SUPPORTING INFORMATION

7,8-Dihydroxycoumarin derivatives: In silico molecular docking and in

vitro anticholinesterase activity

Mücahit Özdemir, Duygu Taşkın, Deniz Ceyhan, Baybars Köksoy, Turgut Taşkın, Mustafa

Bulut, Bahattin Yalçın

Characterization

Figure S1. MALDI-TOF-MS of (5) in the presence of CHCA as a MALDI matrix.

Figure S2. MALDI-TOF-MS of (8) in the presence of CHCA as a MALDI matrix.

 DAD1F,Sig=350,4Ref=360,100(TURGUT\TURGUT20222022-10-1314-45-15\001-0101.D)
mAU

23.302
1000

800

600

400

200

-200
10 20 30 40 50 min

Figure S3. The high-performance liquid chromatography (HPLC) spectrum of coumarin 1.

DAD1B,Sig=260,4Ref=360,100(TURGUT\TURGUT20222022-10-1710-15-57\001-0101.D)
mAU
35.518

1600

1400

1200

1000

800

600

400

200

10 20 30 40 50 min

Figure S4. The high-performance liquid chromatography (HPLC) spectrum of coumarin 2.

 DAD1B,Sig=260,4Ref=360,100(TURGUT\TURGUT20222022-10-1910-09-21\001-0101.D)
mAU

34.584
100

80

60

40

20

10 20 30 40 50 min

Figure S5. The high-performance liquid chromatography (HPLC) spectrum of coumarin 3.

DAD1F,Sig=350,4Ref=360,100(TURGUT\TURGUT20222022-10-1713-08-58\001-0101.D)
mAU
1000
39.599

800

600

400

200

10 20 30 40 50 min

Figure S6. The high-performance liquid chromatography (HPLC) spectrum of coumarin 4.

 DAD1F,Sig=350,4Ref=360,100(TURGUT\TURGUT20222022-10-1713-08-58\002-0201.D)
mAU

38.373
1000

800

600

400

200

10 20 30 40 50 min

Figure S7. The high-performance liquid chromatography (HPLC) spectrum of coumarin 5.

DAD1C,Sig=280,4Ref=360,100(TURGUT\TURGUT20222022-10-1314-45-15\001-0101.D)
mAU
23.316

600

400

200

-200

10 20 30 40 50 min

Figure S8. The high-performance liquid chromatography (HPLC) spectrum of coumarin 6.

 DAD1F,Sig=350,4Ref=360,100(TURGUT\TURGUT20222022-10-1910-09-21\001-0101.D)
mAU

34.588
700

600

500

400

300

200

100

10 20 30 40 50 min

Figure S8. The high-performance liquid chromatography (HPLC) spectrum of coumarin 7.

DAD1H,Sig=374,4Ref=360,100(TURGUT\TURGUT20222022-10-1911-14-43\001-0101.D)
mAU

39.203

400

300

200

100

10 20 30 40 50 min

Figure S10. The high-performance liquid chromatography (HPLC) spectrum of coumarin 8.

Table S1. Docking scores of coumarin derivatives (1-8) and standards on the NADPH,

cytochrome P450, myeloperoxidase, lipoxygenase, and xanthine oxidase enzymes.

Sample NADPH Cytochrome Myeloperoxidase Lipoxygenase Xanthine

P450 oxidase
Docking Score (kcal/mol)
[Ligand Efficiency]
-7.1 -6.6 -7.4 -6.9 -8.0
[0.55] [0.51] [0.57] [0.53] [0.62]

-7.4 -7.6 -7.7 -6.8 -8.5

[0.53] [0.54] [0.55] [0.49] [0.61]

-7.1 -6.9 -7.4 -7.0 -8.2

[0.51] [0.49] [0.53] [0.50] [0.59]

-8.5 -9.4 -8.7 -7.9 -9.7

[0.41] [0.45] [0.41] [0.38] [0.46]

-8.5 -7.8 -10.0 -7.9 -9.4

[0.41] [0.37] [0.48] [0.38] [0.45]

-8.4 -7.9 -9.4 -7.6 -9.5

[0.44] [0.42] [0.50] [0.40] [0.50]

-8.8 -8.1 -8.8 -7.9 -9.8

[0.40] [0.37] [0.40] [0.36] [0.45]

-8.6 -8.3 -8.6 -8.3 -9.7

[0.43] [0.42] [0.43] [0.42] [0.49]

-6.1 -5.5 -6.0 -5.7 -6.2

[0.51] [0.46] [0.50] [0.48] [0.52]

-5.8 -5.8 -5.8 -5.4 -6.3

[0.36] [0.36] [0.36] [0.34] [0.39]
Pharmacokinetic Properties Prediction

Table S2. The ADMET properties of 7,8-dihydroxy-coumarin (1).

Molecule Depiction Molecule Properties

Descriptor Value

Molecular Weight 178.143 g/mol

LogP -3.4573

#Rotatable Bonds 3

#Acceptors 5

#Donors 3

Surface Area 72.66

TPSA 70.67

Volume 144.62

Property Value Unit

Absorption

Water solubility -1.786 Numeric (log mol/L)

Caco2 permeability 1.083 Numeric (log Papp in 10-6 cm/s)

Intestinal absorption (human) 65.583 Numeric (% Absorbed)

Skin Permeability -2.591 Numeric (log Kp)

P-glycoprotein substrate No Categorical (Yes/No)

P-glycoprotein I inhibitor No Categorical (Yes/No)

P-glycoprotein II inhibitor No Categorical (Yes/No)

Distribution

VDss (human) -0.207 Numeric (log L/kg)

Fraction unbound (human) 0.411 Numeric (Fu)

BBB permeability -0.142 Numeric (log BB)

CNS permeability -2.907 Numeric (log PS)

Metabolism

CYP2D6 substrate No Categorical (Yes/No)

CYP3A4 substrate No Categorical (Yes/No)

CYP1A2 inhibitor Yes Categorical (Yes/No)

CYP2C19 inhibitor No Categorical (Yes/No)

CYP2C9 inhibitor No Categorical (Yes/No)

CYP2D6 inhibitor No Categorical (Yes/No)

CYP3A4 inhibitor No Categorical (Yes/No)

Excretion

Total Clearance 0.709 Numeric (log ml/min/kg)

Renal OCT2 substrate No Categorical (Yes/No)

Toxicity

AMES toxicity No Categorical (Yes/No)

Max. tolerated dose (human) 0.601 Numeric (log mg/kg/day)

hERG I inhibitor No Categorical (Yes/No)

hERG II inhibitor No Categorical (Yes/No)

Oral Rat Acute Toxicity (LD50) 2.001 Numeric (mol/kg)

Oral Rat Chronic Toxicity (LOAEL) 2.883 Numeric (log mg/kg_bw/day)

Hepatotoxicity No Categorical (Yes/No)

Skin Sensitisation No Categorical (Yes/No)

T.Pyriformis toxicity 0.552 Numeric (log ug/L)

Minnow toxicity 2.03 Numeric (log mM)

Table S3. The ADMET properties of 7,8-dihydroxy-4-methyl-coumarin (2).
Molecule Depiction Molecule Properties

Descriptor Value

Molecular Weight 192.17 g/mol

LogP 1.51262

#Rotatable Bonds 0

#Acceptors 4

#Donors 2

Surface Area 79.033

TPSA 70.67

Volume 161.18

Property Value Unit

Absorption

Water solubility -2.525 Numeric (log mol/L)

Caco2 permeability 0.244 Numeric (log Papp in 10-6 cm/s)

Intestinal absorption (human) 94.056 Numeric (% Absorbed)

Skin Permeability -2.834 Numeric (log Kp)

P-glycoprotein substrate Yes Categorical (Yes/No)

P-glycoprotein I inhibitor No Categorical (Yes/No)

P-glycoprotein II inhibitor No Categorical (Yes/No)

Distribution

VDss (human) 0.372 Numeric (log L/kg)

Fraction unbound (human) 0.38 Numeric (Fu)

BBB permeability 0.025 Numeric (log BB)

CNS permeability -3.224 Numeric (log PS)

Metabolism

CYP2D6 substrate No Categorical (Yes/No)

CYP3A4 substrate No Categorical (Yes/No)

CYP1A2 inhibitor Yes Categorical (Yes/No)

CYP2C19 inhibitor No Categorical (Yes/No)

CYP2C9 inhibitor No Categorical (Yes/No)

CYP2D6 inhibitor No Categorical (Yes/No)

CYP3A4 inhibitor No Categorical (Yes/No)

Excretion

Total Clearance 0.642 Numeric (log ml/min/kg)

Renal OCT2 substrate No Categorical (Yes/No)

Toxicity

AMES toxicity No Categorical (Yes/No)

Max. tolerated dose (human) 0.494 Numeric (log mg/kg/day)

hERG I inhibitor No Categorical (Yes/No)

hERG II inhibitor No Categorical (Yes/No)

Oral Rat Acute Toxicity (LD50) 1.972 Numeric (mol/kg)

Oral Rat Chronic Toxicity (LOAEL) 1.505 Numeric (log mg/kg_bw/day)

Hepatotoxicity No Categorical (Yes/No)

Skin Sensitisation No Categorical (Yes/No)

T.Pyriformis toxicity 0.619 Numeric (log ug/L)

Minnow toxicity 1.432 Numeric (log mM)

Table S4. The ADMET properties of 4,7,8-trihydroxy coumarin (3).
Molecule Depiction Molecule Properties

Descriptor Value

Molecular Weight 194.142 g/mol

LogP 0.9098

#Rotatable Bonds 0

#Acceptors 5

#Donors 3

Surface Area 77.462

TPSA 90.89

Volume 152.64

Property Value Unit

Absorption

Water solubility -2.357 Numeric (log mol/L)

Caco2 permeability 0.262 Numeric (log Papp in 10-6 cm/s)

Intestinal absorption (human) 83.322 Numeric (% Absorbed)

Skin Permeability -2.833 Numeric (log Kp)

P-glycoprotein substrate Yes Categorical (Yes/No)

P-glycoprotein I inhibitor No Categorical (Yes/No)

P-glycoprotein II inhibitor No Categorical (Yes/No)

Distribution

VDss (human) 0.193 Numeric (log L/kg)

Fraction unbound (human) 0.45 Numeric (Fu)

BBB permeability -0.908 Numeric (log BB)

CNS permeability -3.346 Numeric (log PS)

Metabolism

CYP2D6 substrate No Categorical (Yes/No)

CYP3A4 substrate No Categorical (Yes/No)

CYP1A2 inhibitor Yes Categorical (Yes/No)

CYP2C19 inhibitor No Categorical (Yes/No)

CYP2C9 inhibitor No Categorical (Yes/No)

CYP2D6 inhibitor No Categorical (Yes/No)

CYP3A4 inhibitor No Categorical (Yes/No)

Excretion

Total Clearance 0.602 Numeric (log ml/min/kg)

Renal OCT2 substrate No Categorical (Yes/No)

Toxicity

AMES toxicity No Categorical (Yes/No)

Max. tolerated dose (human) 0.472 Numeric (log mg/kg/day)

hERG I inhibitor No Categorical (Yes/No)

hERG II inhibitor No Categorical (Yes/No)

Oral Rat Acute Toxicity (LD50) 1.78 Numeric (mol/kg)

Oral Rat Chronic Toxicity (LOAEL) 1.187 Numeric (log mg/kg_bw/day)

Hepatotoxicity No Categorical (Yes/No)

Skin Sensitisation No Categorical (Yes/No)

T.Pyriformis toxicity 0.414 Numeric (log ug/L)

Minnow toxicity 2.074 Numeric (log mM)

Table S5. The ADMET properties of 7,8-dihydroxy-3-(2-methoxyphenyl)-coumarin (4).
Molecule Depiction Molecule Properties

Descriptor Value

Molecular Weight 284.267 g/mol

LogP 2.8798

#Rotatable Bonds 2

#Acceptors 5

#Donors 2

Surface Area 119.203

TPSA 79.90

Volume 241.58

Property Value Unit

Absorption

Water solubility -3.301 Numeric (log mol/L)

Caco2 permeability 0.911 Numeric (log Papp in 10-6 cm/s)

Intestinal absorption (human) 96.306 Numeric (% Absorbed)

Skin Permeability -2.754 Numeric (log Kp)

P-glycoprotein substrate Yes Categorical (Yes/No)

P-glycoprotein I inhibitor No Categorical (Yes/No)

P-glycoprotein II inhibitor No Categorical (Yes/No)

Distribution

VDss (human) -0.184 Numeric (log L/kg)

Fraction unbound (human) 0.109 Numeric (Fu)

BBB permeability -0.301 Numeric (log BB)

CNS permeability -2.241 Numeric (log PS)

Metabolism

CYP2D6 substrate No Categorical (Yes/No)

CYP3A4 substrate No Categorical (Yes/No)

CYP1A2 inhibitor Yes Categorical (Yes/No)

CYP2C19 inhibitor Yes Categorical (Yes/No)

CYP2C9 inhibitor Yes Categorical (Yes/No)

CYP2D6 inhibitor Yes Categorical (Yes/No)

CYP3A4 inhibitor Yes Categorical (Yes/No)

Excretion

Total Clearance 0.649 Numeric (log ml/min/kg)

Renal OCT2 substrate No Categorical (Yes/No)

Toxicity

AMES toxicity Yes Categorical (Yes/No)

Max. tolerated dose (human) 0.724 Numeric (log mg/kg/day)

hERG I inhibitor No Categorical (Yes/No)

hERG II inhibitor Yes Categorical (Yes/No)

Oral Rat Acute Toxicity (LD50) 2.284 Numeric (mol/kg)

Oral Rat Chronic Toxicity (LOAEL) 1.812 Numeric (log mg/kg_bw/day)

Hepatotoxicity No Categorical (Yes/No)

Skin Sensitisation No Categorical (Yes/No)

T.Pyriformis toxicity 0.446 Numeric (log ug/L)

Minnow toxicity 0.5 Numeric (log mM)

Table S6. The ADMET properties of 7,8-dihydroxy-3-(3-methoxyphenyl)-coumarin (5).
Molecule Depiction Molecule Properties

Descriptor Value

Molecular Weight 284.267 g/mol

LogP 2.8798

#Rotatable Bonds 2

#Acceptors 5

#Donors 2

Surface Area 119.203

TPSA 79.90

Volume 241.58

Property Value Unit

Absorption

Water solubility -3.231 Numeric (log mol/L)

Caco2 permeability 0.91 Numeric (log Papp in 10-6 cm/s)

Intestinal absorption (human) 96.027 Numeric (% Absorbed)

Skin Permeability -2.761 Numeric (log Kp)

P-glycoprotein substrate Yes Categorical (Yes/No)

P-glycoprotein I inhibitor No Categorical (Yes/No)

P-glycoprotein II inhibitor No Categorical (Yes/No)

Distribution

VDss (human) -0.172 Numeric (log L/kg)

Fraction unbound (human) 0.106 Numeric (Fu)

BBB permeability -0.288 Numeric (log BB)

CNS permeability -2.238 Numeric (log PS)

Metabolism

CYP2D6 substrate No Categorical (Yes/No)

CYP3A4 substrate No Categorical (Yes/No)

CYP1A2 inhibitor Yes Categorical (Yes/No)

CYP2C19 inhibitor Yes Categorical (Yes/No)

CYP2C9 inhibitor Yes Categorical (Yes/No)

CYP2D6 inhibitor Yes Categorical (Yes/No)

CYP3A4 inhibitor Yes Categorical (Yes/No)

Excretion

Total Clearance 0.585 Numeric (log ml/min/kg)

Renal OCT2 substrate No Categorical (Yes/No)

Toxicity

AMES toxicity Yes Categorical (Yes/No)

Max. tolerated dose (human) 0.718 Numeric (log mg/kg/day)

hERG I inhibitor No Categorical (Yes/No)

hERG II inhibitor Yes Categorical (Yes/No)

Oral Rat Acute Toxicity (LD50) 2.225 Numeric (mol/kg)

Oral Rat Chronic Toxicity (LOAEL) 2.013 Numeric (log mg/kg_bw/day)

Hepatotoxicity No Categorical (Yes/No)

Skin Sensitisation No Categorical (Yes/No)

T.Pyriformis toxicity 0.456 Numeric (log ug/L)

Minnow toxicity 0.318 Numeric (log mM)

Table S7. The ADMET properties of 7,8-dihydroxy-3-phenyl-coumarin (6).
Molecule Depiction Molecule Properties

Descriptor Value

Molecular Weight 254.241 g/mol

LogP 2.8712

#Rotatable Bonds 1

#Acceptors 4

#Donors 2

Surface Area 107.725

TPSA 70.67

Volume 216.03

Property Value Unit

Absorption

Water solubility -2.957 Numeric (log mol/L)

Caco2 permeability 1.028 Numeric (log Papp in 10-6 cm/s)

Intestinal absorption (human) 93.36 Numeric (% Absorbed)

Skin Permeability -2.764 Numeric (log Kp)

P-glycoprotein substrate No Categorical (Yes/No)

P-glycoprotein I inhibitor No Categorical (Yes/No)

P-glycoprotein II inhibitor No Categorical (Yes/No)

Distribution

VDss (human) 0.051 Numeric (log L/kg)

Fraction unbound (human) 0.161 Numeric (Fu)

BBB permeability -0.118 Numeric (log BB)

CNS permeability -2.004 Numeric (log PS)

Metabolism

CYP2D6 substrate No Categorical (Yes/No)

CYP3A4 substrate Yes Categorical (Yes/No)

CYP1A2 inhibitor Yes Categorical (Yes/No)

CYP2C19 inhibitor Yes Categorical (Yes/No)

CYP2C9 inhibitor Yes Categorical (Yes/No)

CYP2D6 inhibitor No Categorical (Yes/No)

CYP3A4 inhibitor Yes Categorical (Yes/No)

Excretion

Total Clearance 0.706 Numeric (log ml/min/kg)

Renal OCT2 substrate No Categorical (Yes/No)

Toxicity

AMES toxicity Yes Categorical (Yes/No)

Max. tolerated dose (human) 0.317 Numeric (log mg/kg/day)

hERG I inhibitor No Categorical (Yes/No)

hERG II inhibitor Yes Categorical (Yes/No)

Oral Rat Acute Toxicity (LD50) 2.634 Numeric (mol/kg)

Oral Rat Chronic Toxicity (LOAEL) 2.209 Numeric (log mg/kg_bw/day)

Hepatotoxicity No Categorical (Yes/No)

Skin Sensitisation No Categorical (Yes/No)

T.Pyriformis toxicity 0.558 Numeric (log ug/L)

Minnow toxicity 1.003 Numeric (log mM)

Table S8. The ADMET properties of 7,8-dihydroxy-3-(2-benzoic acid)-coumarin (7).
Molecule Depiction Molecule Properties

Descriptor Value

Molecular Weight 298.25 g/mol

LogP 2.5694

#Rotatable Bonds 2

#Acceptors 5

#Donors 3

Surface Area 119.203

TPSA 107.97

Volume 243.03

Property Value Unit

Absorption

Water solubility -3.318 Numeric (log mol/L)

Caco2 permeability 0.274 Numeric (log Papp in 10-6 cm/s)

Intestinal absorption (human) 61.065 Numeric (% Absorbed)

Skin Permeability -2.734 Numeric (log Kp)

P-glycoprotein substrate Yes Categorical (Yes/No)

P-glycoprotein I inhibitor No Categorical (Yes/No)

P-glycoprotein II inhibitor No Categorical (Yes/No)

Distribution

VDss (human) -0.349 Numeric (log L/kg)

Fraction unbound (human) 0.106 Numeric (Fu)

BBB permeability -1.138 Numeric (log BB)

CNS permeability -2.44 Numeric (log PS)

Metabolism

CYP2D6 substrate No Categorical (Yes/No)

CYP3A4 substrate No Categorical (Yes/No)

CYP1A2 inhibitor Yes Categorical (Yes/No)

CYP2C19 inhibitor No Categorical (Yes/No)

CYP2C9 inhibitor No Categorical (Yes/No)

CYP2D6 inhibitor No Categorical (Yes/No)

CYP3A4 inhibitor No Categorical (Yes/No)

Excretion

Total Clearance 0.692 Numeric (log ml/min/kg)

Renal OCT2 substrate No Categorical (Yes/No)

Toxicity

AMES toxicity No Categorical (Yes/No)

Max. tolerated dose (human) 0.853 Numeric (log mg/kg/day)

hERG I inhibitor No Categorical (Yes/No)

hERG II inhibitor No Categorical (Yes/No)

Oral Rat Acute Toxicity (LD50) 2.399 Numeric (mol/kg)

Oral Rat Chronic Toxicity (LOAEL) 1.971 Numeric (log mg/kg_bw/day)

Hepatotoxicity No Categorical (Yes/No)

Skin Sensitisation No Categorical (Yes/No)

T.Pyriformis toxicity 0.357 Numeric (log ug/L)

Minnow toxicity 0.912 Numeric (log mM)

Table S9. The ADMET properties of 7,8-dihydroxy-3-(o-tolyl)-coumarin (8).
Molecule Depiction Molecule Properties

Descriptor Value

Molecular Weight 268.268 g/mol

LogP 3.17962

#Rotatable Bonds 1

#Acceptors 4

#Donors 2

Surface Area 114.090

TPSA 70.67

Volume 232.59

Property Value Unit

Absorption

Water solubility -3.21 Numeric (log mol/L)

Caco2 permeability 1 Numeric (log Papp in 10-6 cm/s)

Intestinal absorption (human) 95.723 Numeric (% Absorbed)

Skin Permeability -2.806 Numeric (log Kp)

P-glycoprotein substrate Yes Categorical (Yes/No)

P-glycoprotein I inhibitor No Categorical (Yes/No)

P-glycoprotein II inhibitor No Categorical (Yes/No)

Distribution

VDss (human) -0.075 Numeric (log L/kg)

Fraction unbound (human) 0.138 Numeric (Fu)

BBB permeability -0.059 Numeric (log BB)

CNS permeability -1.915 Numeric (log PS)

Metabolism

CYP2D6 substrate No Categorical (Yes/No)

CYP3A4 substrate No Categorical (Yes/No)

CYP1A2 inhibitor Yes Categorical (Yes/No)

CYP2C19 inhibitor Yes Categorical (Yes/No)

CYP2C9 inhibitor Yes Categorical (Yes/No)

CYP2D6 inhibitor No Categorical (Yes/No)

CYP3A4 inhibitor Yes Categorical (Yes/No)

Excretion

Total Clearance 0.63 Numeric (log ml/min/kg)

Renal OCT2 substrate No Categorical (Yes/No)

Toxicity

AMES toxicity Yes Categorical (Yes/No)

Max. tolerated dose (human) 0.743 Numeric (log mg/kg/day)

hERG I inhibitor No Categorical (Yes/No)

hERG II inhibitor Yes Categorical (Yes/No)

Oral Rat Acute Toxicity (LD50) 2.11 Numeric (mol/kg)

Oral Rat Chronic Toxicity (LOAEL) 1.266 Numeric (log mg/kg_bw/day)

Hepatotoxicity No Categorical (Yes/No)

Skin Sensitisation No Categorical (Yes/No)

T.Pyriformis toxicity 0.476 Numeric (log ug/L)

Minnow toxicity 0.544 Numeric (log mM)