4-Hydroxycoumarin — ≥99% HPLC, Key Pharmaceutical Intermediate for Anticoagulant Drug Synthesis (Warfarin Precursor)
4-Hydroxycoumarin (CAS 1076-38-6, 4-Hydroxy-2H-chromen-2-one, C₉H₆O₃, MW 162.14 g/mol) — the core heterocyclic scaffold of the most prescribed class of oral anticoagulants in global medicine. Every 4-hydroxycoumarin anticoagulant — warfarin (Coumadin, 20+ million US prescriptions/year), dicoumarol, acenocoumarol, phenprocoumon, brodifacoum — is built upon this single chemical nucleus. The 4-hydroxy group is the essential pharmacophore: it mimics the naphthoquinone of vitamin K, competitively binding VKORC1 (vitamin K epoxide reductase) and blocking the vitamin K recycling cycle — preventing gamma-carboxylation of clotting factors II, VII, IX, X and depleting functional coagulation proteins. Discovered through one of the most storied investigations in pharmaceutical history — the sweet clover disease epidemic (1920s Wisconsin) → dicoumarol isolation (1939, Campbell & Link) → warfarin synthesis (1948, Wisconsin Alumni Research Foundation — WARF-arin) → President Eisenhower’s 1955 treatment → global anticoagulation standard. ISO 9001:2015, ISO 22000, HACCP certified. Bulk manufacturer and wholesale supplier — premium 4-hydroxycoumarin from UPOR Biotech.
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4-Hydroxycoumarin (IUPAC: 4-Hydroxy-2H-chromen-2-one, also known as 4-Hydroxy-2H-1-benzopyran-2-one, Benzotetronic acid, CAS 1076-38-6, C₉H₆O₃, MW 162.14 g/mol) is a heterocyclic organic compound that serves as the foundational chemical scaffold for the most prescribed class of oral anticoagulant drugs in global medicine — the 4-hydroxycoumarin anticoagulants. This single molecular core has produced warfarin (Coumadin), dicoumarol, acenocoumarol (Sintrom), phenprocoumon (Marcoumar), and the super-warfarin rodenticides (brodifacoum, difenacoum, bromadiolone) — a family of drugs that has prevented millions of strokes, pulmonary embolisms, and thromboembolic deaths over 70+ years of clinical use. The molecule consists of a benzopyran-2-one (coumarin) core with a hydroxyl (-OH) group at the C4 position. This 4-hydroxy substituent is pharmacologically essential and structurally non-negotiable: it is the pharmacophore that enables competitive binding to VKORC1 (vitamin K epoxide reductase complex subunit 1) by mimicking the 2-methyl-1,4-naphthoquinone core of vitamin K. The 4-OH group forms critical hydrogen bonds with the catalytic cysteine residues (Cys132, Cys135 in the CXXC redox center) of VKORC1, while the coumarin aromatic ring engages in pi-pi stacking with Tyr139 in the active site — together blocking the reduction of vitamin K 2,3-epoxide (KO) back to vitamin K quinone (K) and subsequently to the active hydroquinone (KH₂) cofactor. Without KH₂, gamma-glutamyl carboxylase (GGCX) cannot carboxylate glutamic acid residues on clotting factors II (prothrombin), VII, IX, X or regulatory proteins C and S — newly synthesized factors are secreted as non-functional descarboxy-proteins (PIVKA — proteins induced by vitamin K absence/antagonism), and as pre-existing functional factors clear from circulation (half-lives: VII ~6h, IX ~24h, X ~40h, II ~60h), the coagulation cascade is progressively impaired, producing the therapeutic anticoagulant effect monitored by PT/INR. The 4-hydroxy group also confers chemical reactivity critical for drug synthesis: the C3 position (adjacent to the enolic 4-OH) is nucleophilic due to keto-enol tautomerism (4-hydroxycoumarin exists predominantly in the 4-hydroxy enol form, stabilized by intramolecular hydrogen bonding with the C2 carbonyl), enabling Michael addition reactions with alpha,beta-unsaturated ketones — the key synthetic step that builds warfarin from 4-hydroxycoumarin + benzalacetone.
The history of 4-hydroxycoumarin is one of the most remarkable stories in pharmaceutical science — a chain of discovery spanning agricultural tragedy, biochemical detective work, academic-industry partnership, and clinical transformation. In the early 1920s, cattle across the northern United States and Canada began dying from a mysterious hemorrhagic disease after consuming moldy sweet clover hay (Melilotus alba and M. officinalis). The condition — known as ‘sweet clover disease’ — caused cattle to hemorrhage uncontrollably from minor wounds, develop massive subcutaneous hematomas, and die from internal bleeding after routine veterinary procedures. In February 1933, a desperate Wisconsin farmer named Ed Carlson drove through a blizzard to the University of Wisconsin-Madison with a dead cow, a milk can filled with blood that would not clot, and 100 pounds of moldy sweet clover hay. He found his way to the laboratory of Dr. Karl Paul Link, an associate professor of agricultural chemistry. Link assigned the problem to his research group, and after 6 years of painstaking isolation work — processing tons of spoiled hay, developing bioassays using rabbits, and employing the newly available technique of column chromatography — Link’s postdoctoral fellow Dr. Harold A. Campbell finally crystallized the hemorrhagic agent in 1939. The compound was dicoumarol (3,3′-methylenebis(4-hydroxycoumarin)), formed by the fungal oxidation of coumarin (naturally present in sweet clover) to 4-hydroxycoumarin by Aspergillus and Penicillium molds, followed by formaldehyde-mediated dimerization. Campbell identified the structure by degradation studies: acid hydrolysis yielded two molecules of 4-hydroxycoumarin, confirming the dimeric bis(4-hydroxycoumarin) structure. Dicoumarol was the first oral anticoagulant ever tested in humans (1941, Mayo Clinic) and was used clinically through the 1940s-1950s, though it had a slow onset and unpredictable pharmacokinetics. Building on this discovery, Link and his team synthesized a series of 4-hydroxycoumarin derivatives, systematically varying the C3 substituent. Compound #42 — synthesized by condensing 4-hydroxycoumarin with benzalacetone — showed the most favorable potency, water solubility as the sodium salt, and pharmacokinetic profile. It was named warfarin, an acronym from WARF (Wisconsin Alumni Research Foundation, which funded and patented the research) + -arin (from coumarin). Warfarin was patented in 1947 and first marketed as a rodenticide in 1948. The transition to human medicine came through an unlikely route: in 1951, a US Army inductee attempted suicide by consuming warfarin rodenticide but was found, treated with vitamin K and blood transfusions, and fully recovered — demonstrating warfarin’s safety and pharmacological reversibility at therapeutic doses. Clinical trials followed, and after President Dwight D. Eisenhower received warfarin following his 1955 myocardial infarction, it became the global standard for oral anticoagulation. At its peak, warfarin was the #1 prescribed oral anticoagulant worldwide with 20+ million US prescriptions annually. The 4-hydroxycoumarin scaffold thus represents one of the most impactful chemical structures in medical history — transforming cardiovascular medicine and preventing millions of premature deaths from thromboembolic disease.
As a leading 4-hydroxycoumarin manufacturer and bulk supplier, UPOR Biotech provides high-purity 4-hydroxycoumarin (≥99% HPLC, Pharma Intermediate Grade) for B2B pharmaceutical API manufacturers, contract research organizations (CROs), academic medicinal chemistry laboratories, and fine chemical distributors worldwide. 4-Hydroxycoumarin is the key starting material (KSM) for warfarin sodium and warfarin sodium clathrate API synthesis — the global warfarin API market produces over 100 metric tons annually, serving millions of patients requiring long-term anticoagulation for atrial fibrillation, venous thromboembolism, mechanical heart valve thromboprophylaxis, and antiphospholipid syndrome. Beyond warfarin, 4-hydroxycoumarin is the common intermediate for acenocoumarol, phenprocoumon, and multiple second-generation anticoagulant rodenticides (brodifacoum, difenacoum, bromadiolone, flocoumafen) — collectively a multi-thousand-ton annual market for pest control and crop protection. Additionally, 4-hydroxycoumarin serves as the precursor for fluorescent probe chemistry: 7-hydroxy-4-methylcoumarin (4-methylumbelliferone) and its derivatives are among the most widely used fluorogenic substrates in biochemistry — their fluorescence is quenched when the 7-OH is conjugated (e.g., to phosphate, sulfate, or glycoside) and restored upon enzymatic cleavage, enabling real-time fluorescent detection of phosphatase, sulfatase, glycosidase, and esterase activities. OEM and custom synthesis of 4-hydroxycoumarin derivatives available with flexible MOQ. Free sample (5-10g) available for qualified B2B buyers. Every shipment includes full quality documentation: COA, MSDS, HPLC chromatogram, and regulatory certificates.
The 4-Hydroxycoumarin Anticoagulant Family — From Sweet Clover Disease to the #1 Prescribed Oral Anticoagulant Globally: Why the 4-OH Pharmacophore Is Irreplaceable for VKORC1 Inhibition
Every 4-hydroxycoumarin anticoagulant drug traces its origin to a single chemical core — and a single historic investigation. The 4-hydroxy group is the essential pharmacophore: it mimics the 2-methyl-1,4-naphthoquinone of vitamin K, binding VKORC1 through hydrogen bonds with Cys132/Cys135 and pi-pi stacking with Tyr139 — competitively inhibiting vitamin K epoxide reduction and depleting active KH₂ cofactor. Without KH₂, GGCX cannot gamma-carboxylate clotting factors → non-functional PIVKA proteins are secreted → functional factors clear according to their half-lives (VII 6h, IX 24h, X 40h, II 60h) → PT/INR rises → therapeutic anticoagulation. The six major drugs derived from this scaffold: (1) Warfarin (Coumadin) — 4-hydroxycoumarin + benzalacetone Michael adduct. #1 oral anticoagulant globally: 20+ million US Rx/year, 100+ million worldwide. Racemic (S-warfarin 3-5× more potent, metabolized by CYP2C9 — the basis for pharmacogenetic dosing guidelines). Named WARF-arin for Wisconsin Alumni Research Foundation (1948). (2) Dicoumarol (Dicumarol) — the natural bis(4-hydroxycoumarin) isolated from moldy sweet clover (1939, Campbell & Link). The molecule that started it all — sweet clover disease → fungal oxidation of coumarin → 4-hydroxycoumarin → formaldehyde dimerization → dicoumarol. First oral anticoagulant used in humans (1941, Mayo Clinic). (3) Acenocoumarol (Sintrom, Sinthrome) — 4-hydroxycoumarin + 4-nitrobenzalacetone. Widely used in Europe/Asia. Shorter t½ (~8-11h). CYP2C9-dependent metabolism. (4) Phenprocoumon (Marcoumar, Marcumar, Falithrom) — 4-hydroxycoumarin + 1-phenyl-1-buten-3-one. Longest t½ (~160h, 5-6 days). Preferred in Germany/Netherlands for stable long-term anticoagulation. Multi-CYP metabolism reduces pharmacogenetic sensitivity. (5) Brodifacoum — ‘Super-Warfarin’ Rodenticide — 4-hydroxycoumarin + 3-(4′-bromobiphenyl-4-yl)-1,2,3,4-tetrahydro-1-naphthyl-benzalacetone. Extended lipophilic side chain increases VKORC1 affinity ~100× and hepatic half-life to 60-120+ days (vs warfarin 1-2 days). A single bait block contains enough anticoagulant for 30+ days of effect in a 70 kg human. Treatment requires prolonged high-dose vitamin K1 (50-200 mg/day for weeks to months). (6) Difenacoum, Bromadiolone, Flocoumafen — additional second-generation 4-hydroxycoumarin rodenticides with varying potency and species selectivity, all sharing the same VKORC1 inhibition mechanism. The unifying chemical principle: the 4-hydroxycoumarin nucleus is irreplaceable. The 4-OH group establishes the essential VKORC1 hydrogen bond network. The lactone carbonyl at C2 positions the molecule in the active site. The C3 substituent modulates potency, half-life, and species selectivity. Modify the 4-OH and anticoagulant activity is abolished entirely — a structure-activity relationship validated across 80+ years of medicinal chemistry. From a Wisconsin dairy farm in 1933 to the most prescribed anticoagulant on Earth — the 4-hydroxycoumarin story is one of the most consequential drug discoveries in history, and its pharmacophore remains as essential today as when Campbell first crystallized dicoumarol from moldy hay.
Technical Specifications
| Property | Specification |
|---|---|
| Product Name | 4-Hydroxycoumarin — ≥99% HPLC, Key Pharmaceutical Intermediate for Anticoagulant Drug Synthesis |
| IUPAC Name | 4-Hydroxy-2H-chromen-2-one; 4-Hydroxy-2H-1-benzopyran-2-one |
| Common Synonyms | 4-Hydroxycoumarin; 4-Hydroxy-2H-chromen-2-one; 4-Hydroxy-2H-1-benzopyran-2-one; 4-Hydroxy-2-oxo-2H-chromene; Benzotetronic acid; 4-Hydroxy-2H-benzo[b]pyran-2-one; 4-Coumarinol; Warfarin precursor; 4-Hydroxycoumarin anticoagulant scaffold |
| CAS Number | 1076-38-6 |
| EINECS | 214-060-2 |
| Molecular Formula | C₉H₆O₃ |
| Molecular Weight | 162.14 g/mol |
| Chemical Class | Heterocyclic compound — benzopyran-2-one (coumarin) derivative; enolic beta-keto lactone; vinylogous carboxylic acid (4-OH is acidically activated by conjugation with the C2 carbonyl). The 4-hydroxy group renders the C3 position nucleophilic via keto-enol tautomerism — the key reactivity exploited in warfarin and all 4-hydroxycoumarin anticoagulant syntheses. |
| Pharmacophore | The 4-hydroxy group is the essential pharmacophore for VKORC1 inhibition — it mimics the 2-methyl-1,4-naphthoquinone core of vitamin K, forming critical hydrogen bonds with VKORC1 active-site cysteine residues (Cys132, Cys135 in the CXXC redox motif) while the coumarin aromatic ring engages in pi-pi stacking with Tyr139. Removal, methylation, or acylation of the 4-OH abolishes anticoagulant activity completely — the fundamental structure-activity relationship established across 80+ years of 4-hydroxycoumarin medicinal chemistry. |
| Key Synthetic Reactivity | Keto-enol tautomerism (4-hydroxy enol form predominates — stabilized by intramolecular H-bond with C2 carbonyl oxygen). The enolic C3 carbon is activated toward electrophilic attack — the key synthetic transformation: 4-hydroxycoumarin + alpha,beta-unsaturated ketone (e.g., benzalacetone) → Michael addition at C3 → warfarin or warfarin analog. Also reacts with aldehydes to form bis(4-hydroxycoumarin) derivatives (e.g., dicoumarol from formaldehyde). |
| pKa | 4-OH: pKa₁ ~4.1 (enolic hydroxyl — relatively acidic due to vinylogous carboxylic acid character with resonance stabilization of the conjugate base through the C2 carbonyl). pKa₂ (lactone ring opening) ~9.5. The acidity of the 4-OH is critical for forming water-soluble sodium salts of 4-hydroxycoumarin anticoagulants (warfarin sodium, pKa of warfarin enol ~5.0). |
| Appearance | White to off-white crystalline powder; free-flowing |
| Assay — Pharma Intermediate Grade | ≥99.0% (HPLC, anhydrous basis, UV detection at 280-310 nm) |
| Assay — Research Grade | ≥98.0% (HPLC, anhydrous basis) |
| Identification — HPLC | Retention time matches 4-hydroxycoumarin reference standard (USP/EP); single dominant peak with symmetrical peak shape (USP tailing factor ≤2.0) |
| Identification — UV-Vis | Characteristic coumarin chromophore: λmax ~280-310 nm (pi → pi* transition of the benzopyran-2-one system). The enolic 4-OH causes a bathochromic shift vs unsubstituted coumarin (λmax ~275 nm) due to extended conjugation. Absorbance ratio A₂₈₀/A₃₁₀ conforms to reference standard. |
| Identification — FT-IR | Characteristic bands: lactone C=O stretch ~1700-1720 cm⁻¹ (strong); enolic O-H stretch ~3100-3400 cm⁻¹ (broad, hydrogen-bonded); aromatic C=C ring stretch ~1600-1610 cm⁻¹; C-O (lactone) stretch ~1240-1260 cm⁻¹; aromatic C-H out-of-plane bending ~750-770 cm⁻¹ (characteristic of ortho-disubstituted benzene in the coumarin ring) |
| Identification — ¹H-NMR (DMSO-d₆) | δ 5.68 (1H, s, H-3 — the vinylic C3 proton, characteristic singlet of the 4-hydroxycoumarin enol tautomer); δ 7.30-7.42 (2H, m, H-6 and H-8); δ 7.65-7.72 (1H, m, H-7); δ 7.94 (1H, dd, J = 8.0, 1.5 Hz, H-5); δ ~12.6 (1H, broad s, 4-OH — enolic proton, exchangeable with D₂O, downfield shifted due to strong intramolecular H-bonding with C2 carbonyl). The absence of the H-3 singlet at δ 6.4 (coumarin) and presence at δ 5.68 confirms the 4-hydroxy substitution pattern. |
| Melting Point | 211 – 215°C (literature: 212-214°C, with decomposition above 215°C) |
| Solubility | Slightly soluble in cold water (~0.5 g/L at 25°C); soluble in hot water (~5 g/L at 80°C); soluble in ethanol (~30 g/L), methanol, acetone, ethyl acetate, chloroform, DMSO, DMF; freely soluble in aqueous alkaline solutions (pH >8 — deprotonation of the 4-OH enolic hydroxyl to form the water-soluble phenolate anion); practically insoluble in hexane and non-polar solvents. The water solubility at neutral pH reflects the enolic nature of the 4-OH — the vinylogous carboxylic acid character provides some aqueous solubility even without deprotonation. |
| Loss on Drying | ≤0.5% (105°C, 2 hours) |
| Residue on Ignition | ≤0.2% |
| Heavy Metals (Total) | ≤10 ppm (as Pb) |
| Elemental Impurities | Pb ≤2 ppm; As ≤1 ppm; Hg ≤1 ppm; Cd ≤1 ppm; Cr (from synthesis) ≤5 ppm (ICH Q3D / USP <232> / EP <5.20> compliant) |
| Microbial Limits | TAMC ≤1000 CFU/g; TYMC ≤100 CFU/g (USP <61> / EP <2.6.12>); E. coli — Absent in 1g; Salmonella — Absent in 25g; Staphylococcus aureus — Absent in 1g; Pseudomonas aeruginosa — Absent in 1g (USP <62> / EP <2.6.13>) |
| Residual Solvents | ICH Q3C / USP <467> Class 2/3 compliant; methanol ≤3000 ppm; acetone ≤5000 ppm; ethyl acetate ≤5000 ppm; ethanol ≤5000 ppm; dichloromethane ≤600 ppm (Class 2); any other Class 2 solvent within ICH limits |
| Related Substances (HPLC) | Any individual unspecified impurity ≤0.10%; Total unspecified impurities ≤0.50%; Coumarin ≤0.10% (starting material / degradation product); 4-Hydroxycoumarin dimer (dicoumarol-related) ≤0.15%; Any single specified impurity ≤0.15%. Reporting threshold: 0.05%. |
| Assay — Water Content (Karl Fischer) | ≤0.5% (coulometric or volumetric KF) |
| Tautomeric Form | Predominantly the 4-hydroxy enol tautomer (>95% in solid state and in solution at neutral pH) — stabilized by intramolecular hydrogen bonding between the 4-OH proton and the carbonyl oxygen at C2, forming a six-membered pseudo-ring. This tautomeric preference is the basis for C3 nucleophilicity exploited in all 4-hydroxycoumarin drug syntheses. |
| Grade / Standards | Pharma Intermediate Grade (≥99% HPLC) — suitable as Key Starting Material (KSM) for anticoagulant API synthesis per ICH Q7 GMP guidelines; Research Grade (≥98% HPLC) — suitable for academic research, method development, and analytical reference use |
| Certifications | ISO 9001:2015, ISO 22000, HACCP, FDA Facility Registration, Non-GMO, BSE/TSE-Free, Allergen-Free Statement, Conflict-Free Minerals Statement |
| Packaging | 100 g / 500 g / 1 kg amber glass bottles with PTFE-lined caps under nitrogen; 5 kg / 10 kg HDPE drums with double PE liner and nitrogen headspace; 25 kg fiber drums with double PE liner, nitrogen flush, and desiccant |
| Storage | 2-8°C recommended for long-term storage; room temperature (15-25°C) acceptable for short-term (≤6 months); tightly sealed in original container; protect from light (amber packaging) and moisture; store under inert gas (N₂ or Ar) for extended shelf life. 4-Hydroxycoumarin is chemically stable under recommended storage — the enolic 4-OH does not undergo spontaneous oxidation, and the lactone ring is resistant to hydrolysis at neutral pH. |
| Shelf Life | 3 years from date of manufacture under recommended storage (2-8°C, nitrogen headspace, amber glass, protected from light). Retest date assigned at 36 months with full specification testing per ICH Q1A(R2) stability protocol. |
Key Benefits — 4-Hydroxycoumarin
The Core Scaffold of the Most Prescribed Oral Anticoagulant Class Globally
4-Hydroxycoumarin is the irreplaceable chemical nucleus from which warfarin (Coumadin — the #1 oral anticoagulant worldwide, 20+ million US prescriptions/year), acenocoumarol, phenprocoumon, and dicoumarol are synthesized. The global 4-hydroxycoumarin anticoagulant API market exceeds 100 metric tons annually. No other chemical scaffold has been approved as an oral anticoagulant with the same VKORC1-targeting mechanism — 80+ years of medicinal chemistry have confirmed the 4-hydroxycoumarin nucleus is structurally non-negotiable for this pharmacological class.
Warfarin PrecursorEssential 4-OH Pharmacophore — Mechanism-Based VKORC1 Inhibition
The 4-hydroxy group is the pharmacophore for competitive VKORC1 inhibition: it mimics the naphthoquinone core of vitamin K, forming hydrogen bonds with active-site cysteine residues (Cys132/Cys135) and pi-pi stacking with Tyr139. This blocks vitamin K epoxide → quinone → hydroquinone recycling, depleting the KH₂ cofactor required for GGCX-catalyzed gamma-carboxylation of clotting factors. Methylate or acylate the 4-OH and all anticoagulant activity is abolished — confirmed across decades of structure-activity relationship studies.
VKORC1 PharmacophoreRich Synthetic Versatility — Michael Acceptor Chemistry at C3
Keto-enol tautomerism activates the C3 position for Michael addition and condensation reactions — the key synthetic transformations for building all 4-hydroxycoumarin drugs. React with benzalacetone → warfarin. React with 4-nitrobenzalacetone → acenocoumarol. React with 1-phenyl-1-buten-3-one → phenprocoumon. React with 4′-bromobiphenyl-substituted benzalacetones → brodifacoum and second-generation rodenticides. One intermediate → an entire drug class.
Synthetic Versatility≥99% Pharma Intermediate Grade — Key Starting Material for API Manufacturing
Pharma Intermediate Grade 4-hydroxycoumarin at ≥99% HPLC purity with full ICH Q7-compliant documentation — suitable as a validated Key Starting Material (KSM) for anticoagulant API manufacturing, DMF filings, and regulatory submissions. Rigorous impurity control (individual unspecified ≤0.10%, total ≤0.50%) with HPLC, IR, NMR, and UV-Vis identity confirmation. ISO 9001:2015, ISO 22000, HACCP certified.
≥99% HPLCApplications
Warfarin & Warfarin Sodium API Synthesis — #1 Oral Anticoagulant Globally
4-Hydroxycoumarin is the key starting material (KSM) for warfarin sodium API manufacturing. Condensation with benzalacetone (Claisen-Schmidt condensation product of benzaldehyde + acetone) via Michael addition at C3 yields racemic warfarin. Subsequent resolution (chiral separation) or enantioselective synthesis provides S-warfarin (3-5× more potent enantiomer) and R-warfarin. The global warfarin API market exceeds 100 metric tons/year — serving 100+ million patients on long-term oral anticoagulation worldwide. Pharma Intermediate Grade (≥99%) with full DMF support documentation.
Acenocoumarol & Phenprocoumon API Synthesis — European Anticoagulant Standards
4-Hydroxycoumarin as the common intermediate for acenocoumarol (Sintrom, Sinthrome) — widely used in Europe and Asia, and phenprocoumon (Marcoumar, Marcumar, Falithrom) — the preferred European long-term anticoagulant with the longest half-life (~160h) of any 4-hydroxycoumarin drug. Both synthesized via the same Michael addition chemistry with different alpha,beta-unsaturated ketone partners. One scaffold → multiple commercial APIs.
Second-Generation Anticoagulant Rodenticide Synthesis — Brodifacoum & Related
4-Hydroxycoumarin as the precursor for ‘super-warfarin’ second-generation anticoagulant rodenticides: brodifacoum, difenacoum, bromadiolone, and flocoumafen. These incorporate extended lipophilic side chains (4′-bromobiphenyl, biphenyl, etc.) at C3 that increase VKORC1 binding affinity ~100× and dramatically extend hepatic half-life (60-120+ days). Multi-thousand-ton annual market for global pest control and crop protection. The 4-hydroxycoumarin core is identical — only the C3 substituent differentiates therapeutic anticoagulants from rodenticides.
VKORC1 Enzymology & Anticoagulant Drug Discovery Research
High-purity 4-hydroxycoumarin (≥98-99% HPLC) as a pharmacological probe and scaffold for academic and pharmaceutical research: VKORC1 enzyme kinetics and inhibition studies, structure-activity relationship (SAR) exploration of C3-substituted 4-hydroxycoumarin derivatives, rational design of novel anticoagulants with improved therapeutic windows, investigation of VKORC1 genetic polymorphisms affecting warfarin sensitivity, and discovery of non-anticoagulant 4-hydroxycoumarin bioactivities (anti-inflammatory, anticancer, antimicrobial).
Fluorescent Probe Chemistry — Umbelliferone & 4-Methylumbelliferone Derivatives
4-Hydroxycoumarin as the precursor for fluorogenic probe synthesis. While 4-hydroxycoumarin itself has modest fluorescence, its 7-hydroxy derivative (umbelliferone, 7-hydroxycoumarin) and 7-hydroxy-4-methyl derivative (4-methylumbelliferone, 4-MU) are among the most widely used fluorogenic substrates in biochemistry. 4-MU conjugated to phosphate, sulfate, galactoside, glucuronide, or other enzyme-cleavable groups is non-fluorescent — enzymatic cleavage releases the highly fluorescent 4-MU (λex 360 nm, λem 450 nm, quantum yield ~0.70), enabling real-time detection of phosphatase, sulfatase, glycosidase, glucuronidase, and esterase activities. 4-Hydroxycoumarin serves as the starting material for these essential biochemical reagents.
Dicoumarol & Bis(4-Hydroxycoumarin) Synthesis — The Original Anticoagulant
4-Hydroxycoumarin as the precursor for dicoumarol (3,3′-methylenebis(4-hydroxycoumarin)) — the original oral anticoagulant discovered from moldy sweet clover (1939, Campbell & Link). Synthesized by formaldehyde-mediated dimerization of 4-hydroxycoumarin. While largely replaced by warfarin clinically, dicoumarol remains important as a biochemical tool for VKORC1 research and as a historical reference compound. The bis(4-hydroxycoumarin) structure also serves as the template for designing bivalent VKORC1 inhibitors with enhanced potency and extended duration.
Frequently Asked Questions
4-Hydroxycoumarin (CAS 1076-38-6, 4-hydroxy-2H-chromen-2-one, C₉H₆O₃, MW 162.14 g/mol) is a heterocyclic organic compound that serves as the core chemical scaffold for all 4-hydroxycoumarin anticoagulant drugs — the most prescribed class of oral anticoagulants in medical history. Its discovery story is one of the most consequential in pharmaceutical science. In the early 1920s, cattle across North America began dying from a mysterious hemorrhagic disease after consuming moldy sweet clover hay (Melilotus alba and M. officinalis) — ‘sweet clover disease.’ In February 1933, Wisconsin farmer Ed Carlson brought a dead cow, a milk can of unclotted blood, and 100 pounds of moldy hay to Dr. Karl Paul Link’s laboratory at the University of Wisconsin-Madison. After 6 years of painstaking isolation, Link’s postdoctoral fellow Dr. Harold A. Campbell crystallized the hemorrhagic agent in 1939 — dicoumarol (3,3′-methylenebis(4-hydroxycoumarin)), formed when coumarin naturally present in sweet clover is oxidized by Aspergillus/Penicillium molds to 4-hydroxycoumarin, which then dimerizes with formaldehyde. Building on dicoumarol’s structure, Link’s team synthesized warfarin in 1948 — named WARF-arin after the Wisconsin Alumni Research Foundation that funded the research. Warfarin was patented in 1947, first marketed as a rodenticide (1948), and transitioned to human medicine after a 1951 suicide attempt demonstrated its safety and vitamin K reversibility at therapeutic doses. After President Eisenhower received warfarin following his 1955 myocardial infarction, it became the global standard for oral anticoagulation — reaching 20+ million US prescriptions annually. 4-Hydroxycoumarin thus stands as the foundational chemical intermediate whose discovery transformed cardiovascular medicine, preventing millions of strokes and thromboembolic deaths over 70+ years.
The anticoagulant mechanism of 4-hydroxycoumarin-derived drugs centers on competitive inhibition of VKORC1 (vitamin K epoxide reductase complex subunit 1), the enzyme responsible for recycling vitamin K in the body. The process proceeds through a six-step cascade: (1) Vitamin K-dependent gamma-glutamyl carboxylase (GGCX) carboxylates glutamic acid (Glu) to gamma-carboxyglutamic acid (Gla) on clotting factors II, VII, IX, X and proteins C/S — this post-translational modification enables calcium-dependent phospholipid membrane binding essential for coagulation. During each carboxylation, the reduced vitamin K hydroquinone (KH₂) cofactor is oxidized to vitamin K 2,3-epoxide (KO). (2) VKORC1 catalyzes the regeneration of KO → vitamin K quinone (K) → KH₂ — completing the vitamin K cycle. This recycling is essential because the total body vitamin K pool is small (~1 μg/kg) and must be reused ~2000 times daily. (3) 4-Hydroxycoumarin anticoagulants bind VKORC1 and competitively inhibit its reductase activity — the 4-hydroxy group is structurally essential: it mimics the naphthoquinone core of vitamin K, forming hydrogen bonds with VKORC1 catalytic cysteines (Cys132, Cys135) and pi-pi stacking with Tyr139. (4) VKORC1 inhibition blocks KO → K → KH₂ recycling, depleting intracellular KH₂ within 2-4 hours. Without KH₂, GGCX produces non-functional descarboxy-proteins (PIVKA — proteins induced by vitamin K absence). (5) Anticoagulation develops over 24-72 hours as functional clotting factors clear according to their half-lives: VII (~6h), IX (~24h), X (~40h), II (~60h) — explaining warfarin’s delayed onset and the need for heparin bridging. (6) Effect is monitored by PT/INR (prothrombin time / international normalized ratio), titrated to INR 2.0-3.0 for most indications. VKORC1 gene polymorphisms (e.g., -1639G>A, rs9923231) reduce enzyme expression and increase warfarin sensitivity — explaining ~25% of inter-individual dose variability. The VKORC1 crystal structure (2004, Li et al., Nature) with bound warfarin confirmed the competitive binding mechanism and provided the structural basis for rational anticoagulant design — all centered on the 4-hydroxycoumarin pharmacophore.
The 4-hydroxycoumarin scaffold has produced six major anticoagulant drugs spanning therapeutic and rodenticide applications — the most prescribed oral anticoagulant class in history. (1) Warfarin (Coumadin, Jantoven) — synthesized by condensing 4-hydroxycoumarin with benzalacetone. #1 oral anticoagulant globally: 20+ million US Rx/year, 100+ million worldwide. Racemic (S-warfarin 3-5× more potent, CYP2C9-metabolized). Indications: atrial fibrillation, DVT/PE, mechanical heart valves, post-MI, antiphospholipid syndrome. Named WARF-arin for Wisconsin Alumni Research Foundation (1948). (2) Dicoumarol (Dicumarol) — the original anticoagulant from moldy sweet clover (1939, Campbell & Link). Naturally formed bis(4-hydroxycoumarin) via fungal oxidation + formaldehyde dimerization. First oral anticoagulant in humans (1941, Mayo Clinic). Now primarily a VKORC1 biochemical probe. (3) Acenocoumarol (Sintrom, Sinthrome) — 4-nitrophenyl substituent at C3. Widely used in Europe/Asia. Shorter half-life (~8-11h). CYP2C9-dependent metabolism. (4) Phenprocoumon (Marcoumar, Marcumar, Falithrom) — 1-phenylpropyl at C3. Longest half-life (~160h, 5-6 days). Preferred in Germany/Netherlands for stable long-term anticoagulation. Multi-CYP metabolism reduces pharmacogenetic sensitivity. (5) Brodifacoum — ‘Super-Warfarin’ — 4′-bromobiphenyl-4-yl extended side chain at C3. VKORC1 affinity ~100× warfarin, hepatic half-life 60-120+ days. Second-generation rodenticide. A single bait block provides 30+ days of anticoagulation in humans — treatment requires high-dose vitamin K1 for weeks to months. (6) Difenacoum, Bromadiolone, Flocoumafen — additional second-generation 4-hydroxycoumarin rodenticides with varying potency and species selectivity. All six share the identical mechanism: 4-OH group binds VKORC1 → competitive inhibition → vitamin K recycling blocked → clotting factor depletion → anticoagulation. The critical structure-activity relationship: modify the 4-OH and all anticoagulant activity is abolished. The C3 substituent determines potency, half-life, and therapeutic vs rodenticide use — but the 4-hydroxycoumarin nucleus is invariant across the entire drug class.
UPOR Biotech offers 4-Hydroxycoumarin in two purity grades with full regulatory documentation: (1) Pharma Intermediate Grade — ≥99% by HPLC (anhydrous basis). Intended for use as a validated Key Starting Material (KSM) in anticoagulant API manufacturing per ICH Q7 GMP guidelines. This grade provides: HPLC purity ≥99.0% with full related-substance impurity profile, individual unspecified impurities ≤0.10% and total ≤0.50%, coumarin (starting material/degradant) ≤0.10%, 4-hydroxycoumarin dimer ≤0.15%, residual solvents per ICH Q3C / USP <467> (methanol ≤3000 ppm, acetone ≤5000 ppm, ethyl acetate ≤5000 ppm, dichloromethane ≤600 ppm Class 2), elemental impurities per ICH Q3D / USP <232> (Pb ≤2 ppm, As ≤1 ppm, Hg ≤1 ppm, Cd ≤1 ppm, Cr ≤5 ppm), microbial limits per USP <61>/<62> (TAMC ≤1000 CFU/g, TYMC ≤100 CFU/g, pathogens absent), Karl Fischer water content ≤0.5%, and comprehensive identity confirmation by HPLC, UV-Vis, FT-IR, and/or ¹H-NMR. Full documentation supports drug master file (DMF) filings and regulatory submissions. (2) Research Grade — ≥98% by HPLC (anhydrous basis). Suitable for academic research, VKORC1 enzymology studies, anticoagulant drug discovery, fluorescent probe methodology, coumarin chemistry, and analytical reference standard use. Both grades supplied as white to off-white crystalline powder with complete COA, MSDS, signed and dated HPLC chromatogram, certificate of origin, ISO certificates, and complete lot traceability. Free sample (5-10g) available for qualified B2B buyers. MOQ: 1 kg (Pharma Grade), 100g (Research Grade). Additional GMP documentation and DMF support letters available for pharmaceutical customers upon signing of Quality Agreement and CDA.
Every shipment of UPOR Biotech 4-Hydroxycoumarin includes a comprehensive quality documentation package: Certificate of Analysis (COA) with HPLC purity assay (≥99% Pharma Grade / ≥98% Research Grade), full related-substance impurity profile (individual unspecified ≤0.10%, total ≤0.50%, coumarin ≤0.10%, dimer ≤0.15%), heavy metals ≤10 ppm (Pb ≤2 ppm, As ≤1 ppm, Hg ≤1 ppm, Cd ≤1 ppm, Cr ≤5 ppm per ICH Q3D / USP <232>), residual solvents per ICH Q3C Class 3 / USP <467> (methanol ≤3000 ppm, acetone ≤5000 ppm, ethyl acetate ≤5000 ppm, dichloromethane ≤600 ppm, ethanol ≤5000 ppm), microbial limits per USP <61>/<62> (TAMC ≤1000 CFU/g, TYMC ≤100 CFU/g, E. coli/Salmonella/S. aureus/P. aeruginosa absent), Karl Fischer water content ≤0.5%, loss on drying ≤0.5%, residue on ignition ≤0.2%, melting point 211-215°C, and identity confirmation (HPLC RRT, UV-Vis λmax 280-310 nm, FT-IR carbonyl/lactone/enolic OH bands, optionally ¹H-NMR). Also provided: Material Safety Data Sheet (MSDS), Signed and dated HPLC Chromatogram with peak integration, UV-Vis Absorption Spectrum, FT-IR Spectrum (optional ¹H-NMR spectrum for Pharma Grade), ISO 9001:2015 Certificate, ISO 22000 Certificate, HACCP Certificate, FDA Facility Registration, Non-GMO Statement, Allergen Statement, BSE/TSE-Free Statement, Certificate of Origin, Stability Data (25°C/60%RH 36-month real-time and 40°C/75%RH 6-month accelerated per ICH Q1A(R2)), and Complete Lot Traceability from raw material to finished product with assignable batch records. All documents provided in English. Free sample (5-10g) for qualified B2B buyers. MOQ: 1 kg (Pharma Grade), 100g (Research Grade). Additional GMP documentation, DMF support letters, and technical dossiers available for pharmaceutical intermediate customers upon signing of a Quality Agreement and CDA.