguanine and MGMT has been cleverly exploited to design new methods for detecting and quantifying the enzyme. For example, by synthesizing 6-O-benzylguanine with a radioactive tritium label ([³H]BG), researchers developed an assay to measure active MGMT levels in cell extracts or tumor biopsies. The assay works by incubating the sample with [³H]BG and then measuring the amount of radioactivity that becomes covalently attached to the MGMT protein.[47]

More recently, this principle has been extended to create sophisticated fluorescence-based probes. By attaching a "clickable" chemical handle (like a propargyl group) to a guanine analog, researchers can label active MGMT inside cells. After the transfer reaction, a fluorescent dye can be attached to the modified MGMT via a highly specific "click chemistry" reaction, allowing for the direct visualization and quantification of active MGMT using microscopy.[48] This enables dynamic studies of MGMT activity and inactivation in living cells.

Furthermore, this concept is being translated for in vivo clinical applications. Derivatives of 6-O-benzylguanine have been labeled with positron-emitting isotopes, such as Carbon-11 (11C) or Fluorine-18 (18F), to create probes for Positron Emission Tomography (PET) imaging. The goal of this research is to develop a non-invasive method to measure MGMT levels in a patient's tumor before treatment. Such a tool could be invaluable for personalizing medicine, allowing clinicians to predict which patients are most likely to respond to alkylating agent chemotherapy.[49]

Foundation for SNAP-tag and CLIP-tag Technology

Perhaps the most widespread and impactful legacy of 6-O-benzylguanine research is its role as the chemical foundation for the SNAP-tag and CLIP-tag protein labeling technologies.[12] These powerful tools are now staples in molecular biology laboratories around the world. The technology is based on a genetically engineered, mutant form of the human MGMT protein that acts as the "tag" (the SNAP-tag). This tag is genetically fused to a protein of interest that a researcher wishes to study.

The SNAP-tag protein retains its ability to react with 6-O-benzylguanine derivatives but has been optimized for this purpose. Researchers can then treat the cells expressing the fusion protein with a substrate consisting of an O⁶-benzylguanine molecule covalently linked to a probe of their choice, such as a fluorescent dye, a biotin molecule, or a bead. The SNAP-tag on the target protein will specifically and covalently react with the O⁶-benzylguanine substrate, thereby permanently attaching the probe to the protein of interest. This allows for highly specific, covalent, and versatile labeling of proteins in living cells for a vast array of applications, including fluorescence microscopy, protein localization studies, and pull-down experiments.[12]

The story of 6-O-benzylguanine serves as a powerful illustration of how a compound that fails in the clinic can nevertheless become a triumph for science. The journey of this molecule highlights that the value of a chemical probe is not always measured by its therapeutic index. The very properties that made 6-O-benzylguanine a problematic drug—its potent, specific, and irreversible inhibition of a single, vital protein—are the exact qualities that make it an exemplary research tool. While its on-target systemic toxicity prevented it from becoming a successful cancer therapy, its on-target specificity has provided researchers with an unparalleled instrument. This instrument has been used to dissect a fundamental DNA repair pathway, to design novel diagnostic assays, and, in a stroke of scientific ingenuity, to provide the chemical basis for a universal protein labeling technology that has enabled countless discoveries far beyond the original scope of oncology. In this way, a clinical failure was repurposed into a fundamental and enduring scientific success.

Regulatory Status and History

The regulatory and developmental history of 6-O-benzylguanine reflects its journey as a promising but ultimately unapproved therapeutic agent. Its status is firmly in the investigational realm, and its timeline of development can be traced through key publications and clinical trial registrations.

Regulatory Status

6-O-benzylguanine is an investigational drug. It has not been approved for commercial marketing as a therapeutic agent by the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA). Its use in humans has been restricted to the context of formal clinical trials.

As part of its development, it was assigned an Investigational New Drug (IND) application number by the National Cancer Institute: 45789.[6] An IND is a submission to the FDA that is required before a new drug can be administered to human subjects. It allows the sponsor to legally ship an unapproved drug across state lines to clinical investigators for the purpose of conducting clinical trials.[50] The existence of an IND and the progression of 6-O-benzylguanine through multiple phases of clinical testing—including Phase I, Phase II, and up to Phase III trials for some indications—confirm its status as a significant investigational agent.[1] However, the data generated from these trials have not been sufficient to support a New Drug Application (NDA), which is the formal request for FDA approval to market a new drug.[53]

Historical Development Timeline

The scientific and clinical history of 6-O-benzylguanine can be broadly divided into distinct periods of discovery, preclinical validation, clinical evaluation, and subsequent repurposing.

• Early 1990s: Foundational Discovery and Mechanistic Studies. This period saw the initial reports on 6-O-benzylguanine as a potent inactivator of MGMT. Foundational research published between 1990 and 1994 detailed its mechanism of action as a suicide inhibitor, demonstrated its ability to rapidly deplete cellular MGMT activity, and provided the first evidence of its capacity to sensitize cancer cells to alkylating agents in vitro. Early investigations into its metabolism and the synthesis of various chemical analogues also began during this time.[8]
• Mid-to-Late 1990s: Preclinical Validation and Entry into the Clinic. Building on the strong mechanistic foundation, the mid-to-late 1990s were characterized by extensive preclinical validation in animal xenograft models, which consistently showed significant enhancement of antitumor activity. This success led to the initiation of the first Phase I clinical trials in humans. Key publications from 1997-1998 described the role of 6-O-benzylguanine in chemotherapy and reported the initial clinical findings, including the determination of the biologically active dose required for tumor MGMT depletion.[4] Parallel research also began to explore innovative strategies to counteract its toxicity, such as the creation of 6-O-benzylguanine-resistant MGMT mutants for gene therapy applications.[56]
• 2000s: Pivotal Clinical Efficacy Trials. This decade was marked by the execution of larger and more definitive clinical trials aimed at evaluating the therapeutic efficacy of 6-O-benzylguanine in combination with chemotherapy. Phase I/II and Phase II trials, particularly in patients with recurrent malignant glioma, were completed. Pharmacokinetic studies were also extended to pediatric populations.[21] However, the results from these pivotal trials, especially the 2009 publication detailing the lack of efficacy in glioblastoma, were largely disappointing and signaled the end of its mainstream clinical development as a chemosensitizer.[9]
• 2010s to Present: Repurposing and Legacy Applications. With its path as a therapeutic agent effectively closed, research in the last decade has shifted to capitalize on its value as a scientific tool. This period has seen the continued refinement and use of 6-O-benzylguanine-based technologies, including its application in developing PET imaging agents for non-invasive MGMT detection and its central role in advanced gene therapy strategies aimed at protecting hematopoietic stem cells.[12] Its use as a standard laboratory reagent to study DNA repair remains a constant.

Conclusion and Future Outlook

The comprehensive evaluation of 6-O-benzylguanine reveals a molecule with a rich and instructive history, embodying both the promise of rational drug design and the formidable challenges of translating a potent biological mechanism into a safe and effective therapy. Its journey from a promising chemosensitizer to an indispensable research tool offers critical lessons for the field of oncology and drug development.

Summary of 6-O-benzylguanine's Journey

6-O-benzylguanine was conceived from a clear understanding of a key cancer resistance mechanism: the DNA repair protein MGMT. Its design as a potent, irreversible suicide inhibitor was a triumph of medicinal chemistry, and its ability to dramatically enhance the efficacy of alkylating agents in preclinical models was unequivocal. This created enormous optimism for its potential to improve outcomes for patients with aggressive, treatment-resistant cancers like glioblastoma. However, this preclinical promise did not translate into clinical success. The pivotal clinical trials revealed that the drug's powerful mechanism was a double-edged sword, leading to an unacceptable level of on-target toxicity in healthy tissues that ultimately negated its therapeutic potential.

The Unresolved Challenge of On-Target Toxicity

The story of 6-O-benzylguanine serves as a powerful case study on the challenge of on-target toxicity. Unlike off-target toxicities, which can sometimes be engineered out of a molecule, the dose-limiting myelosuppression seen with 6-O-benzylguanine was a direct and unavoidable consequence of its intended mechanism of action. Because MGMT is a ubiquitously expressed and fundamentally protective enzyme, its systemic inhibition rendered all rapidly dividing cells, particularly those in the bone marrow, vulnerable to the co-administered chemotherapy. This experience underscores a crucial principle in drug development: for therapies that target fundamental cellular defense mechanisms, achieving a sufficient therapeutic window—a dose that is toxic to the tumor but tolerable for the patient—can be exceedingly difficult without a strategy to selectively protect healthy tissues.

Future Directions and the Legacy of O6-BG

While the systemic administration of 6-O-benzylguanine as a simple chemosensitizer is no longer a viable strategy, its failure has directly inspired more sophisticated and targeted therapeutic approaches that aim to solve the problem of host toxicity.

• Advanced Therapeutic Strategies: The most promising of these next-generation strategies involves the use of gene therapy to "shield" the bone marrow. This approach entails harvesting a patient's hematopoietic stem cells, genetically modifying them ex vivo to express a mutant form of MGMT (such as P140K or G156A) that is resistant to inhibition by 6-O-benzylguanine, and then re-infusing these protected stem cells into the patient.[4] In theory, this would create a patient whose bone marrow is resistant to the sensitizing effects of 6-O-benzylguanine, while their tumor remains sensitive. This could reopen the therapeutic window, allowing for the safe administration of higher, more therapeutically effective doses of both 6-O-benzylguanine and the alkylating agent. Clinical trials exploring this very concept are underway and represent the most direct and hopeful evolution of the original therapeutic idea.[12]
• Biomarker Development: The legacy of 6-O-benzylguanine also continues in the field of diagnostics. The ongoing development of PET imaging agents based on its structure holds the potential to provide a non-invasive, real-time method for quantifying MGMT protein levels in patient tumors.[49] Such a tool would be invaluable for patient stratification, helping to identify individuals most likely to benefit from alkylating agents and potentially guiding the use of future MGMT-targeted therapies.
• Enduring Value as a Research Standard: Ultimately, the most certain and impactful legacy of 6-O-benzylguanine is its secure place in the toolkit of biomedical research. It remains the undisputed gold-standard inhibitor for studying the function of MGMT and has provided the chemical blueprint for powerful technologies like SNAP-tag that have enabled discoveries across countless fields of biology. While it may never be a standard-of-care cancer drug in its original form, its contribution to our fundamental understanding of DNA repair and its role in spawning new research technologies ensure that its impact on science will be both profound and lasting.

Works cited

1. 6-(Phenylmethoxy)-9H-purin-2-amine | C12H11N5O | CID 4578 - PubChem, accessed September 2, 2025, https://pubchem.ncbi.nlm.nih.gov/compound/4578
2. O6-Benzylguanine - Wikipedia, accessed September 2, 2025, https://en.wikipedia.org/wiki/O6-Benzylguanine
3. Regulation of expression of O6-methylguanine-DNA methyltransferase and the treatment of glioblastoma (Review) - Spandidos Publications, accessed September 2, 2025, https://www.spandidos-publications.com/10.3892/ijo.2015.3026
4. (PDF) O6-Benzylguanine and its role in chemotherapy - ResearchGate, accessed September 2, 2025, https://www.researchgate.net/publication/13470435_O6-Benzylguanine_and_its_role_in_chemotherapy
5. The O6-Methyguanine-DNA Methyltransferase Inhibitor O6-Benzylguanine Enhanced Activity of Temozolomide + Irinotecan Against Models of High-Risk Neuroblastoma - PMC, accessed September 2, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC9255907/
6. Definition of O6-benzylguanine - NCI Drug Dictionary - NCI, accessed September 2, 2025, https://www.cancer.gov/publications/dictionaries/cancer-drug/def/o6-benzylguanine
7. O6-Methylguanine-DNA Methyltransferase, O6-Benzylguanine, and ..., accessed September 2, 2025, https://aacrjournals.org/clincancerres/article/11/7/2747/188190/O6-Methylguanine-DNA-Methyltransferase-O6
8. Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents. | PNAS, accessed September 2, 2025, https://www.pnas.org/doi/10.1073/pnas.87.14.5368
9. Phase II Trial of Temozolomide Plus O6-Benzylguanine in Adults With Recurrent, Temozolomide-Resistant Malignant Glioma - PMC - PubMed Central, accessed September 2, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC2667825/
10. Applied molecular evolution of O6-benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells | PNAS, accessed September 2, 2025, https://www.pnas.org/doi/10.1073/pnas.091601198
11. 6-O-benzylguanine: Uses, Interactions, Mechanism of Action ..., accessed September 2, 2025, https://go.drugbank.com/drugs/DB11919
12. Benzylguanine - Drug Targets, Indications, Patents - Patsnap Synapse, accessed September 2, 2025, https://synapse.patsnap.com/drug/5eafd8cc966149eb994ab47e9ad573c9
13. O6-Benzylguanine | CAS 19916-73-5 | SCBT, accessed September 2, 2025, https://www.scbt.com/p/o6-benzylguanine-19916-73-5
14. O6-Benzylguanine | 99.91%(HPLC) | In Stock | DNA alkylator inhibitor - Selleck Chemicals, accessed September 2, 2025, https://www.selleckchem.com/products/o6-benzylguanine.html
15. O~6~-Benzylguanine 19916-73-5 | Tokyo Chemical Industry (India) Pvt. Ltd., accessed September 2, 2025, https://www.tcichemicals.com/IN/en/p/B4208
16. O6-Benzylguanine =98 TLC,solid 19916-73-5 - Sigma-Aldrich, accessed September 2, 2025, https://www.sigmaaldrich.com/US/en/product/sigma/b2292
17. Multifaceted roles of alkyltransferase and related proteins in DNA repair, DNA damage, resistance to chemotherapy, and research tools - Penn State, accessed September 2, 2025, https://pure.psu.edu/en/publications/multifaceted-roles-of-alkyltransferase-and-related-proteins-in-dn
18. O 6 -Methylguanine-DNA methyltransferase inactivation and chemotherapy - Oxford Academic, accessed September 2, 2025, https://academic.oup.com/bmb/article/85/1/17/290808
19. The Versatile Attributes of MGMT: Its Repair Mechanism, Crosstalk with Other DNA Repair Pathways, and Its Role in Cancer - PubMed Central, accessed September 2, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC10814553/
20. Regulatory mechanisms of O6-methylguanine methyltransferase expression in glioma cells, accessed September 2, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC12174773/
21. Pharmacokinetics of O-benzylguanine in Pediatric Patients with Central Nervous System Tumors | Request PDF - ResearchGate, accessed September 2, 2025, https://www.researchgate.net/publication/8414497_Pharmacokinetics_of_O-benzylguanine_in_Pediatric_Patients_with_Central_Nervous_System_Tumors
22. O6-Methylguanine-DNA Methyltransferase (MGMT): Challenges and New Opportunities in Glioma Chemotherapy - Frontiers, accessed September 2, 2025, https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2019.01547/full
23. New Gene Therapy Clinical Trial for Patients Newly Diagnosed with Glioma, accessed September 2, 2025, https://braintumorcenter.ucsf.edu/news/new-gene-therapy-clinical-trial-patients-newly-diagnosed-glioma
24. O6-Benzylguanine | MGMT Inhibitor | MedChemExpress, accessed September 2, 2025, https://www.medchemexpress.com/o6-benzylguanine.html
25. Phase I trial of O6-benzylguanine for patients undergoing surgery for malignant glioma - PubMed, accessed September 2, 2025, https://pubmed.ncbi.nlm.nih.gov/9817277/
26. O6-benzylguanine potentiates the in vivo toxicity and clastogenicity of temozolomide and BCNU in mouse bone marrow - PubMed, accessed September 2, 2025, https://pubmed.ncbi.nlm.nih.gov/9057638/
27. The therapeutic potential of O 6 -alkylguanine DNA alkyltransferase inhibitors, accessed September 2, 2025, https://www.tandfonline.com/doi/abs/10.1517/13543784.16.10.1573
28. O6-methylguanine DNA methyltransferase (MGMT) expression in U1242 glioblastoma cells enhances in vitro clonogenicity, tumor implantation in vivo, and sensitivity to alisertib-carboplatin combination treatment - Frontiers, accessed September 2, 2025, https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2025.1552015/full
29. (PDF) The O6-methyguanine-DNA methyltransferase inhibitor O6-benzylguanine enhanced activity of temozolomide + irinotecan against models of high-risk neuroblastoma - ResearchGate, accessed September 2, 2025, https://www.researchgate.net/publication/347410061_The_O6-methyguanine-DNA_methyltransferase_inhibitor_O6-benzylguanine_enhanced_activity_of_temozolomide_irinotecan_against_models_of_high-risk_neuroblastoma
30. O6-benzylguanine and its role in chemotherapy - PubMed, accessed September 2, 2025, https://pubmed.ncbi.nlm.nih.gov/9815757/
31. PRE SURGICAL O6 BENZYLGUANINE TREATMENT OF PTS W/ MALIGNANT GLIOMA, accessed September 2, 2025, https://pure.psu.edu/en/projects/pre-surgical-o6-benzylguanine-treatment-of-pts-w-malignant-glioma-2
32. O(6)-Benzylguanine in Treating Patients With Malignant Glioma | ClinicalTrials.gov, accessed September 2, 2025, https://clinicaltrials.gov/study/NCT00002971
33. Phase I trial of O6-benzylguanine for patients undergoing surgery for malignant glioma., accessed September 2, 2025, https://ascopubs.org/doi/10.1200/JCO.1998.16.11.3570
34. O6-benzylguanine in humans: metabolic, pharmacokinetic, and ..., accessed September 2, 2025, https://ascopubs.org/doi/10.1200/JCO.1998.16.5.1803
35. Phase I trial of single-dose temozolomide and continuous administration of O>6>-benzylguanine in children with brain tumors, accessed September 2, 2025, https://utsouthwestern.elsevierpure.com/en/publications/phase-i-trial-of-single-dose-temozolomide-and-continuous-administ
36. A Phase II Study of O6-Benzylguanine and Temozolomide in Pediatric Patients with Recurrent or Progressive High Grade Gliomas and Brainstem Gliomas - PubMed Central, accessed September 2, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC3518022/
37. 6-O-benzylguanine Terminated Phase 1 Trials for Unspecified Adult Solid Tumor, Protocol Specific Treatment - DrugBank, accessed September 2, 2025, https://go.drugbank.com/drugs/DB11919/clinical_trials?conditions=DBCOND0028453&phase=1&purpose=treatment&status=terminated
38. O6-Benzylguanine-Mediated Tumor Sensitization With Chemoprotected Autologous Stem Cell in Treating Patients With Malignant Gliomas - Meghan M Johnson Memorial Scholarship Fund - Clinical Trial Finder, accessed September 2, 2025, https://meghan-johnson-scholarship-fund.clinicaltrialconnect.com/trials/NCT00669669
39. O6-benzylguanine Followed by Surgery in Treating Patients With Solid Tumors That Can Be Removed During Surgery | ClinicalTrials.gov, accessed September 2, 2025, https://clinicaltrials.gov/study/NCT00003766
40. Pharmacokinetics of O6-benzylguanine (NSC637037) and its metabolite, 8-oxo-O6 ... - PubMed, accessed September 2, 2025, https://pubmed.ncbi.nlm.nih.gov/12953345/
41. Metabolism of O6-Benzylguanine, an Inactivator of O6-Alkylguanine-DNA Alkyltransferase1, accessed September 2, 2025, https://aacrjournals.org/cancerres/article/54/19/5123/500330/Metabolism-of-O6-Benzylguanine-an-Inactivator-of
42. O6-benzylguanine in humans: metabolic, pharmacokinetic, and pharmacodynamic findings - PubMed, accessed September 2, 2025, https://pubmed.ncbi.nlm.nih.gov/9586894/
43. SAFETY DATA SHEET - Sigma-Aldrich, accessed September 2, 2025, https://www.sigmaaldrich.com/BE/en/sds/sigma/b2292
44. SAFETY DATA SHEET, accessed September 2, 2025, https://www.sigmaaldrich.cn/CN/en/sds/sigma/b2292
45. 6-O-Benzylguanine - Safety Data Sheet - ChemicalBook, accessed September 2, 2025, https://www.chemicalbook.com/msds/6-o-benzylguanine.htm
46. O6-Benzylguanine | STEMCELL Technologies, accessed September 2, 2025, https://www.stemcell.com/products/o6-benzylguanine.html
47. Development of an O6-alkylguanine—DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O6-benzylguanine to the protein - PMC, accessed September 2, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC2773450/
48. A ”Clickable” Probe for Active MGMT in Glioblastoma Demonstrates ..., accessed September 2, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC7072665/
49. WO2018008311A1 - 11c-labelled o6-benzylguanine, positron emission tomography (pet) probe capable of visualizing o6-methyl guanine methyl-transferase activity, and production methods therefor - Google Patents, accessed September 2, 2025, https://patents.google.com/patent/WO2018008311A1/en
50. Investigational New Drug Applications (INDs) for CBER-Regulated Products | FDA, accessed September 2, 2025, https://www.fda.gov/vaccines-blood-biologics/development-approval-process-cber/investigational-new-drug-applications-inds-cber-regulated-products
51. Investigational New Drug (IND) Application - FDA, accessed September 2, 2025, https://www.fda.gov/drugs/types-applications/investigational-new-drug-ind-application
52. 21 CFR Part 312 -- Investigational New Drug Application - eCFR, accessed September 2, 2025, https://www.ecfr.gov/current/title-21/chapter-I/subchapter-D/part-312
53. New Drug Application (NDA) - FDA, accessed September 2, 2025, https://www.fda.gov/drugs/types-applications/new-drug-application-nda
54. Substituted O6-Benzylguanine Derivatives and Their Inactivation of Human O6-Alkylguanine-DNA Alkyltransferase | Journal of Medicinal Chemistry - ACS Publications, accessed September 2, 2025, https://pubs.acs.org/doi/10.1021/jm00029a005
55. Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine | Biochemistry - ACS Publications, accessed September 2, 2025, https://pubs.acs.org/doi/10.1021/bi00096a009
56. Creation of Human Alkyltransferases Resistant to O6-Benzylguanine1 - AACR Journals, accessed September 2, 2025, https://aacrjournals.org/cancerres/article/57/10/2007/503167/Creation-of-Human-Alkyltransferases-Resistant-to