Synthesis and anti-inflammatory activities of 4H-chromene and chromeno[2,3-b]pyridine derivatives

Article abstract of DOI:10.1007/s11164-015-2081-7

Several derivatives of 4H-chromene and chromeno[2,3-b]pyridine were efficiently prepared under microwave irradiation in a one-pot reaction, and their anti-inflammatory activities were evaluated. Six synthetic products (1b, 1c, 1h, 2d, 2j, and 2l) exhibited more powerfully inhibited the production of tumor necrosis factor-α-induced nitric oxide (NO) than quercetin and exhibited comparable cell viability in both human and porcine chondrocytes. In particular, 2d at dosages of 10 and 20 mg/kg had a very potent anti-inflammatory effect by suppressing the formation of carrageenan-induced rat paw edema and prostaglandin E₂. The results herein suggest that these compounds may have potential as structural templates in the design and development of new anti-inflammatory drugs.

Full text of DOI:10.1007/s11164-015-2081-7

Res Chem Intermed
DOI 10.1007/s11164-015-2081-7
Synthesis and anti-inflammatory activities of 4H-
chromene and chromeno[2,3-b]pyridine derivatives
Shu-Ting Chung1 Wen-Hsin Huang1,2 Chih-Kuo Huang2
•
•
•
Feng-Cheng Liu3 Ren-Yeong Huang4 Chin-Chen Wu5
An-Rong Lee1,2
•
•
•
Received: 3 March 2015 / Accepted: 24 April 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Several derivatives of 4H-chromene and chromeno[2,3-b]pyridine were
efficiently prepared under microwave irradiation in a one-pot reaction, and their
anti-inflammatory activities were evaluated. Six synthetic products (1b, 1c, 1h, 2d,
2j, and 2l) exhibited more powerfully inhibited the production of tumor necrosis
factor-a-induced nitric oxide (NO) than quercetin and exhibited comparable cell
viability in both human and porcine chondrocytes. In particular, 2d at dosages of 10
and 20 mg/kg had a very potent anti-inflammatory effect by suppressing the for-
mation of carrageenan-induced rat paw edema and prostaglandin E2. The results
herein suggest that these compounds may have potential as structural templates in
the design and development of new anti-inflammatory drugs.
Keywords 4H-chromene Á Chromeno[2,3-b]pyridine Á NO production Á
Carrageenan-induced rat paw edema Á Anti-inflammatory
&
An-Rong Lee
416806@gmail.com
1
2
3
Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan
School of Pharmacy, National Defense Medical Center, Taipei, Taiwan
Division of Rheumatology/Immunology and Allergy, Tri-Service General Hospital, National
Defense Medical Center, Taipei, Taiwan
4
5
Department of Periodontology, School of Dentistry, Tri-Service General Hospital, National
Defense Medical Center, Taipei, Taiwan
Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan
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S.-T. Chung et al.
Introduction
Inflammation is a general response to infection and injury and has been associated with
such diseases arthritis, asthma, allergy, diabetes, atherosclerosis, and cancer, which
affect people globally [1–6]. Patients with rheumatoid arthritis and osteoarthritis
commonly describe swelling, pain, and stiffness as significant symptoms, which
reduce health-related quality of life [7]. Many investigations have demonstrated that
certain important inflammatory mediators, such as cytokines, nitric oxide (NO) and
prostaglandin E2 (PGE2), have a significant pathophysiological role in the develop-
ment of inflammation [8–10]. Numerous anti-inflammatory drugs have been approved
for treating inflammation, including aspirin, corticosteroids, indomethacin, diclofe-
nac, ibuprofen, pranoprofen, celecoxib, and roficoxib [7, 11]. Of these, nonsteroidal
anti-inflammatory drugs (NSAIDs) are effective anti-inflammatory agents and
analgesics that inhibit the biosynthesis or release of prostaglandins from arachidonic
acid by suppressing cyclooxygenases [7]. Unfortunately, since these drugs inhibit the
production of prostaglandins, they can cause serious side effects, such as gastroin-
testinal irritation, ulceration, hepatotoxicity, acute renal failure, hypertension, and
even heart failure [12–15]. The need to develop new and safer therapeutic agents
motivated the design and synthesis herein of two series of substituted 4H-chromene
and chromeno[2,3-b]pyridine derivatives as potential anti-inflammatory agents.
Recently, derivatives of 4H-chromene and chromeno[2,3-b]pyridine (Fig. 1)
have been found to have various potential biological activities, including anti-
cancerous, anti-proliferative, anti-inflammatory, anti-rheumatic, anti-allergic, apop-
tosis-inducing, antibacterial, and anti-tubercular activities [16–23]. For example,
pranoprofen, a safe and effective NSAID with a chromeno[2,3-b]pyridine scaffold,
has been approved to treat conjunctivitis topically by inhibiting prostaglandin
synthesis [24–26]. Pranoprofen has also determined to be as active as diclofenac
sodium in reducing pain and inflammation following strabismus surgery [25, 26].
Interestingly, derivatives of both 4H-chromene and chromeno[2,3-b]pyridine
comprise a benzopyran nucleus, and most have a 2-amino-3-nitrilo functionality.
To the best of our knowledge, only a few studies have focused on structural
modifications of such compounds that are associated with potential anti-inflamma-
tory activity. The conventional syntheses of those compounds by the condensation
of aromatic aldehydes, malononitrile and phenols with or without a catalyst in a
solvent or neat are typically time-consuming (normally [24 h) [27, 28]. Recent
investigations have presented the use of a multicomponent reaction (MCR) to
synthesize certain pharmacologically important heterocyclic compounds in a few
steps or in a one-pot procedure [19, 29]. Accordingly, MCR has been identified as
time-effective and economically favorable for a large number of manufacturing
processes. Microwave methods have enabled in the rapid synthesis of target
compounds, the optimization of chemical reactions, and green chemistry. The
combination of MCR and microwave-assisted synthesis appears to be a fascinating
means of preparing active pharmaceutical ingredients.
Our earlier efforts to design and prepare novel anti-inflammatory agents has led to
the discovery of some unique 4H-chromene derivatives synthesized by MCRs, with
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Synthesis and anti-inflammatory activities of 4H-chromene…
Fig. 1 Derivatives of 4H-chromene and chromeno[2,3-b]pyridine as privileged medicinal scaffolds
Scheme 1 Synthesis of compounds 1a–j
moderate anti-inflammatory activity [30]. In this work, two novel series of substituted
4H-chromenes (1a–j, Scheme 1) and chromeno[2,3-b]pyridines (2a–l, Scheme 2)
were obtained by one-pot, microwave-assisted synthesis in 5–30 min with good
yields. A systematic investigation of the structures of the aforementioned 4H-
chromenes and chromeno[2,3-b]pyridines revealed that all of the synthetic products,
1a–j and 2a–l, significantly inhibited the production of NO in both human and porcine
chondrocytes in vitro. The anti-inflammatory activity of selected products, evaluated
using a carrageenan-induced rat hind paw edema assay [31, 32], indicated that 2d
exhibited very potent anti-inflammatory effect in vivo.
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S.-T. Chung et al.
Scheme 2 Synthesis of compounds 2a–l
Experimental
Chemistry
All the chemicals were purchased from Aldrich-Sigma Chemical Company (St.
Louis, MO, USA) and Alfa-Aesar Chemical Company (Heysham, LA32XY,
England) and purified before use if necessary. All reactions were carried out in a
CEM Discover Benchmate TM microwave apparatus (CEM Corp., Italy). All
reactions were routinely monitored by TLC on Merck F254 silica gel plates.
Melting points were measured on a Bu¨chi-530 melting point apparatus. UV–Vis
spectra were recorded on a Shimazu UV-160 A UV–visible recording spectropho-
tometer. IR spectra were registered on a Perkin-Elmer FTIR 1610 series infrared
spectrophotometer in KBr discs. The 1H- and 13C-NMR spectra were determined on
a Varian Gemini-400 NMR instrument in DMSO-d6 unless otherwise noted.
Chemical shifts (d) were reported as parts per million (ppm) downfield from
tetramethylsilane (TMS) as the internal standard and coupling constants (J) were
given in hertz (Hz). High-resolution mass spectra (HR-MS) were performed in the
Instrument Center of the National Science Counsel at the National Tsing-Hua
University, Taiwan, using a Finnigan MAT-95XL.
General procedure for synthesis of 4H-chromene derivatives 1a–j
To a solution of 3,4,5-trimethoxybenzaldehyde (1.0 mmol), malononitrile (1.0 m-
mol) and substituted phenols (1.0 mmol) in propanol (3.0 mL) was added piperidine
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Synthesis and anti-inflammatory activities of 4H-chromene…
(0.5 mmol). The mixture was subject to microwave irradiation (300 W) at 80 or
120 °C for 5–30 min. The residue was filtered and washed with ethanol and hexane.
Re-crystallization from ethanol afforded pure 1a–j.
2,7-Diamino-4-(3,4,5-trimethoxyphenyl)-4H-chromene-3-carbonitrile (1a)
Conditions: 80 °C, 5 min. yield: 85 %. mp: 208–210 °C (lit. 205–208 °C) [19]. UV
(MeOH): 269 (5.64), 214 (5.21). IR (KBr): 3416, 3356, 3329, 3175 (NH2), 2195
1
(CN), 1647. H-NMR (400 MHz, DMSO-d6): 3.61 (s, 3H), 3.70 (s, 6H), 4.48 (s,
1H), 5.21 (s, 2H), 6.18 (s, 1H), 6.27 (dd, J = 8.4, 2.0 Hz, 1H), 6.44 (s, 2H), 6.69 (d,
1H), 6.75 (s, 2H). 13C-NMR (100 MHz, DMSO-d6): 55.8, 56.1, 59.9, 99.9, 104.5,
109.8, 111.0, 120.9, 129.3, 136.1, 142.4, 148.7, 148.8, 152.8, 160.5. HR-EI-MS:
353.1377 (M?, C19H19N3O4?; calc. 353.1376).
2-Amino-7-(dimethylamino)-4-(3,4,5-trimethoxyphenyl)-4H-chromene-3-
carbonitrile (1b)
Conditions: 80 °C, 5 min. yield: 89 %. mp: 180–182 °C (lit. 179–181 °C) [27]. UV
(MeOH): 268 (5.68). IR (KBr): 3460, 3327, 3198 (NH2), 2193 (CN), 1663. H-
1
NMR (400 MHz, DMSO-d6): 2.85 (s, 6H), 3.61 (s, 3H), 3.70 (s, 6H), 4.55 (s, 1H),
6.21 (sd, J = 2.4 Hz, 1H), 6.44 (sd, J = 2.4 Hz, 1H), 6.47 (s, 2H), 6.79 (s, 2H),
6.86 (d, J = 8.4 Hz, 1H). 13C-NMR (100 MHz, DMSO-d6): 55.8, 56.0, 59.9, 98.5,
104.6, 109.3, 110.3, 120.9, 129.4, 136.2, 142.2, 148.8, 150.2, 152.9, 160.5. HR-EI-
MS: 381.1681 (M?, C21H23N3O4?; calc. 381.1689).
Amino-7-(diethylamino)-4-(3,4,5-trimethoxyphenyl)-4H-chromene-3-carbonitrile
(1c)
Conditions: 80 °C, 5 min. yield: 92 %. mp: 169–171 °C. UV (MeOH): 267 (5.73).
IR (KBr): 3456, 3360, 3198 (NH2), 2189 (CN), 1653. 1H-NMR (400 MHz, DMSO-
d6): 1.04 (t, J = 7.0 Hz, 6H), 3.27 (q, J = 7.2 Hz, 4H), 3.61 (s, 3H), 3.71 (s, 6H),
4.53 (s, 1H, CH), 6.14 (s, 2H), 6.39 (dd, J = 8.4, 2.0 Hz, 1H), 6.48 (s, 2H), 6.77 (s,
2H), 6.85 (d, J = 8.4 Hz, 1H). 13C-NMR (100 MHz, DMSO-d6): 12.3, 43.7, 55.8,
56.0, 60.0, 97.5, 104.6, 108.5, 109.2, 120.9, 129.6, 136.2, 142.2, 147.3, 149.1,
152.9, 160.6. HR-EI-MS: 409.2000 (M?, C23H27N3O4?; calc. 409.2002).
Amino-7-hydroxy-4-(3,4,5-trimethoxyphenyl)-4H-chromene-3-carbonitrile (1d)
Conditions: 80 °C, 5 min. yield: 94 %. mp: 243–245 °C. UV (MeOH): 270 (5.65),
210 (5.13). IR (KBr): 3410, 3333, 3219 (NH2), 2193 (CN), 1653. 1H-NMR
(400 MHz, DMSO-d6): 3.61 (s, 3H), 3.71 (s, 6H), 4.57 (s, 1H), 6.38 (sd, J = 2.4,
Hz, 1H), 6.46 (s, 2H), 6.49 (dd, J = 8.4, 2.0 Hz, 1H), 6.84 (s, 2H), 6.88 (d,
J = 8.4 Hz, 1H), 9.67 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 55.8, 56.0, 60.0,
102.1, 104.6, 112.3, 113.5, 120.7, 129.8, 136.2, 141.9, 148.7, 152.9, 157.0, 160.3.
HR-EI-MS: 354.1217 (M?, C19H18N2O5?; calc. 354.1216).
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S.-T. Chung et al.
2-Amino-7-methoxy-4-(3,4,5-trimethoxyphenyl)-4H-chromene-3-carbonitrile (1e)
Conditions: 120 °C, 30 min. yield: 80 %. mp: 226–227 °C (lit. 228–230 °C) [19].
UV (MeOH): 269 (5.67), 211 (5.17). IR (KBr): 3422, 3337, 3215 (NH2), 2183 (CN),
1651. 1H-NMR (400 MHz, DMSO-d6): 3.61 (s, 3H), 3.71 (s, 6H), 3.72 (s, 3H), 4.63
(s, 1H), 6.48 (s, 2H), 6.54 (sd, J = 2.8, Hz, 1H), 6.66 (dd, J = 8.8, 2.8 Hz, 1H),
6.89 (s, 2H), 7.00 (d, J = 8.8 Hz, 1H). 13C-NMR (100 MHz, DMSO-d6): 55.4, 55.8,
55.9, 59.9, 100.9, 104.6, 111.2, 115.2, 120.6, 129.9, 136.3, 141.8, 148.7, 152.9,
158.9, 160.3. HR-EI-MS: 368.1378 (M?, C20H20N2O5?; calc. 368.1372).
6-Amino-8-(3,4,5-trimethoxyphenyl)-8H-[1,3]dioxolo[4,5-g]chromene-7-
carbonitrile (1f)
Conditions: 80 °C, 5 min. yield: 97 %. mp: 247–249 °C. UV (MeOH): 271 (5.67).
IR (KBr): 3441, 3327, 3209 (NH2), 2186 (CN), 1661. 1H-NMR (400 MHz, DMSO-
d6): 3.62 (s, 3H), 3.72 (s, 6H), 4.56 (s, 1H), 5.94, 5.99 (both d, on J = 0.8 Hz, 1H),
6.49 (s, 2H), 6.63 (s, 1H), 6.66 (s, 1H), 6.86 (s, 2H). 13C-NMR (100 MHz, DMSO-
d6): 40.9, 55.4, 55.8, 59.9, 97.7, 101.6, 104.5, 107.3, 115.4, 120.6, 136.3, 141.6,
142.5, 144.0, 146.7, 153.0, 160.5. HR-EI-MS: 382.1159 (M?, C20H18N2O6?; calc.
382.1165).
2-Amino-4-(3,4,5-trimethoxyphenyl)-4H-benzo[g]chromene-3-carbonitrile (1g)
Conditions: 120 °C, 30 min. yield: 85 %. mp: 181–183 °C. UV (MeOH): 228
(5.20). IR (KBr): 3477, 3356, 3196 (NH2), 2187 (CN), 1655. H-NMR (400 MHz,
1
DMSO-d6): 3.57 (s, 3H), 3.63 (s, 6H), 5.25 (s, 1H), 6.46 (s, 2H), 6.95 (s, 2H), 7.33
(d, J = 8.8 Hz, 1H), 7.41–7.49 (m, 2H), 7.90–7.94 (m, 3H). 13C-NMR (100 MHz,
DMSO-d6): 55.8, 57.8, 59.9, 104.3, 115.5, 116.8, 120.5, 123.7, 125.0, 127.1, 128.4,
129.5, 130.3, 130.8, 136.2, 141.4, 146.8, 152.9, 159.8. HR-EI-MS: 388.1420 (M?,
C23H20N2O4?; calc. 388.1423).
2-Amino-4-(3,4,5-trimethoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile (1h)
Conditions: 80 °C, 5 min. yield: 95 %. mp: 184–185 °C (lit. 185–186 °C) [33]. UV
(MeOH): 224 (5.30). IR (KBr): 3396, 3321, 3198 (NH2), 2185 (CN), 1663. H-
1
NMR (400 MHz, DMSO-d6): 3.61 (s, 3H), 3.70 (s, 6H), 4.86 (s, 1H), 6.57 (s, 2H),
7.13 (s, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.54–7.64 (m, 3H), 7.89 (d, J = 8.0 Hz, 1H),
8.22 (d, J = 8.4 Hz, 1H). 13C-NMR (100 MHz, DMSO-d6): 41.1, 55.9, 56.0, 59.9,
105.0, 117.7, 120.5, 120.8, 122.7, 123.8, 126.2, 126.6, 126.8, 127.7, 132.7, 136.4,
141.3, 142.5, 153.0, 160.3. HR-EI-MS: 388.1430 (M?, C23H20N2O4?; calc.
388.1423).
2-Amino-4-(3,4,5-trimethoxyphenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1i)
Conditions: 120 °C, 25 min. yield: 80 %. mp: 226–228 °C. UV (MeOH): 234
(5.19). IR (KBr): 3447, 3377, 3198 (NH2), 2195 (CN), 1664. H-NMR (400 MHz,
1
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Synthesis and anti-inflammatory activities of 4H-chromene…
DMSO-d6): 3.62 (s, 3H), 3.70 (s, 6H), 4.92 (s, 1H), 6.59 (s, 2H), 7.14 (s, 2H), 7.30
(d, J = 8.4 Hz, 1H), 7.58–7.61 (m, 1H), 7.65 (d, J = 8.4 Hz, 1H), 8.33 (d,
J = 7.6 Hz, 1H), 8.92 (sd, J = 3.2 Hz, 1H). 13C-NMR (100 MHz, DMSO-d6):
41.3, 55.7, 59.9, 60.0, 105.0, 120.5, 121.8, 122.2, 123.5, 126.9, 127.7, 136.0, 136.5,
?
137.5, 141.2, 142.8, 150.2, 153.0, 160.4. HR-EI-MS: 389.1382 (M?, C22H19N3O4
calc. 389.1376).
;
2-Amino-7-morpholino-4-(3,4,5-trimethoxyphenyl)-4H-chromene-3-carbonitrile
(1j)
Conditions: 120 °C, 15 min. yield: 90 %. mp: 227–229 °C. UV (MeOH): 216
(5.25). IR (KBr): 3431, 3331, 3209 (NH2), 2199 (CN), 1666. H-NMR (400 MHz,
1
DMSO-d6): 3.06 (s, 4H), 3.61 (s, 3H), 3.69 (s, 4H), 3.71 (s, 6H), 4.59 (s, 1H), 6.45
(s, 1H), 6.48 (s, 2H), 6.69 (d, J = 8.8 Hz, 1H), 6.85 (s, 2H), 6.93 (d, J = 8.8 Hz,
1H). 13C-NMR (100 MHz, DMSO-d6): 48.0, 55.8, 59.9, 66.0, 101.4, 104.6, 111.9,
113.4, 120.8, 129.4, 136.2, 142.0, 148.7, 150.9, 152.9, 160.5. HR-EI-MS: 423.1788
(M?, C23H25N3O5?; calc. 423.1794).
General procedure for synthesis of chromeno[2,3-b]pyridine derivatives 2a–l
Two drops of TEA were added to a mixture of substituted salicylaldehydes
(1.0 mmol), substituted phenols (1.0 mmol), and malononitrile (2.0 mmol) in
propanol (5.0 mL). The mixture was subjected to microwave irradiation (300 W) at
150 °C for 5–30 min. The residue was filtered and washed with ethanol and hexane.
Re-crystallization from ethanol afforded pure 2a–l.
2,4-Diamino-5-(4-amino-2-hydroxyphenyl)-5H-chromeno[2,3-b]pyridine-3-
carbonitrile (2a)
Conditions: 10 min. yield: 87 %. mp: 297–299 °C. UV (MeOH): 215 (4.63). IR
(KBr): 3444, 3358, 3348, 3241, 3208 (NH2), 2203 (CN), 1617. 1H-NMR (400 MHz,
DMSO-d6): 4.91 (s, 2H), 5.17 (s, 1H), 5.90 (d, J = 8.4 Hz, 1H), 6.06 (s, 1H), 6.16
(s, 2H), 6.35–6.38 (m, 3H), 6.97–7.18 (m, 4H), 9.72 (s, 1H). 13C-NMR (100 MHz,
DMSO-d6): 70.6, 91.4, 100.2 106.9, 116.1, 116.5, 119.6, 123.9, 125.7, 127.4, 129.3,
129.6, 148.3, 150.0, 153.1, 156.6, 158.6, 159.2. HR-EI-MS: 345.1230 (M?,
C19H15N5O2?; calc. 345.1226).
2,4-Diamino-5-(4-(dimethylamino)-2-hydroxyphenyl)-5H-chromeno[2,3-
b]pyridine-3-carbonitrile (2b)
Conditions: 30 min. yield: 92 %. mp: 286–288 °C. UV (MeOH): 229 (4.72). IR
(KBr): 3497, 3433, 3390, 3348, 3241 (NH2), 2192 (CN), 1653, 1636, 1624. H-
1
NMR (400 MHz, DMSO-d6): 2.76 (s, 6H), 5.22 (s, 1H), 6.08 (dd, J = 8.4, 2.4 Hz,
1H), 6.15–6.16 (m, 3H), 6.36 (s, 2H), 6.55 (d, J = 8.8 Hz, 1H), 6.97–7.19 (m, 4H),
9.86 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 40.0, 70.6, 91.2, 98.9, 105.4, 116.1,
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S.-T. Chung et al.
116.5, 120.1, 123.9, 125.4, 127.5, 129.3, 129.6, 150.0, 150.1, 153.1, 156.6, 158.7,
159.2. HR-EI-MS: 373.1532 (M?, C21H19N5O2?; calc. 373.1539).
2,4-Diamino-5-(4-(diethylamino)-2-hydroxyphenyl)-5H-chromeno[2,3-b]pyridine-
3-carbonitrile (2c)
Conditions: 10 min. yield: 85 %. mp: 278–279 °C. UV (MeOH): 231 (4.71). IR
(KBr): 3497, 3444, 3390, 3348, 3241 (NH2), 2192 (CN), 1662, 1617. H-NMR
1
(400 MHz, DMSO-d6): 1.01 (t, J = 6.8 Hz, 6H), 3.18 (q, J = 6.8 Hz, 4H), 5.19 (s,
1H), 6.00 (dd, J = 8.8, 2.4 Hz, 1H), 6.11 (s, 1H), 6.12 (s, 2H), 6.35 (s, 2H), 6.50 (d,
J = 8.8 Hz, 1H), 6.97–7.19 (m, 4H), 9.75 (s, 1H). 13C-NMR (100 MHz, DMSO-
d6): 12.6, 43.6, 70.6, 91.3, 97.7, 104.3, 116.2, 116.5, 118.9, 123.9, 125.6, 127.5,
129.3, 129.8, 147.1, 150.0, 153.4, 156.6, 158.7, 159.2. HR-EI-MS: 401.1855 (M?,
C23H23N5O2?; calc. 401.1852).
2,4-Diamino-5-(1-hydroxynaphthalen-2-yl)-5H-chromeno[2,3-b]pyridine-3-
carbonitrile (2d)
Conditions: 5 min. yield: 93 %. mp: 285–287 °C (lit. 283–284 °C) [28]. UV
(MeOH): 229 (5.19). IR (KBr): 3410, 3358, 3250 (NH2), 2203 (CN), 1614, 1601.
1H-NMR (400 MHz, DMSO-d6): 5.73 (s, 1H), 6.20 (s, 2H), 6.45 (s, 2H), 6.92–8.30
(m, 10H), 10.08 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 70.7, 90.2, 116.3, 116.4,
121.3, 122.3, 124.1, 124.5, 125.5, 126.0, 126.3, 127.3, 127.6, 128.0, 129.3, 133.0,
146.9, 150.0, 156.7, 158.9, 159.5. HR-EI-MS: 380.1269 (M?, C23H16N4O2?; calc.
380.1273).
2,4-Diamino-5-(4-amino-2-hydroxyphenyl)-9-methoxy-5H-chromeno[2,3-
b]pyridine-3-carbonitrile (2e)
Conditions: 10 min. yield: 83 %. mp: 310–312 °C. UV (MeOH): 235 (4.74). IR
(KBr): 3485, 3443, 3368, 3229 (NH2), 2203 (CN), 1614. 1H-NMR (400 MHz,
DMSO-d6): 3.81 (s, 3H), 4.90 (s, 2H), 5.14 (s, 1H), 5.89 (dd, J = 8.4, 2.0 Hz, 1H),
6.05 (sd, J = 2.0 Hz, 1H), 6.15 (s, 2H), 6.33 (s, 2H), 6.35–6.93 (m, 4H), 9.71 (s,
1H). 13C-NMR (100 MHz, DMSO-d6): 55.8, 70.6, 91.5, 100.3, 107.0, 110.0, 116.6,
119.6, 120.5, 123.6, 126.3, 129.6, 139.6, 147.4, 148.3, 153.1, 156.6, 158.6, 159.2.
HR-EI-MS: 375.1323 (M?, C20H17N5O3?; calc. 375.1331).
2,4-Diamino-5-(4-(dimethylamino)-2-hydroxyphenyl)-9-methoxy-5H-
chromeno[2,3-b]pyridine-3-carbonitrile (2f)
Conditions: 30 min. yield: 90 %. mp: 306–308 °C. UV (MeOH): 232 (4.85). IR
(KBr): 3476, 3337, 3229, 3165 (NH2), 2203 (CN), 1624. 1H-NMR (400 MHz,
DMSO-d6): 2.76 (s, 6H), 3.81 (s, 3H), 5.19 (s, 1H), 6.07–6.15 (m, 4H), 6.35 (s, 2H),
6.53–6.93 (m, 4H), 9.85 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 40.0, 55.7, 70.6,
91.1, 98.9, 105.5, 110.0, 116.5, 120.0, 120.4, 123.5, 126.0, 129.6, 139.5, 147.3,
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Synthesis and anti-inflammatory activities of 4H-chromene…
150.1, 153.0, 156.6, 158.6, 159.2. HR-EI-MS: 403.1651 (M?, C22H21N5O3?; calc.
403.1644).
2,4-Diamino-5-(4-(diethylamino)-2-hydroxyphenyl)-9-methoxy-5H-chromeno[2,3-
b]pyridine-3-carbonitrile (2g)
Conditions: 20 min. yield: 80 %. mp: 296–298 °C. UV (MeOH): 232 (5.02), 216
(3.96). IR (KBr): 3425, 3325, 3229 (NH2), 2214 (CN), 1618. H-NMR (400 MHz,
1
DMSO-d6): 1.01 (t, J = 6.8 Hz, 6H), 3.18 (q, J = 6.8 Hz, 4H), 3.81 (s, 3H), 5.16 (s,
1H), 6.00 (d, J = 8.0 Hz, 1H), 6.10 (s, 1H), 6.15 (s, 2H), 6.34 (s, 2H), 6.48–6.93 (m,
4H), 9.74 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 12.6, 43.7, 55.8, 70.6, 91.4,
97.7, 104.4, 110.0, 116.6, 118.8, 120.5, 123.6, 126.2, 129.8, 139.6, 147.2, 147.4,
153.3, 156.6, 158.7, 159.2. HR-EI-MS: 431.1962 (M?, C24H25N5O3?; calc.
431.1957).
2,4-Diamino-5-(1-hydroxynaphthalen-2-yl)-9-methoxy-5H-chromeno[2,3-
b]pyridine-3-carbonitrile (2h)
Conditions: 5 min. yield: 92 %. mp: 300–302 °C (lit. 319–321 °C) [28]. UV
(MeOH): 234 (5.18), 213 (4.19). IR (KBr): 3443, 3358, 3240 (NH2), 2214 (CN),
1614. 1H-NMR (400 MHz, DMSO-d6): 3.83 (s, 3H), 5.70 (s, 1H), 6.19 (s, 2H), 6.43
(s, 2H), 6.71–8.29 (m, 9H), 10.06 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 55.8,
70.7, 90.2, 110.5, 116.4, 120.5, 121.3, 122.4, 123.8, 125.2, 125.5, 125.6, 126.0,
126.4, 127.3, 127.7, 133.1, 139.6, 146.9, 147.5, 156.7, 158.9, 159.5. HR-EI-MS:
410.1374 (M?, C24H18N4O3?; calc. 410.1379).
9,11-Diamino-12-(4-amino-2-hydroxyphenyl)-12H-benzo-[5,6]chromeno[2,3-
b]pyridine-10-carbonitrile (2i)
Conditions: 10 min. yield: 84 %. mp: 303–305 °C. UV (MeOH): 237 (5.01). IR
(KBr): 3443, 3368, 3240 (NH2), 2214 (CN), 1614. 1H-NMR (400 MHz, DMSO-d6):
4.89 (s, 2H), 5.61 (s, 1H), 5.82 (dd, J = 8.4, 2.0 Hz, 1H), 6.09 (s, 1H), 6.35–6.38
(m, 3H), 6.56 (s, 2H), 7.33–8.03 (m, 6H), 10.33 (s, 1H). 13C-NMR (100 MHz,
DMSO-d6): 70.7, 92.3, 99.7, 107.3, 116.6, 117.5, 117.9, 118.9, 123.0, 124.5, 126.9,
128.5, 128.6, 129.6, 130.5, 130.9, 148.0, 148.3, 151.9, 156.8, 158.1, 159.1. HR-EI-
MS: 395.1392 (M?, C23H17N5O2?; calc. 395.1382).
9,11-Diamino-12-(4-(dimethylamino)-2-hydroxyphenyl)-12H-
benzo[5,6]chromeno[2,3-b]pyridine-10-carbonitrile (2j)
Conditions: 30 min. yield: 91 %. mp: 337–339 °C. UV (MeOH): 233 (5.12). IR
(KBr): 3443, 3346, 3240 (NH2), 2203 (CN), 1612. 1H-NMR (400 MHz, DMSO-d6):
2.72 (s, 6H), 5.67 (s, 1H), 6.01 (dd, J = 8.8, 2.4 Hz, 1H), 6.18 (sd, J = 8.8 Hz, 1H),
6.39 (s, 2H), 6.52–6.54 (m, 3H), 7.35–8.04 (m, 6H), 10.49 (s, 1H). 13C-NMR
(100 MHz, DMSO-d6): 39.9, 70.7, 92.1, 98.2, 105.85, 116.5, 117.5, 117.7, 119.2,
123

S.-T. Chung et al.
122.9, 124.5, 126.9, 128.5, 128.7, 129.7, 130.5, 130.9, 148.0, 150.0, 151.9, 156.8,
158.1, 159.1. HR-EI-MS: 423.1687 (M?, C25H21N5O2?; calc. 423.1695).
9,11-Diamino-12-(4-(diethylamino)-2-hydroxyphenyl)-12H-
benzo[5,6]chromeno[2,3-b]pyridine-10-carbonitrile (2k)
Conditions: 20 min. yield: 83 %. mp: 327–329 °C. UV (MeOH): 235 (5.06). IR
(KBr): 3476, 3358, 3231 (NH2), 2203 (CN), 1620. 1H-NMR (400 MHz, DMSO-d6):
0.97 (t, J = 6.8 Hz, 6H), 3.13 (q, J = 6.8 Hz, 4H), 5.65 (s, 1H), 5.93 (dd, J = 8.8,
2.0 Hz, 1H), 6.14 (sd, J = 2.0 Hz, 1H), 6.39 (s, 2H), 6.49 (d, J = 8.4 Hz, 1H), 6.55
(s, 2H), 7.35–8.05 (m, 6H), 10.36 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 12.5,
43.5, 70.7, 92.2, 96.9, 104.7, 116.5, 117.5, 117.8, 118.0, 123.0, 124.5, 126.9, 128.5,
128.6, 129.9, 130.5, 130.9, 147.1, 148.0, 152.2, 156.7, 158.1, 159.1. HR-EI-MS:
451.2006 (M?, C27H25N5O2?; calc. 451.2008).
9,11-Diamino-12-(3-hydroxynaphthalen-2-yl)-12H-benzo[5,6]chromeno[2,3-
b]pyridine-10-carbonitrile (2l)
Conditions: 5 min. yield: 97 %. mp: 315–317 °C (lit. 317–319 °C) [28]. UV
(MeOH): 237 (5.20). IR (KBr): 3497, 3352, 3379, 3250 (NH2), 2214 (CN), 1618.
1H-NMR (400 MHz, DMSO-d6): 6.16 (s, 1H), 6.47 (s, 2H), 6.57 (s, 2H), 6.95–8.36
(m, 12H), 10.79 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 70.8, 91.1, 116.3, 116.9,
117.6, 121.4, 122.1, 122.8, 124.4, 124.6, 125.6, 125.9, 126.0, 126.5, 127.1, 127.7,
128.6, 129.2, 130.5, 130.9, 133.0, 145.9, 148.3, 156.9, 158.4, 159.4. HR-EI-MS:
430.1432 (M?, C27H18N4O2?; calc. 430.1430).
Biological activity
Isolation and culture of human and porcine chondrocytes
Human cartilage from osteoarthritis patients receiving total knee joint replacement
was obtained aseptically with prior approval of the Institutional Review Board
(IRB), Tri-Service General Hospital, Taipei, Taiwan. The porcine cartilage was
obtained from the hind leg joints of pigs. The preparation of chondrocytes from
cartilage was performed according to our previous report [34]. After enzymatic
digestion of articular cartilage with 2 mg/mL protease in serum-free Dulbecco’s
modified Eagle’s medium (DMEM) containing antibiotics and 10 % fetal bovine
serum (FBS), the specimens were then digested overnight with 2 mg/mL
collagenase I and 0.9 mg/mL hyaluronidase in DMEM/antibiotics. The cells were
collected, passed through a cell strainer (Beckton Dickinson, Mountain View, CA,
USA) and cultured in DMEM containing 10 % FBS and antibiotics for 3–4 days
before use.
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Synthesis and anti-inflammatory activities of 4H-chromene…
In vitro cytotoxicity analysis
Potential cytotoxic effects of 1a–2l were evaluated by using 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assays, as described
elsewhere [34]. Briefly, chondrocytes were incubated in the presence or absence of
1a–2l for 48 h. Then, 25 lL of MTT (1 mg/mL in H2O) was added and cells were
incubated at 37 °C for 5 h, prior to adding 100 lL of lysis buffer containing 20 %
sodium dodecyl sulfate and 50 % dimethylformamide. Following incubation at
37 °C for another 2 h, 100 lL of dimethyl sulfoxide (DMSO) was added and the
absorbance was measured at a wavelength of 590 nm using an ELISA reader
(Dynatech, Chantilly, VA, USA).
Measurement of NO production
The release of NO was measured determining the concentration of its stable end
product, nitrite, in the supernatants [34]. The Griess reaction was performed and the
obtained concentrations of nitrite were measured by a spectrophotometer. In brief,
an aliquot (100 lL) of culture supernatant was incubated with 50 lL of 0.1 %
sulfanilamide in 5 % phosphoric acid and 50 lL of 0.1 % N-1-naphthylethylene-
diamine dihydrochloride. After 10 min of incubation at room temperature, the
absorbance was measured at a wavelength of 550 nm using a plate reader (Tecan,
Grodig, Australia).
Animals
Adult male Wister rats (290 20 g) were provided by BioLasco Taiwan Co., Ltd.
(Taipei, Taiwan) and housed in the Laboratory Animal Center of National Defense
Medical Center, Taipei, Taiwan. Rats were maintained on a 12 h light/dark cycle, at
a temperature of 23 2 °C and a humidity of 60 5 %; they were given free
access to water and food. The rats were cared for according to the Guide for the
Care and Use of Laboratory Animals, and the relevant ethics regulations of our
Medical Center.
In vivo anti-inflammatory activity
The in vivo anti-inflammatory activity of the synthetic products was evaluated using
the carrageenan-induced rat hind paw edema assay [32, 33]. Rats were separated at
random into five groups (n = 6). A suspension of the tested compounds 1h and 2d
and reference drugs (quercetin and ibuprofen) in 1 % DMSO was administered
intraperitoneally (10 and 20 mg/kg) 1 h before 0.05 mL of carrageenan (1 %
solution in normal saline) was injected into the plantar tissue of the right hind paw.
Control animals were similarly treated with 1 mL of 1 % DMSO. The volume of
paw edema was measured hourly for 5 h using a plethysmometer (Ugo Basile Srl,
Italy). The edema rate (E %) was calculated as E % = (Vt – V0)/V0 9 100, where
V0 denotes the mean volume of the hind paw before the administering of 1 %
carrageenan and Vt is the mean volume of the hind paw t h after the administering of
123

S.-T. Chung et al.
1 % carrageenan. The percentage inhibition (I %) was given by determined as
E % = (Ec – Et)/Ec 9 100, where Ec is the edema rate of the control group and Et
is the edema rate of the test groups at t h.
Assay of PGE2 in inflammatory rat paw
The paws of rats were cut 0.5 cm away from the ankle joints immediately after the
rats had been euthanized by an intraperitoneal injection of chloral hydrate (400 mg/
kg). The paws were pulverized in liquid nitrogen using a mortar and pestle. The
tissue homogenates [35] were centrifuged at 12,000 rpm at 4 °C for 30 min, and the
resulting supernatant was collected and maintained at -80 °C until further analysis.
Protein levels were quantified by the Bradford protein assay (Bio-Rad, 500-0006).
PGE2 concentrations were measured using an ELISA kit (MyBioSource, San Diego,
CA, USA).
Statistical analysis
Results are expressed as mean SD. Unpaired Student’s t test was used to
determine statistical differences. p \ 0.05 was considered to indicate statistical
significance, unless noted otherwise.
Results and discussion
Chemistry
Schemes 1 and 2 present the synthesis and molecular structures of the titled
compounds. Of the products, 1a, 1b, and 1e are reportedly obtainable with mediocre
yields by conventional synthesis by heating at reflux or stirring at room temperature
[19, 27]. In the preparation herein of those compounds, one-pot microwave-assisted
synthesis was used, and all yields exceeded 80 %. The one-pot preparation of 1h
from 3,4,5-trimethoxybenzaldehyde, malononitrile, and 1-naphthol in the presence
of 4-dimethylaminopyridine as a catalyst under microwave irradiation (MWI) gave
an 89 % yield [33], which, in the modified method, was improved to 95 %
substituting 4-dimethylaminopyridine with piperidine. The conventional synthesis
of compounds 2d, 2h, and 2l involves heating at 110 °C under solvent- and catalyst-
free conditions [28]. However, bumping frequently occurs during the reactions,
even in the presence of boiling chips, and the resulting charring of mixtures
rendered their purification extremely time-consuming. On the contrary, our rather
mild reaction conditions of MWI afforded 2d, 2h, and 2l in a relatively short time
(5–30 min). Table 1 compares the modified reaction conditions and yields of the
known products obtained herein with those of the aforementioned conventional
methods. In addition, none of the aforementioned compounds have ever had
reported anti-inflammatory activity. This study demonstrates that 1h and 2d have
highly potent anti-inflammatory effects, both in vitro and in vivo.
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Synthesis and anti-inflammatory activities of 4H-chromene…
123

S.-T. Chung et al.
123

Synthesis and anti-inflammatory activities of 4H-chromene…
Fig. 2 Structures of 1k and 1l
As outlined in Scheme 1, the derivatives of 4H-chromene were prepared using
equimolar 3,4,5-trimethoxybenzaldehyde, malononitrile, and various substituted
phenols in a one-pot reaction in propanol in the presence of piperidine under MWI
(80 or 120 °C, 300 W, 5–30 min) to yield target compounds 1a–j in favorable
overall yields (80–97 %). The desired final targets were formed in the Knoevenagel
condensation of malononitrile with 3,4,5-trimethoxybenzaldehyde, followed by the
Michael addition of phenols, followed by intramolecular cyclization. A singlet peak
1
at 4.48–5.25 ppm in H-NMR was associated with pyran-CH in 1a–j.
Interestingly, when the reactions were carried out at or below 80 °C, the isolated
products were consistently proved to be the intermediate 2-(3,4,5-trimethoxyben-
zylidene)malononitrile (1k, Fig. 2). Only when conducted in the range of
approximately 80–120 °C did the reaction run to completion. The structure of 1k
1
was elucidated using H-NMR (DMSO-d6: d 3.80, s, 3H; 3.81, s, 6H; 7.36, s, 2H;
8.38, s, 1H) and HR-EI-MS: 244.0842 (M?, C13H12N2O3?; calc. 244.0848).
When 4-aminophenol was used as a Michael addition reagent, after 1 h of
reaction under 300 W of MWI, regardless of temperature and even under neat
conditions, the synthesis failed and an unexpected product was obtained, the
structure of which was (E)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (1l, Fig. 2),
determined by spectral analysis by 1H-NMR (DMSO-d6: d 3.68, s, 3H; 3.81, s, 6H;
6.81, d, J = 16.0 Hz, 1H; 7.05, s, 2H; 7.55, d, J = 16.0 Hz, 1H) and HR-EI-MS:
219.0892 (M?, C12H13NO3?; calc. 219.0895). While the mechanism of formation of
1l is yet to be determined, the intermediate that firstly formed might have quickly
degraded to (E)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (1l), preventing it from
reacting further. The microwave irradiation power appeared to be critical in this
one-pot reaction, as a similar reaction in the literature that used a higher power
(540–720 W) gave yields of 70–80 % [29].
To identify the most favorable conditions for synthesizing chromeno[2,3-
b]pyridine derivatives, the microwave-assisted reaction of substituted salicylalde-
hydes, substituted phenols, and malononitrile in a ratio of 1:1:2 was performed neat
or in one of various solvents, such as H2O, toluene, EtOH, or propanol, using one of
two catalysts (piperidine or TEA) under MWI with a maximum power of 300 W.
The reaction in propanol in the presence of TEA at 150 °C for 5–30 min turned out
to be most satisfactory, so these conditions were used for all reactions hereafter. To
the best of our knowledge, the proposed method is the first efficient and convenient
method for preparing the derivatives of chromeno[2,3-b]pyridine. This one-pot
reaction may have proceeded via an initial Knoevenagel condensation of
salicylaldehyde and malononitrile, followed by subsequent intramolecular cycliza-
tion (the Pinner reaction), the product of which was then attacked by phenol to form
123

S.-T. Chung et al.
Table 2 In vitro IC50 of 1a–j in human and porcine chondrocytes by MTT assay and NO production
Compound
R
MTT
NO
IC50 (lM)
Human
IC50 (lM)
Human
Porcine
Porcine
1a
7-NH2
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
13.4 2.9
9.6 0.1
9.1 1.8
12.5 2.0
10.4 0.2
10.4 1.7
15.3 0.8
8.1 0.6
13.1 0.2
14.0 1.4
65.7 3.5
19.9 1.7
9.5 0.3
8.7 0.5
20.8 2.0
17.0 1.5
18.6 1.1
15.8 0.6
8.0 0.2
12.4 1.9
24.1 1.7
63.4 5.5
1b
7-N(CH3)2
7-N(C2H5)2
7-OH
1c
1d
1e
7-OCH3
1f
6,7-OCH2O–
6,7-C6H4–
7,8-C6H4–
7,8-C3H3N–
7-NC4H8O–
1g
1h
1i
1j
Quercetin
Cell lines were treated with different concentrations of the compounds for 48 h as described in the
experimental section
IC50 (lM) values are indicated as the mean standard deviation (n = 6)
a 4H-chromene intermediate, which subsequently reacted with a second molecule of
1
malononitrile to generate the final products. A singlet peak at 5.14–6.16 ppm in H
NMR was characteristic of the pyran-CH in 2a–l.
All of the crude compounds were purified by re-crystallization from ethanol. The
structures of the end products were established spectroscopically.
Cytotoxicity and inhibition of NO production in human and porcine
chondrocytes
The cytotoxic effects of the two new series compounds were tested by performing
the MTT assay in human and porcine chondrocytes [34]. Tables 2 and 3 present the
resulting IC50 values of cytotoxicity. None of these compounds exhibited significant
cytotoxicity in either human or porcine chondrocytes.
After stimulation of human and porcine chondrocytes with TNF-a for 12 h, the
inhibitory effects of 1a–j and 2a–l on the production of NO were measured by
123

Synthesis and anti-inflammatory activities of 4H-chromene…
123

S.-T. Chung et al.
Table 4 Anti-inflammatory activity of 1h and 2d using carrageenan-induced paw edema in rats
Compound Dose
(mg/kg)
Anti-inflammatory activity (% inhibition)
1 h
2 h
3 h
4 h
0
5 h
0
Control
0
0
0
0
1h
10
20
10
20
10
20
10
20
22.8 0.9*
36.3 1.0* 54.4 1.6** 56.1 0.6** 64.3 0.5**
46.2 2.9** 55.7 1.3* 62.9 2.7** 64.3 2.0** 69.0 0.5**
36.3 0.1** 45.9 1.5* 67.3 6.2** 69.0 4.6** 75.4 6.2**
36.2 0.7** 59.4 0.7* 64.3 0.3** 76.6 1.8** 85.4 0.7**
2d
Quercetin
Ibuprofen
6.7 0.9
38.7 1.5
9.8 0.8
19.5 1.8*
19.6 1.4
40.1 0.1* 53.3 1.4** 53.8 0.4** 52.2 0.8*
39.0 0.9* 46.2 0.8** 48.5 1.3* 46.6 1.5*
44.7 7.1* 55.9 0.9** 54.8 1.2** 52.0 0.2**
36.7 6.1*
31.5 1.8
30.3 1.2
Each value represents the mean standard deviation (n = 6)
Each statistically significant difference from control was expressed as * p \ 0.05 and ** p \ 0.01
analyzing the nitrite concentrations using the Griesis reaction [34]. The results thus
obtained revealed that all derivatives of 4H-chromene and chromeno[2,3-b]pyridine
behaved similarly in both human and porcine chondrocytes and that their potencies
in inhibiting NO production exceeded that of the positive control quercetin.
Generally, the derivatives of 4H-chromene exhibited still higher potency than those
of chromeno[2,3-b]pyridine; the most powerful were 1b, 1c, 1h, 2d, 2j, and 2l, with
IC50 values of around 6.9–9.6 lM in human and 6.1–9.5 lM in porcine
chondrocytes. Of the series of 4H-chromene derivatives, compounds with amino
groups substituted at C(7) (1a, 1b, 1c) were more potent than the other compounds.
Introducing an N,N-dimethyl (1b) or an N,N-diethyl group (1c) resulted in the
particularly favorable inhibition of NO, with an activity that was approximately
eight times that of quercetin. The most potent compound in this series was 1h,
which had a benzene ring that was fused to C(7)–C(8) and was more potent than the
1i, in which a pyridine ring was fused at the corresponding C(7)–C(8). However, 1g,
the constitutional isomer of 1h, did not exhibit a potency comparable with that of 1h
in either human or porcine chondrocytes. Of the derivatives of chromeno[2,3-
b]pyridine, compounds in which a benzene ring was fused to C(30)–C(40) (2d) or
C(6)–C(7) (2j) or both (2l) displayed the most potent activity, with IC50 values of
approximately 6.9–7.6 lM in human and 6.1–8.8 lM in porcine chondrocytes.
Of the two series of products, 1h and 2d were selected for further in vivo studies.
Anti-inflammatory activity in carrageenan-induced rat paw edema
Compounds 1h and 2d were tested in vivo using a carrageenan-induced edema
method to evaluate their anti-inflammatory activities; the method was developed by
Winter and modified as described in our previous report [32, 33]. Test groups (1h
and 2d) and reference groups (quercetin and ibuprofen) of Wistar rats received an
intraperitoneal injection at a dose of 10 and 20 mg/kg 1 h before the injection of
123

Synthesis and anti-inflammatory activities of 4H-chromene…
Fig. 3 PGE2 levels (pg/mL) of test groups (1h, 2d) and reference groups (quercetin and ibuprofen). Each
value represents the mean standard deviation (n = 6). Each statistically significant difference from
control was expressed as **p \ 0.01
0.05 mL of a 1 % solution of carrageenan in normal saline into the right hind paw.
The paw volumes were measured at intervals of 1 h for up to 5 h. Table 4 reveals
that both 1h and 2d exhibited significant and dose-dependent anti-inflammatory
activities that were sustained and intensified, respectively, throughout the time
course, whereas the anti-inflammatory activities of references quercetin and
ibuprofen reached their plateaus at about the third hour, subsequently declining
gradually. Additionally, 2d presented extremely potent anti-inflammatory activity
with an inhibitory effect against carrageen-induced edema of 67.3 % (3 h), 69.0 %
(4 h), and 75.4 % (5 h) at 10 mg/kg and 64.3 % (3 h), 76.6 % (4 h), and 85.4 %
(5 h) at 20 mg/kg.
Inhibition of PGE2 production in carrageenan-induced rat paw edema
Levels of PGE2 were evaluated in vivo using the same carrageenan-induced paw
edema in rats [35]. In Fig. 3, both 1h and 2d produced significantly less PGE2 than
did the reference quercetin, once again, in a dose-dependent manner. Compound 2d
displayed greater PGE2-suppressing activity (191.7 pg/mL at 10 mg/kg; 112.5 pg/
mL at 20 mg/kg) than either 1h (369.2 pg/mL at 10 mg/kg; 245.0 pg/mL at 20 mg/
kg) or ibuprofen (285.4 pg/mL at 10 mg/kg; 167.5 pg/mL at 20 mg/kg).
Conclusion
Novel derivatives of 4H-chromenes and chromeno[2,3-b]pyridines were success-
fully prepared by a one-pot, microwave-assisted, three- or four-component
condensation; their anti-inflammatory activities both in vitro and in vivo were
evaluated. Among them, six compounds (1b, 1c, 1h, 2d, 2j, and 2l) exhibited
123

S.-T. Chung et al.
significant anti-inflammatory activities in vitro in both human and porcine
chondrocytes. Moreover, 1h and 2d exhibited a persistent ability to protect against
carrageenan-induced rat paw edema. Compound 2d even demonstrated superiority
over the clinically used ibuprofen in both potency and sustainability. The
advantages of the ease of synthesis, simple purification, potent activity and
favorable safety profile of 2d renders it promising for further use in developing
novel anti-inflammatory agents. Detailed pharmacological and pharmacokinetic
studies are in progress and will be published in the near future.
Acknowledgments We gratefully acknowledge the research grant, NSC 103-2320-B-016-009,
supported from the National Science Council of the Republic of China. The authors would like to
thank Professor Ted Knoy for his editorial assistance.
Conflict of interest The authors declare no conflicts of interest.
Ethical standard This study was performed in accordance with the criteria of the National Academy of
Sciences, and approved by the Institutional Animal Care and Use Committee of National Defense
Medical Center, Taipei, Taiwan (Approval Number: IACUC-13-178). All patients enrolled with informed
consent in this study were purposively sampled under Tri-Service General Hospital Institution Review
Board approval (Approval Number: 1-102-05-091).
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