Synthesis and Anti-Inflammatory Evaluation of 4H-Chromene and Chromeno[2,3-b]pyridine Derivatives

Study Overview and Abstract

• The research details the efficient preparation of several derivatives of $4H$-chromene and chromeno[2,3-$b$]pyridine using microwave irradiation in a one-pot reaction.

• The study evaluates the anti-inflammatory activities of these synthetic products.

• Key findings include six synthetic products (1b, 1c, 1h, 2d, 2j, and 2l) that demonstrated a more powerful inhibition of tumor necrosis factor-$\alpha$-induced (TNF-$\alpha$-induced) nitric oxide (NO) production compared to quercetin.

• These compounds showed comparable cell viability in both human and porcine chondrocytes.

• Compound 2d, at dosages of $10\,mg/kg$ and $20\,mg/kg$, exhibited a potent anti-inflammatory effect by suppressing carrageenan-induced rat paw edema and prostaglandin E2 (PGE2) formation.

• The results suggest these compounds serve as potential structural templates for the design and development of new anti-inflammatory drugs.

• Keywords: $4H$-chromene, chromeno[2,3-$b$]pyridine, NO production, Carrageenan-induced rat paw edema, Anti-inflammatory.

Introduction to Inflammation and Pharmacological Context

• Inflammation is a general response to infection and injury linked to diseases such as arthritis, asthma, allergy, diabetes, atherosclerosis, and cancer.

• Symptoms of rheumatoid arthritis and osteoarthritis, such as swelling, pain, and stiffness, significantly reduce quality of life.

• Key inflammatory mediators include cytokines, nitric oxide (NO), and prostaglandin E2 (PGE2), all of which play pathophysiological roles in the development of inflammation.

• Approved anti-inflammatory drugs include:

  • Aspirin

  • Corticosteroids

  • Indomethacin

  • Diclofenac

  • Ibuprofen

  • Pranoprofen

  • Celecoxib

  • Roficoxib

• Nonsteroidal anti-inflammatory drugs (NSAIDs) function as analgesics and anti-inflammatory agents by inhibiting the biosynthesis or release of prostaglandins from arachidonic acid via the suppression of cyclooxygenases.

• Side effects of current NSAIDs include:

  • Gastrointestinal irritation and ulceration

  • Hepatotoxicity

  • Acute renal failure

  • Hypertension

  • Heart failure

Scaffolds and Synthetic Rationale

• Derivatives of $4H$-chromene and chromeno[2,3-$b$]pyridine are recognized as privileged medicinal scaffolds with biological activities including anticancerous, anti-proliferative, anti-inflammatory, anti-rheumatic, anti-allergic, apoptosis-inducing, antibacterial, and anti-tubercular activities.

• Pranoprofen is a specific approved NSAID utilizing the chromeno[2,3-$b$]pyridine scaffold, used topically for conjunctivitis and post-strabismus surgery inflammation.

• Structural commonality: Both series comprise a benzopyran nucleus, with most featuring a 2-amino-3-nitrilo functionality.

• Conventional synthesis challenges: Often time-consuming (typically >24\,h) involving condensation of aromatic aldehydes, malononitrile, and phenols.

• Solution: Multicomponent reaction (MCR) combined with microwave-assisted synthesis allows for rapid (5–30 min) one-pot preparation, chemical reaction optimization, and green chemistry.

Experimental Chemistry and Instrumentation

• Chemicals: Purchased from Aldrich-Sigma Chemical Company (St. Louis, MO, USA) and Alfa-Aesar Chemical Company (Heysham, LA32XY, England).

• Microwave Apparatus: CEM Discover Benchmate™ (CEM Corp., Italy).

• Monitoring: TLC on Merck F254 silica gel plates.

• Melting Point Measurement: Büchi-530 melting point apparatus.

• UV–Vis Spectra: Shimazu UV-160 A UV–visible recording spectrophotometer.

• IR Spectra: Perkin-Elmer FTIR 1610 series infrared spectrophotometer in KBr discs.

• NMR Spectroscopy: Varian Gemini-400 instrument ($400\,MHz$ for $^1H$; $100\,MHz$ for $^{13}C$) in DMSO-$d_6$.

  • Chemical shifts ($\delta$) in ppm downfield from tetramethylsilane (TMS).

  • Coupling constants ($J$) in hertz ($Hz$).

• Mass Spectrometry: Finnigan MAT-95XL high-resolution mass spectra (HR-MS) performed at the Instrument Center of the National Science Counsel at National Tsing-Hua University, Taiwan.

Synthesis of $4H$-chromene Derivatives 1a–j

• General Procedure:

  • Mix 3,4,5-trimethoxybenzaldehyde ($1.0\,mmol$), malononitrile ($1.0\,mmol$), and substituted phenols ($1.0\,mmol$) in propanol ($3.0\,mL$).

  • Add piperidine ($0.5\,mmol$) as a catalyst.

  • Apply microwave irradiation ($300\,W$) at $80\,^{\circ}C$ or $120\,^{\circ}C$ for 5–30 min.

  • Residue filtered and washed with ethanol and hexane; re-crystallization from ethanol.

• Reaction Mechanism: Knoevenagel condensation of malononitrile with 3,4,5-trimethoxybenzaldehyde, followed by Michael addition of phenols and intramolecular cyclization.

• Characteristic Signal: Pyran-CH singlet peak at $\delta\,4.48$ to $5.25\,ppm$ in $^1H$-NMR.

• Intermediate Discovery: At or below $80\,^{\circ}C$, the intermediate 2-(3,4,5-trimethoxybenzylidene)malononitrile (1k) was isolated.

• Unusual Result with 4-Aminophenol: Reaction failed and yielded (E)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (1l) instead of the expected chromene.

Characterization Data for Selected 1 Series Compounds

• 1a (2,7-Diamino-4-(3,4,5-trimethoxyphenyl)-$4H$-chromene-3-carbonitrile):

  • Conditions: $80\,^{\circ}C$, 5 min. Yield: 85%. mp: 208–$210\,^{\circ}C$.

  • IR (KBr): $3416, 3356, 3329, 3175\,(NH_2)$, $2195\,(CN)$.

• 1b (2-Amino-7-(dimethylamino)-4-(3,4,5-trimethoxyphenyl)-$4H$-chromene-3-carbonitrile):

  • Conditions: $80\,^{\circ}C$, 5 min. Yield: 89%. mp: 180–$182\,^{\circ}C$.

• 1c (Amino-7-(diethylamino)-4-(3,4,5-trimethoxyphenyl)-$4H$-chromene-3-carbonitrile):

  • Conditions: $80\,^{\circ}C$, 5 min. Yield: 92%. mp: 169–$171\,^{\circ}C$.

• 1h (2-Amino-4-(3,4,5-trimethoxyphenyl)-$4H$-benzo[$h$]chromene-3-carbonitrile):

  • Conditions: $80\,^{\circ}C$, 5 min. Yield: 95%. mp: 184–$185\,^{\circ}C$.

• 1j (2-Amino-7-morpholino-4-(3,4,5-trimethoxyphenyl)-$4H$-chromene-3-carbonitrile):

  • Conditions: $120\,^{\circ}C$, 15 min. Yield: 90%. mp: 227–$229\,^{\circ}C$.

Synthesis of Chromeno[2,3-$b$]pyridine Derivatives 2a–l

• General Procedure:

  • Mixture of substituted salicylaldehydes ($1.0\,mmol$), substituted phenols ($1.0\,mmol$), and malononitrile ($2.0\,mmol$) in propanol ($5.0\,mL$).

  • Add two drops of triethylamine (TEA) as a catalyst.

  • Microwave irradiation ($300\,W$) at $150\,^{\circ}C$ for 5–30 min.

  • Residue filtered, washed, and re-crystallized from ethanol.

• Reaction Mechanism: Initial Knoevenagel condensation of salicylaldehyde and malononitrile $\rightarrow$ Intramolecular cyclization (Pinner reaction) $\rightarrow$ Nucleophilic attack by phenol to form a $4H$-chromene intermediate $\rightarrow$ Reaction with a second malononitrile molecule.

• Characteristic Signal: Pyran-CH singlet peak at $\delta\,5.14$ to $6.16\,ppm$ in $^1H$-NMR.

• Comparison with Conventional Method: Literature method for 2d, 2h, 2l required $110\,^{\circ}C$ without solvent, resulting in bumping and charring; microwave method is faster and cleaner.

Characterization Data for Selected 2 Series Compounds

• 2a (2,4-Diamino-5-(4-amino-2-hydroxyphenyl)-$5H$-chromeno[2,3-$b$]pyridine-3-carbonitrile):

  • Conditions: 10 min. Yield: 87%. mp: 297–$299\,^{\circ}C$.

• 2d (2,4-Diamino-5-(1-hydroxynaphthalen-2-yl)-$5H$-chromeno[2,3-$b$]pyridine-3-carbonitrile):

  • Conditions: 5 min. Yield: 93%. mp: 285–$287\,^{\circ}C$.

• 2j (9,11-Diamino-12-(4-(dimethylamino)-2-hydroxyphenyl)-$12H$-benzo[5,6]chromeno[2,3-$b$]pyridine-10-carbonitrile):

  • Conditions: 30 min. Yield: 91%. mp: 337–$339\,^{\circ}C$.

• 2l (9,11-Diamino-12-(3-hydroxynaphthalen-2-yl)-$12H$-benzo[5,6]chromeno[2,3-$b$]pyridine-10-carbonitrile):

  • Conditions: 5 min. Yield: 97%. mp: 315–$317\,^{\circ}C$.

Biological Activity Protocols

• Chondrocyte Isolation:

  • Human cartilage obtained from osteoarthritis patients under Tri-Service General Hospital IRB approval.

  • Porcine cartilage from hind leg joints.

  • Enzymatic digestion using $2\,mg/mL$ protease in serum-free DMEM, followed by overnight digestion with $2\,mg/mL$ collagenase I and $0.9\,mg/mL$ hyaluronidase.

• In Vitro Cytotoxicity (MTT Assay):

  • Cells incubated with compounds 1a–2l for 48 h.

  • Added 25 $\mu L$ of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, $1\,mg/mL$ in $H_2O$).

  • Measured absorbance at $590\,nm$.

• Nitric Oxide (NO) Production:

  • Measured via nitrite concentration using the Griess reaction.

  • Supernatant incubated with 0.1% sulfanilamide (in 5% phosphoric acid) and 0.1% $N$-1-naphthylethylenediamine dihydrochloride.

  • Measured absorbance at $550\,nm$.

• Animal Subjects:

  • Adult male Wistar rats ($290 \pm 20\,g$) housed at 23 $\pm 2\,^{\circ}C$, 60 $\pm 5\%$ humidity, 12 h light/dark cycle.

• In Vivo Anti-inflammatory Assay:

  • Carrageenan-induced rat hind paw edema (Winter's method modified).

  • Compounds 1h, 2d, quercetin, or ibuprofen administered intraperitoneally ($10$ and $20\,mg/kg$) 1 h before injecting carrageenan (0.05 mL of 1% solution).

  • Edema rate ($E\%$) calculation: $E\% = \frac{V_t - V_0}{V_0} \times 100$.

  • Percentage inhibition ($I\%$) calculation: $I\% = \frac{E_c - E_t}{E_c} \times 100$.

• PGE2 Assay:

  • Rat paws cut, pulverized in liquid nitrogen, and homogenized.

  • Centrifuged at $12,000\,rpm$ at $4\,^{\circ}C$ for 30 min.

  • PGE2 measured using ELISA kit; protein levels quantified by Bradford protein assay.

Summary of In Vitro Results

• Cytotoxicity:

  • All synthetic compounds (1a–2l) showed IC_{50} > 100\,\mu M in both human and porcine chondrocytes, indicating low toxicity.

• NO Inhibition Results (Selected Compounds):

  • Quercetin (Control): Human $IC_{50} = 65.7 \pm 3.5\,\mu M$; Porcine $IC_{50} = 63.4 \pm 5.5\,\mu M$.

  • 1b: Human $IC_{50} = 9.6 \pm 0.1\,\mu M$; Porcine $IC_{50} = 9.5 \pm 0.3\,\mu M$.

  • 1c: Human $IC_{50} = 9.1 \pm 1.8\,\mu M$.

  • 1h: Human $IC_{50} = 8.1 \pm 0.6\,\mu M$; Porcine $IC_{50} = 8.0 \pm 0.2\,\mu M$.

  • 2d: Human $IC_{50} = 6.9 \pm 0.3\,\mu M$; Porcine $IC_{50} = 6.1 \pm 1.0\,\mu M$.

  • 2j: Human $IC_{50} = 7.6 \pm 1.3\,\mu M$.

  • 2l: Human $IC_{50} = 7.6 \pm 0.1\,\mu M$.

• Key Potency Observation: Compounds with amino groups at C(7) and fused benzene rings (1h, 2d, 2l) were significantly more potent than others.

Summary of In Vivo Results

• Anti-inflammatory Activity (Paw Edema Inhibition at 5 h):

  • 1h ($20\,mg/kg$): 69.0% inhibition.

  • 2d ($20\,mg/kg$): 85.4% inhibition (clinical superiority over references).

  • Quercetin ($20\,mg/kg$): 52.2% inhibition.

  • Ibuprofen ($20\,mg/kg$): 52.0% inhibition.

• PGE2 Suppression in Rat Paws:

  • 2d exhibited the greatest PGE2-suppressing activity: $191.7\,pg/mL$ (at $10\,mg/kg$) and $112.5\,pg/mL$ (at $20\,mg/kg$).

  • Reference Ibuprofen results: $285.4\,pg/mL$ (at $10\,mg/kg$) and $167.5\,pg/mL$ (at $20\,mg/kg$).

  • Reference Quercetin results: Significantly higher PGE2 levels compared to test groups.

Conclusions and Implications

• Efficiency: One-pot microwave synthesis proved highly efficient compared to conventional methods for producing these scaffolds.

• Potency: Compound 2d demonstrated superior potency and sustainability in anti-inflammatory effects compared to the clinically used drug ibuprofen.

• Safety: Compounds exhibited favorable safety profiles with minimal in vitro cytotoxicity.

• Future Work: Detailed pharmacological and pharmacokinetic studies are ongoing to further investigate 2d as a novel anti-inflammatory lead.

• Ethical Compliance: Study performed in accordance with National Academy of Sciences criteria and approved by the IACUC (Number: IACUC-13-178) and IRB (Number: 1-102-05-091).