Heme oxygenase inhibition by 2-Oxy-substituted 1-Azolyl-4-phenylbutanes: Effect of variation of the azole moiety. X-ray crystal structure of human heme oxygenase-1 in complex with 4-phenyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone

ª 2009 John Wiley & Sons A/S

doi: 10.1111/j.1747-0285.2009.00909.x

Chem Biol Drug Des 2010; 75: 68–90

Research Article

Heme Oxygenase Inhibition by 2-Oxy-substituted

1-Azolyl-4-phenylbutanes: Effect of Variation of

the Azole Moiety. X-Ray Crystal Structure of

Human Heme Oxygenase-1 in Complex with

4-Phenyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone^†

Gheorghe Roman¹, Mona N. Rahman²,

the triazole moiety, whereas the phenyl group is

stabilized by hydrophobic interactions from resi-

dues within the binding pocket.

Dragic Vukomanovic³, Zongchao Jia²,

Kanji Nakatsu³and Walter A. Szarek^1,*

Key words: azole-based inhibitors, carbon monoxide, heme, heme

oxygenase, heme oxygenase–inhibitor complex, structure–activity rela-

tionship, X-ray crystallography

¹Department of Chemistry, Queen's University, Kingston, ON K7L

3N6, Canada

²Department of Biochemistry, Queen's University, Kingston, ON K7L

3N6, Canada

Received 19 June 2009, revised 24 September 2009 and accepted for

publication 26 September 2009

³Department of Pharmacology & Toxicology, Queen's University,

Kingston, ON K7L 3N6, Canada

^ÃCorresponding author: Walter A. Szarek,

szarekw@chem.queensu.ca

Heme oxygenase (HO)-1 (HO-1) has emerged as an important regu-

latory molecule and may be important in a number of settings such

as cardiovascular diseases (1), lung injury (2), and neuroprotection

(3). The initial apparent raison d'Þtre of this enzyme was the

'housekeeping degradation' of heme with the simultaneous release

of bilirubin, carbon monoxide (CO), and iron, a collection of products

that was viewed as mere waste. Subsequently, the endogenously

generated CO has been shown to possess intriguing signaling prop-

erties affecting numerous critical cellular functions including inflam-

mation, cellular proliferation, and apoptosis (4), whereas bilirubin is

now recognized as being a potent endogenous antioxidant (5). Pro-

gress in understanding the physiologic roles of the stress-induced

HO-1, its related constitutive isozyme HO-2, and of CO has been

hampered by the use of metalloporphyrins as inhibitors of the

CO ⁄ HO system. These analogs of heme have drawn criticism

because they interfere with the normal functioning of other regula-

tory hemoenzymes such as nitric oxide synthase (6) or soluble guan-

ylyl cyclase (7). Although the use of metalloporphyrins at cautiously

monitored concentrations is still a viable option (8), the limited

range of concentrations in which selectivity is observed proscribes

the utility of metalloporphyrins and has raised criticism about the

validity of data obtained by employing these inhibitors. A major

step forward in the comprehension of the function of the CO ⁄ HO

system could be achieved through the use of significantly more-

selective HO inhibitors. The program developed by our group with

the aim of designing HO inhibitors that are not based on the por-

phyrin ring system was triggered by the discovery of the mamma-

lian lanosterol 14a-demethylase inhibitor (9), azalanstat (1)

(Figure 1), as a drug inhibiting the activity of HO in vitro (10) and

in vivo (11). The initial study has introduced a series of azalanstat

analogs for which the modulation of HO inhibitory activity was

Coordinates and structure factors of the ternary complex of human

HO-1, heme and 4-phenyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone 7 have

been deposited in the Protein Data Bank as entry # 3K4F.

A

series of 1-azolyl-4-phenyl-2-butanones was

designed and synthesized for the inhibition of heme

oxygenases (heme oxygenase-1 and heme oxygen-

ase-2). The replacement of imidazole by other

azoles led to the discovery of novel 1H-1,2,4-triaz-

ole- and 1H-tetrazole-based inhibitors equipotent to

a lead imidazole-based inhibitor. The inhibitors fea-

turing 2H-tetrazole or 1H-1,2,3-triazole as the phar-

macophore were less potent. Monosubstitution at

position 2 or 4(5), or identical disubstitution at posi-

tions 4 and 5 of imidazole by a variety of electron-

withdrawing or electron-donating, small or bulky

groups, as well as the replacement of the tradi-

tional imidazole pharmacophore by an array of 3- or

5-substituted triazoles, identically 3,5-disubsti-

tuted triazoles, 5-substituted-1H- and 5-substi-

tuted-2H-tetrazoles proved to be detrimental to the

inhibition of HO, with a few exceptions. The azole-

dioxolanes and the azole-alcohols derived from the

active azole-ketones were synthesized also, but

these inhibitors were less active than the corre-

sponding imidazole-based analogs. The ﬁrst

reported X-ray crystal structure of human heme

oxygenase-1 in complex with a 1,2,4-triazole-based

inhibitor, namely 4-phenyl-1-(1H-1,2,4-triazol-1-yl)-

2-butanone, was also determined. The inhibitor

binds to the human heme oxygenase-1 distal pocket

through the coordination of heme iron by the N⁴in

68

Azole-Based Heme Oxygenase Inhibitors

Methods and Materials

3-Methylthio-1H-1,2,4-triazole was obtained from Oakwood Products,

Inc. (West Columbia, SC, USA). Dimethyl 1H-imidazole-4,5-dicarboxy-

late was a product of Acros Organics (Geel, Belgium). Ethyl 1H-imida-

zole-2-carboxylate, 2-nitro-1H-imidazole, 4-phenyl-1H-imidazole, and

methyl 1H-1,2,4-triazole-3-carboxylate were purchased from Alfa

Aesar (Ward Hill, MA, USA). All other chemical reagents were

obtained from Sigma-Aldrich (Oakville, ON, Canada). Column chroma-

tography was performed on Silicycle (Quebec City, QC, Canada) silica

gel (230–400 mesh, 60 ꢀ). Analytical thin-layer chromatography was

performed on glass-backed Silicycle precoated silica gel 60 F254

plates, and the compounds were visualized by UV illumination

(254 nm). Melting points were taken on a Mel-Temp II apparatus

(Laboratory Devices, Inc., Holliston, MA, USA) and are uncorrected. ¹H

and ¹³C NMR spectra were recorded on a Bruker Avance 400-MHz

spectrometer (Bruker BioSpin Ltd., Milton, ON, Canada). The signals

owing to residual protons in the deuterated solvents were used as

internal standards for the ¹H NMR spectra. The chemical shifts

for the carbon atoms are given relative to CDCl₃(d = 77.16 ppm),

CD₃OD (d = 49.00 ppm), or d₆-dimethyl sulphoxide (DMSO) (d = 39.52

ppm). High-resolution mass spectra (HRMS) were obtained on an

Applied Biosystems/MDS Sciex QSTAR XL spectrometer (Applied Bio-

systems, Inc., Foster City, CA, USA) equipped with an Agilent HP1100

Cap-LC system either in electron ionization (EI) mode or electrospray

ionization (ESI) mode. The starting materials 2-methylthio-1H-imidaz-

ole (17), 3-phenyl-1H-1,2,4-triazole (18), 3,5-dibromo-1H-1,2,4-triazole

(19), 3,5-diphenyl-1H-1,2,4-triazole (20), and 1-bromo-4-phenyl-2-buta-

none (4) (15) were prepared according to the reported procedures.

1-(1H-Imidazol-1-yl)-4-phenyl-2-butanone (6), 1-((2-phenylethyl)-1,3-

dioxolan-2-yl)methyl)-1H-imidazole (49), and (€)-1-(1H-imidazol-1-yl)-

4-phenyl-2-butanol (57) are known compounds (15).

Figure 1: Azalanstat (1) and the selective heme oxygenase-1

inhibitor (2R,4R)-2-(2-(4-chlorophenyl)ethyl)-2-((1H-imidazol-1-yl)methyl)-

4-methyl-1,3-dioxolane (2).

achieved through the altering of the position of the amino group

attached to the aromatic ring of the phenylthio moiety in com-

pounds having different configurations of the stereogenic center in

the dioxolane moiety (12). A thorough investigation of the effect, on

HO inhibition, of substitution in the phenylthio moiety as well as

the replacement of the phenylthio by a phenoxy moiety in azalan-

stat analogs resulted in a plethora of excellent inhibitors of both

HO-1 and HO-2 (13), whereas the replacement of the phenylthio

moiety with several small functional groups led to the first selective

HO-1 inhibitors, with (2R,4R)-2-(2-(4-chlorophenyl)ethyl)-2-((1H-imi-

dazol-1-yl)methyl)-4-methyl-1,3-dioxolane (2) (Figure 1) being the

most selective (14). The structure–activity relationship study con-

ducted with a series of simple imidazole-dioxolanes, the related

imidazole-ketones, and the corresponding imidazole-alcohols

revealed that the replacement of the chlorine atom in the lead com-

pound by other halogens such as bromine or iodine, supposedly

more able to undergo polar–polar interactions with an unidentified

hydrogen-bonding group at the active site of the enzyme, may con-

tribute to the fine tuning of HO inhibitory activity (15). It was

noticed also that the imidazole-ketones were generally as potent as

the corresponding imidazole-dioxolanes (15). Although some of

these compounds were shown to have little or no effect on rat-

brain nitric oxide synthase and rat lung soluble guanylyl cyclase

(16), the in vitro activities of several cytochrome P450 enzymes

were strongly inhibited, prompting the undertaking of further struc-

ture–activity studies with the view of identifying inhibitors having

improved selectivity. To that point, all the non-porphyrin HO inhibi-

tors that were designed in our group contained an unsubstituted

imidazole ring as the part of the inhibitor that binds to heme.

Therefore, it was considered that a departure from the prototypical

imidazole pharmacophore with the development of HO inhibitors

based on azoles other than imidazole, along with the investigation

of the substitution pattern in these novel azole-based inhibitors, not

only would allow insight into the structural requirements for binding

to heme in HOs, but also could lead potentially to more-potent and

more-selective HO inhibitors.

Brain and spleen tissue were obtained from adult male Sprague–

Dawley rats (250–300 g) purchased from Charles River, Inc. (Mon-

treal, QC, Canada). Rats were maintained on 12 h light cycles and ad

libitum access to water and standard Ralston Purina laboratory chow

5001 (Ren's Feed Supplies, Ltd, Oakville, ON, Canada). All of the ani-

mals were maintained in accordance with principles and guidelines

of the Canadian Council on Animal Care and experimental protocols

approved by the Queen's University Animal Care Committee.

Synthesis of inhibitors

General procedure for the N-alkylation of

azoles using 1-bromo-4-phenyl-2-butanone

A mixture of bromoketone 4 (681 mg, 3 mmol), the azole (3 mmol),

and anhydrous K₂CO₃(414 mg, 3 mmol) in dry N,N-dimethylforma-

mide (DMF) (5 mL) was stirred at room temperature under a nitrogen

atmosphere for 2 h. The mixture was diluted then with water

(60 mL), extracted with ethyl acetate (2 · 20 mL) and then the com-

bined organic phases were washed with water (2 · 20 mL), and

dried over anhydrous Na₂SO₄. The solvent was removed under

reduced pressure to afford a residue that was purified as described.

4-Phenyl-1-(1H-pyrazol-1-yl)-2-butanone (5). Column chromatography

(silica gel, hexanes–ethyl acetate 4:1 v ⁄ v, then ethyl acetate)

afforded a yellowish solid (334 mg, 52%), mp 74–75 ꢀC, R_f0.26

Chem Biol Drug Des 2010; 75: 68–90

69

Roman et al.

1

(hexanes–ethyl acetate 3:1 v ⁄ v). H NMR (400 MHz, CDCl₃): d 2.67

(t, J = 7.4 Hz, 2H), 2.90 (t, J = 7.6 Hz, 2H), 4.87 (s, 2H), 6.33 (t,

J = 2.2. Hz, 1H), 7.12–7.23 (m, 3H), 7.25–7.31 (m, 2H), 7.36 (d,

J = 2.0 Hz, 1H), 7.57 (d, J = 1.6 Hz, 1H). ¹³C NMR (100 MHz,

CDCl₃): d 29.4, 41.2, 60.8, 106.8, 126.5, 128.5, 128.7, 130.7, 140.5,

204.0. HRMS (ESI) calcd for C₁₃H₁₅N₂O: 215.1184 [M+H]⁺; found:

215.1176.

J = 7.6 Hz), 5.32 (s, 2H), 7.17–7.26 (m, 5H), 7.27–7.33 (m, 2H),

7.36–7.41 (m, 1H), 7.63–7.68 (m, 1H), 8.07 (s, 1H). ¹³C NMR

(100 MHz, d₆-DMSO): d 28.7, 40.5, 52.7, 110.4, 119.3, 121.5, 122.3,

126.0, 128.2, 128.4, 134.4, 140.8, 143.0, 144.6, 203.7. HRMS (EI)

calcd for C₁₇H₁₆N₂O: 264.1263 (M⁺); found: 264.1270.

1-(1H-Benzotriazol-1-yl)-4-phenyl-2-butanone (13) and 1-(2H-ben-

zotriazol-2-yl)-4-phenyl-2-butanone (14). Column chromatography

(silica gel, hexanes–ethyl acetate 3:1 v ⁄ v) afforded compound 14

as a white solid (135 mg, 17%), mp 110–111 ꢀC, R_f0.52 (hexanes–

ethyl acetate 3:1 v ⁄ v). ¹H NMR (400 MHz, d₆-DMSO): d 2.85 (t,

J = 7.6 Hz, 2H), 2.97 (t, J = 7.4 Hz, 2H), 5.92 (s, 2H), 7.15–7.25 (m,

3H), 7.25–7.32 (m, 2H), 7.55 (dd, J = 6.4 Hz, J = 2.8 Hz, 2H), 7.93

(dd, J = 6.6 Hz, J = 2.6 Hz, 2H). ¹³C NMR (100 MHz, d₆-DMSO): d

28.4, 40.7, 64.1, 117.9, 126.0, 126.6, 128.4, 128.3, 140.7, 144.0,

201.8. HRMS (EI) calcd for C₁₆H₁₅N₃O: 265.1215 (M⁺); found:

265.1212. Further elution with hexanes–ethyl acetate (3:1 v ⁄ v)

yielded compound 13 as a white solid (454 mg, 57%), mp 142–

143 ꢀC, R_f0.24 (hexanes–ethyl acetate 3:1 v ⁄ v). ¹H NMR

(400 MHz, d₆-DMSO): d 2.87 (t, J = 7.6 Hz, 2H), 3.04 (t, J = 7.6 Hz,

2H), 5.88 (s, 2H), 7.17–7.34 (m, 5H), 7.41 (dd, J = 7.6 Hz, 1H), 7.53

(dd, J = 7.6 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 8.4 Hz,

1H). ¹³C NMR (100 MHz, d₆-DMSO): d 28.5, 40.6, 55.9, 110.7,

119.1, 123.9, 126.0, 127.3, 128.2, 128.4, 133.6, 140.7, 145.0, 202.5.

HRMS (EI) calcd for C₁₆H₁₅N₃O: 265.1215 (M⁺); found: 265.1219.

4-Phenyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone hydrochloride (7). The

crude material separated after the processing was transformed

directly into the hydrochloride by treating its solution in 2-propanol

(12 mL) with 37% HCl (700 mg) to yield a solid that was recrystal-

lized from 2-propanol to give white crystals (362 mg, 48%), mp

1

167–168 ꢀC. H NMR (400 MHz, CD₃OD): d 2.92–3.03 (m, 4H), 5.53

(s, 2H), 7.15–7.31 (m, 5H), 8.90 (s, 1H), 9.75 (s, 1H). ¹³C NMR

(100 MHz, CD₃OD): d 30.0, 42.3, 60.4, 127.3, 129.4, 129.6, 141.8,

144.4, 144.9, 201.3. HRMS (ESI) calcd for C₁₂H₁₄N₃O: 216.1137

[M+H]⁺; found: 216.1134.

4-Phenyl-1-(1H-1,2,3-triazol-1-yl)-2-butanone (8) and 4-phenyl-1-(2H-

1,2,3-triazol-2-yl)-2-butanone (9). Column chromatography (silica

gel, hexanes–ethyl acetate 2:1 v ⁄ v) afforded firstly compound 9 as

a yellowish solid (220 mg, 34%), mp 74–76 ꢀC, R_f0.60 (hexanes–

ethyl acetate 2:1 v ⁄ v). ¹H NMR (400 MHz, CDCl₃): d 2.63 (t,

J = 7.6 Hz, 2H), 2.90 (t, J = 7.6 Hz, 2H), 5.21 (s, 2H), 7.11–7.30 (m,

5H), 7.70 (s, 2H). ¹³C NMR (100 MHz, CDCl₃): d 29.2, 41.3, 63.0,

125.0, 126.5, 128.4, 128.7, 135.4, 140.4, 202.3. HRMS (EI) calcd for

C₁₂H₁₃N₃O: 215.1059 (M⁺); found: 215.1059. Further elution with

ethyl acetate yielded compound 8 as an off-white solid (329 mg,

1-(2-Methyl-1H-imidazol-1-yl)-4-phenyl-2-butanone hydrochloride (15).

Column chromatography (silica gel, ethyl acetate, then ethyl ace-

tate–methanol 1:1 v ⁄ v) afforded an off-white solid. The solution of

this solid in 2-propanol (5 mL) was treated with 37% HCl (400 mg)

to yield the hydrochloride that was recrystallized from 2-propanol–

diethyl ether to give white crystals (468 mg, 59%), mp 159–160 ꢀC.

¹H NMR (400 MHz, CD₃OD): d 2.93–3.03 (m, 4H), 5.21 (s, 2H),

7.17–7.31 (m, 5H), 7.32 (d, J = 2 Hz, 1H), 7.44 (d, J = 2 Hz, 1H).

¹³C NMR (100 MHz, CD₃OD): d 10.4, 30.2, 42.1, 56.8, 119.0, 124.3,

127.4, 129.4, 129.6, 141.8, 147.0, 202.2. HRMS (ESI) calcd for

C₁₄H₁₇N₂O: 229.1341 [M+H]⁺; found: 229.1330.

1

51%), mp 105–106 ꢀC, R_f0.11 (hexanes–ethyl acetate 2:1 v ⁄ v). H

NMR (400 MHz, CDCl₃): d 2.81 (t, J = 7.4 Hz, 2H), 2.96 (t,

J = 7.4 Hz, 2H), 5.16 (s, 2H), 7.15–7.33 (m, 5H), 7.55 (s, 1H), 7.76

(s, 1H). ¹³C NMR (100 MHz, CDCl₃): d 29.5, 41.6, 58.1, 125.0, 126.7,

128.4, 128.8, 134.3, 140.0, 142.0, 201.0. HRMS (EI) calcd for

C₁₂H₁₃N₃O: 215.1059 (M⁺); found: 215.1065.

4-Phenyl-1-(1H-tetrazol-1-yl)-2-butanone (10) and 4-phenyl-1-(2H-

tetrazol-2-yl)-2-butanone (11). Column chromatography (silica gel,

hexanes–ethyl acetate 2:1 v ⁄ v) afforded firstly compound 11 as an

off-white solid (247 mg, 38%), mp 77–78 ꢀC (2-propanol), R_f0.42

4-Phenyl-1-(2-phenyl-1H-imidazol-1-yl)-2-butanone hydrochloride (16).

Column chromatography (silica gel, ethyl acetate–hexanes 2:1 v ⁄ v)

gave a solid that was transformed into the hydrochloride in the

usual manner to give an off-white solid (216 mg, 22%), mp 139–

140 ꢀC (2-propanol–diethyl ether). ¹H NMR (400 MHz, CD₃OD): d

2.84–2.94 (m, 4H), 5.25 (s, 2H), 7.11–7.27 (m, 5H), 7.45–7.50 (m,

2H), 7.54–7.61 (m, 3H), 7.66–7.72 (m, 2H). ¹³C NMR (100 MHz,

CD₃OD): d 30.0, 42.1, 57.6, 120.4, 123.6, 125.7, 127.4, 129.4, 129.6,

130.3, 130.8, 133.8, 141.7, 147.5, 202.6. HRMS (ESI) calcd for

C₁₉H₁₉N₂O: 291.1497 [M+H]⁺; found: 291.1488.

1

(hexanes–ethyl acetate 2:1 v ⁄ v). H NMR (400 MHz, CDCl₃): d 2.78

(t, J = 7.4 Hz, 2H), 2.96 (t, J = 7.4 Hz, 2H), 5.43 (s, 2H), 7.13–7.25

(m, 3H), 7.27–7.33 (m, 2H), 8.59 (s, 1H). ¹³C NMR (100 MHz, CDCl₃):

d 29.2, 41.7, 60.5, 126.7, 128.4, 128.8, 139.9, 153.5, 199.3. HRMS

(EI) calcd for C₁₁H₁₂N₄O: 216.1011 (M⁺); found: 216.1010. Further

elution with ethyl acetate yielded compound 10 as an off-white

solid (350 mg, 54%), mp 123–124 ꢀC (2-propanol), R_f0.12 (hex-

anes–ethyl acetate 2:1 v ⁄ v). ¹H NMR (400 MHz, CDCl₃): d 2.91

(t, J = 6.6 Hz, 2H), 3.01 (t, J = 6.8 Hz, 2H), 5.20 (s, 2H), 7.16–7.26

(m, 3H), 7.29–7.35 (m, 2H), 8.64 (s, 1H). ¹³C NMR (100 MHz, CDCl₃):

d 29.6, 41.9, 56.0, 126.9, 128.4, 129.0, 139.7, 143.7, 199.1. HRMS

(EI) calcd for C₁₁H₁₂N₄O: 216.1011 (M⁺); found: 216.1018.

1-(2-Methylthio-1H-imidazol-1-yl)-4-phenyl-2-butanone (17). Column

chromatography (silica gel, ethyl acetate) yielded a yellowish solid

(461 mg, 59%), mp 80–81 ꢀC, R_f0.56 (ethyl acetate). ¹H NMR

(400 MHz, CDCl₃): d 2.52 (s, 3H), 2.75 (t, J = 7.4 Hz, 2H), 2.95 (t,

J = 7.4 Hz, 2H), 4.65 (s, 2H), 6.82 (s, 1H), 7.11 (s, 1H), 7.14–7.25

(m, 3H), 7.26–7.32 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): d 17.0,

29.6, 41.4, 55.4, 121.9, 126.6, 128.5, 128.8, 130.0, 140.2, 143.3,

202.8. HRMS (ESI) calcd for C₁₄H₁₇N₂OS: 261.1062 [M+H]⁺; found:

261.1069.

1-(1H-Benzimidazol-1-yl)-4-phenyl-2-butanone (12). The solid result-

ing from dilution of the reaction mixture with water was removed

by filtration, washed thoroughly with water, and recrystallized from

2-propanol to give a white solid (420 mg, 53%), mp 172–173 ꢀC.

¹H NMR (400 MHz, d₆-DMSO): d 2.86 (t, J = 7.2 Hz, 2H), 2.95 (t,

70

Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

Ethyl 1-(2-oxo-4-phenylbut-1-yl)-1H-imidazole-2-carboxylate hydro-

chloride (18). Column chromatography (silica gel, ethyl acetate–

hexanes 2:1 v ⁄ v) yielded a solid from which the hydrochloride was

prepared in the usual manner as a white solid (649 mg, 67%), mp

v ⁄ v) yielded the free base as a greenish solid. The hydrochloride

was prepared in the usual manner, and then the solvent was

removed under reduced pressure. After trituration of the residue

with a small volume of methanol, the hydrochloride separated as a

yellowish solid (72 mg, 8%), mp 104–106 ꢀC. ¹H NMR (400 MHz,

d₆-DMSO): d 2.86 (t, J = 7.4 Hz, 2H), 2.98 (t, J = 7.6 Hz, 2H) 5.41

(s, 2H), 7.16–7.26 (3H), 7.26–7.32 (2H), 8.16 (s, 1H). ¹³C NMR

(100 MHz, d₆-DMSO): d 28.4, 40.5, 55.3, 108.3, 112.4, 113.2, 120.9,

126.0, 128.2, 128.4, 140.5, 143.9, 201.4. HRMS (ESI) calcd for

C₁₅H₁₃N₄O: 265.1089 [M+H]⁺; found: 265.1101.

1

137–138 ꢀC (2-propanol–diethyl ether). H NMR (400 MHz, CD₃OD):

d 1.39 (t, J = 7.2 Hz, 3H), 2.92–2.98 (m, 2H), 2.99–3.04 (m, 2H),

4.46 (q, J = 7.1 Hz, 2H), 4.87 (s, 2H), 7.16–7.31 (m, 5H), 7.72 (d,

J = 2 Hz, 1H), 7.79 (d, J = 2 Hz, 1H). ¹³C NMR (100 MHz, CD₃OD):

d 14.2, 30.0, 42.2, 59.3, 65.3, 122.1, 127.3, 128.7, 129.4, 129.6,

135.1, 141.8, 154.8, 201.4. HRMS (ESI) calcd for C₁₆H₁₉N₂O₃:

287.1396 [M+H]⁺; found: 287.1383.

4-Phenyl-1-(4-phenyl-1H-imidazol-1-yl)-2-butanone hydrochloride (24).

Recrystallization of the crude reaction mixture from 2-propanol gave

the free base, which was converted into the hydrochloride in the

usual manner to give an off-white solid (510 mg, 52%), mp 163–

164 ꢀC (2-propanol–diethyl ether). ¹H NMR (400 MHz, CD₃OD): d

2.95–3.03 (m, 4H), 5.30 (s, 2H), 7.16–7.22 (m, 1H), 7.23–7.32 (m, 4H),

7.46–7.57 (m, 3H), 7.68–7.73 (m, 2H), 7.79 (d, J = 1.2 Hz, 1H), 8.88 (s,

1H). ¹³C NMR (100 MHz, CD₃OD): d 30.1, 42.2, 58.2, 120.2, 126.8,

127.3, 127.6, 129.4, 129.6, 130.6, 131.0, 135.1, 138.0, 141.9, 202.2.

HRMS (ESI) calcd for C₁₉H₁₉N₂O: 291.1497 [M+H]⁺; found: 291.1486.

1-(2-Nitro-1H-imidazol-1-yl)-4-phenyl-2-butanone hydrochloride (19). The

amber solid obtained after the processing was recrystallized twice

from 2-propanol to afford the free base. The treatment of the free

base with a twofold-equimolar amount of 37% HCl in acetone

(7 mL) gave a white solid (515 mg, 58%), mp 153–154 ꢀC (2-propa-

nol–diethyl ether). ¹H NMR (400 MHz, d₆-DMSO): d 2.80–2.93 (m,

4H), 5.09 (s, 2H), 7.02 (d, J = 1.2 Hz, 1H), 7.16–7.25 (m, 4H), 7.25–

7.32 (m, 2H). ¹³C NMR (100 MHz, d₆-DMSO): d 30.2, 42.0, 48.4,

57.4, 121.5, 125.8, 127.4, 129.4, 129.6, 133.4, 141.7, 201.1. HRMS

(EI) calcd for C₁₃H₁₃N₃O₃: 259.0957 (M⁺); found: 259.0945.

1-(4-Bromo-1H-imidazol-1-yl)-4-phenyl-2-butanone (25) and 1-(5-

bromo-1H-imidazol-1-yl)-4-phenyl-2-butanone (28). Column chroma-

tography (silica gel, ethyl acetate) gave firstly compound 25 as a

yellowish solid (616 mg, 70%), mp 104–105 ꢀC, R_f0.54 (ethyl ace-

1-(4,5-Dichloro-1H-imidazol-1-yl)-4-phenyl-2-butanone hydrochloride

(20). The crude separated after the processing was transformed

directly into the hydrochloride by treating its solution in 2-propanol

(10 mL) with 37% HCl (700 mg); recrystallization of the hydrochloride

from 2-propanol afforded white crystals (604 mg, 63%), mp 154–

155 ꢀC. ¹H NMR (400 MHz, CD₃OD): d 2.93–2.99 (m, 4H), 5.16 (s, 2H),

7.14–7.31 (m, 5H), 8.27 (s, 1H). ¹³C NMR (100 MHz, CD₃OD): d 30.2,

42.0, 56.3, 118.6, 121.6, 127.3, 129.4, 129.6, 137.4, 141.8, 201.8.

HRMS (EI) calcd for C₁₃H₁₂Cl₂N₂O: 282.0327 (M⁺); found: 282.0338.

1

tate). H NMR (400 MHz, CDCl₃): d 2.77 (t, J = 7.2 Hz, 2H), 2.96 (t,

J = 7.2 Hz, 2H), 4.55 (s, 2H), 6.74 (d, J = 1.2 Hz, 1H), 7.13–7.19 (m,

2H), 7.20–7.26 (m, 2H), 7.28–7.35 (m, 2H). ¹³C NMR (100 MHz,

CDCl₃): d 29.8, 41.4, 56.0, 115.8, 119.3, 126.8, 128.5, 128.9, 137.5,

139.9, 201.7. HRMS (EI) calcd for C₁₃H₁₃BrN₂O: 292.0211 (M⁺);

found: 292.0215. Further elution with ethyl acetate gave compound

28 as a yellowish solid (79 mg, 9%), mp 76–77 ꢀC, R_f0.28 (ethyl

1

1-(4,5-Diphenyl-1H-imidazol-1-yl)-4-phenyl-2-butanone hydrochloride

(21). Column chromatography (silica gel, ethyl acetate) yielded the

free base as a white solid. The hydrochloride was prepared in the

usual manner and was recrystallized from 2-propanol–diethyl ether as

acetate). H NMR (400 MHz, CDCl₃): d 2.79 (t, J = 7.2 Hz, 2H), 2.97

(t, J = 7.2 Hz, 2H), 4.67 (s, 2H), 7.07 (s, 1H), 7.14–7.25 (m, 3H),

7.26–7.34 (m, 2H), 7.55 (s, 1H). ¹³C NMR (100 MHz, CDCl₃): d 29.6,

41.4, 54.6, 103.9, 126.7, 128.5, 128.8, 129.5, 138.8, 140.0, 201.6.

HRMS (EI) calcd for C₁₃H₁₃BrN₂O: 292.0211 (M⁺); found: 292.0210.

1

a white solid (423 mg, 35%), mp 201–202 ꢀC. H NMR (400 MHz,

CD₃OD): d 2.73–2.84 (m, 4H), 5.11 (s, 2H), 7.09–7.14 (m, 2H),

7.15–7.20 (m, 1H), 7.21–7.29 (m, 4H), 7.32–7.43 (m, 5H), 7.44–7.51

(m, 2H), 7.54–7.60 (m, 1H), 9.05 (s, 1H). ¹³C NMR (100 MHz, CD₃OD):

d 29.9, 42.1, 56.3, 126.5, 127.3, 127.9, 128.7, 129.3, 129.6, 130.2,

130.7, 130.8, 131.6, 131.9, 132.0, 132.1, 137.4, 141.7, 202.4. HRMS

(ESI) calcd for C₂₅H₂₂N₂NaO: 389.1630 [M+Na]⁺; found: 389.1615.

1-(4-Nitro-1H-imidazol-1-yl)-4-phenyl-2-butanone (26). Recrystalliza-

tion of the crude reaction mixture from 2-propanol gave a tan solid

(443 mg, 57%), mp 151–152 ꢀC. ¹H NMR (400 MHz, d₆-DMSO): d

2.81–2.92 (m, 4H), 5.18 (s, 2H), 7.16–7.25 (m, 3H), 7.26–7.33 (m,

2H), 7.71 (s, 1H), 8.23 (d, J = 0.8 Hz, 1H). ¹³C NMR (100 MHz, d₆-

DMSO): d 28.5, 40.5, 55.7, 122.7, 126.1, 128.3, 128.4, 138.2, 140.7,

146.7, 202.3. HRMS (EI) calcd for C₁₃H₁₃N₃O₃: 259.0957 (M⁺); found:

259.0957.

Dimethyl

1-(2-oxo-4-phenylbut-1-yl)-1H-imidazole-4,5-dicarboxylate

hydrochloride (22). Column chromatography (silica gel, ethyl ace-

tate) yielded the free base as a white solid. The hydrochloride was

prepared in the usual manner and was recrystallized from 2-propa-

nol–diethyl ether to give a white solid (627 mg, 57%), mp 81–

82 ꢀC. ¹H NMR (400 MHz, CD₃OD): d 2.93 (bs, 4H), 3.80 (s, 3H),

3.91 (s, 3H), 5.25 (s, 2H), 7.13–7.33 (m, 5H), 7.92 (s, 1H). ¹³C NMR

(100 MHz, CD₃OD): d 30.0, 42.1, 53.1, 53.2, 56.8, 126.4, 127.2,

129.4, 129.5, 135.4, 141.9, 142.0, 160.9, 162.9, 203.0. HRMS (ESI)

calcd for C₁₇H₁₉N₂O₅: 331.1294 [M+H]⁺; found: 331.1292.

Methyl 1-(2-oxo-4-phenylbut-1-yl)-1H-imidazole-4-carboxylate (27) and

methyl 1-(2-oxo-4-phenylbut-1-yl)-1H-imidazole-5-carboxylate (29). Col-

umn chromatography (silica gel, ethyl acetate–hexanes 2:1 v ⁄ v) gave

firstly compound 29 as an off-white solid (261 mg, 32%), mp 121–

1

123 ꢀC, R_f0.52 (ethyl acetate–hexanes 2:1 v ⁄ v). H NMR (400 MHz,

CDCl₃): d 2.87 (t, J = 7.2 Hz, 2H), 2.97 (t, J = 7.4 Hz, 2H), 3.79 (s, 3H),

4.98 (s, 2H), 7.13–7.25 (m, 3H), 7.27–7.34 (m, 2H), 7.44 (s, 1H), 7.75 (s,

1H). ¹³C NMR (100 MHz, CDCl₃): d 29.4, 41.5, 51.7, 55.2, 122.5, 126.5,

128.5, 128.6, 137.6, 140.4, 142.7, 161.0, 201.8. HRMS (EI) calcd for

C₁₅H₁₆N₂O₃: 272.1161 (M⁺); found: 272.1169. Further elution with

1-(4,5-Dicyano-1H-imidazol-1-yl)-4-phenyl-2-butanone hydrochloride

(23). Column chromatography (silica gel, ethyl acetate–hexanes 3:2

Chem Biol Drug Des 2010; 75: 68–90

71

Roman et al.

ethyl acetate gave compound 27 as an off-white solid (335 mg,

41%), mp 147–148 ꢀC, R_f0.28 (ethyl acetate–hexanes 2:1 v ⁄ v). H

NMR (400 MHz, CDCl₃): d 2.80 (t, J = 7.2 Hz, 2H), 2.97 (t, J = 7.2 Hz,

2H), 3.89 (s, 3H), 4.63 (s, 2H), 7.14–7.25 (m, 3H), 7.29–7.34 (m, 3H),

7.47 (d, J = 1.2 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃): d 29.9, 41.5,

51.8, 55.9, 126.2, 128.5, 128.9, 134.1, 138.6, 139.9, 163.2, 201.3.

HRMS (EI) calcd for C₁₅H₁₆N₂O₃: 272.1161 (M⁺); found: 272.1161.

gel column (ethyl acetate), and the fractions containing the fast-

eluting compounds (R_fꢀ0.74) were collected; the solvent was

removed under reduced pressure, and the residue was recrystallized

three times from 2-propanol to give a white solid (507 mg, 58%), mp

1

99–100 ꢀC. H NMR (400 MHz, CDCl₃): d 2.82 (t, J = 7.4 Hz, 2H),

2.96 (t, J = 7.4 Hz, 2H), 4.93 (s, 2H), 7.10–7.33 (m, 5H), 7.35–7.48 (m,

3H), 8.02–8.13 (m, 3H). ¹³C NMR (100 MHz, CDCl₃): d 29.5, 41.7, 58.1,

126.5, 126.7, 128.5, 128.7, 128.8, 129.6, 130.7, 140.1, 145.3, 163.0,

201.6. HRMS (EI) calcd for C₁₈H₁₇N₃O: 291.1372 (M⁺); found:

291.1364.

Methyl 1-(2-oxo-4-phenylbut-1-yl)-1H-1,2,4-triazole-3-carboxylate (30)

and methyl 1-(2-oxo-4-phenylbut-1-yl)-1H-1,2,4-triazole-5-carboxylate

(34). Column chromatography (silica gel, ethyl acetate–hexanes

2:1 v ⁄ v) gave firstly compound 34 as a yellowish solid (82 mg,

10%), mp 70–71 ꢀC, R_f0.58 (ethyl acetate–hexanes 2:1 v ⁄ v). ¹H

NMR (400 MHz, CDCl₃): d 2.86 (t, J = 7.2 Hz, 2H), 2.98 (t,

J = 7.6 Hz, 2H), 3.96 (s, 3H), 5.41 (s, 2H), 7.15–7.25 (m, 3H),

7.27–7.34 (m, 2H), 8.02 (s, 1H). ¹³C NMR (100 MHz, CDCl₃): d

29.3, 41.6, 53.3, 59.7, 126.6, 128.4, 128.8, 140.2, 144.9, 151.2,

158.6, 200.2. HRMS (EI) calcd for C₁₄H₁₅N₃O₃: 273.1113 (M⁺);

found: 273.1115. Further elution with ethyl acetate–hexanes (2:1

v ⁄ v) gave compound 30 as an off-white solid (451 mg, 55%), mp

126–127 ꢀC, R_f0.28 (ethyl acetate–hexanes 2:1 v ⁄ v). ¹H NMR

(400 MHz, CDCl₃): d 2.84 (t, J = 7.2 Hz, 2H), 2.97 (t, J = 7.2 Hz,

2H), 4.00 (s, 3H), 5.01 (s, 2H), 7.14–7.27 (m, 3H), 7.28–7.34 (m,

2H), 8.14 (s, 1H). ¹³C NMR (100 MHz, CDCl₃): d 29.4, 41.8, 53.0,

58.4, 126.7, 128.4, 128.9, 139.9, 146.1, 154.9, 160.1, 200.1. HRMS

(EI) calcd for C₁₄H₁₅N₃O₃: 273.1113 (M⁺); found: 273.1102.

1-(3,5-Dibromo-1H-1,2,4-triazol-1-yl)-4-phenyl-2-butanone (36). Recry-

stallization of the product from 2-propanol gave an off-white

solid (660 mg, 59%), mp 90–91 ꢀC. ¹H NMR (400 MHz, CDCl₃):

d 2.81 (t, J = 7.4 Hz, 2H), 2.98 (t, J = 7.4 Hz, 2H), 4.89 (s, 2H),

7.15–7.25 (m, 3H), 7.27–7.33 (m, 2H). ¹³C NMR (100 MHz, CDCl₃):

d 29.4, 41.6, 58.1, 126.8, 128.5, 128.9, 131.2, 139.9, 140.8,

199.2. HRMS (EI) calcd for C₁₂H₁₁Br₂N₃O: 370.9269 (M⁺); found:

370.9270.

1-(3,5-Diphenyl-1H-1,2,4-triazol-1-yl)-4-phenyl-2-butanone (37). Recry-

stallization of the product from 2-propanol gave a white solid

(816 mg, 74%), mp 119–120 ꢀC. ¹H NMR (400 MHz, CDCl₃): d

2.81 (t, J = 7.2 Hz, 2H), 2.95 (t, J = 7.2 Hz, 2H), 4.97 (s, 2H), 7.12–

7.30 (m, 5H), 7.38–7.56 (m, 8H), 8.13–8.18 (m, 2H). ¹³C NMR

(100 MHz, CDCl₃): d 29.3, 41.7, 58.2, 126.6, 126.7, 127.9, 128.5,

128.7, 128.8 (2C), 129.2, 129.5, 130.5, 131.0, 140.3, 157.0, 162.1,

202.3. HRMS (EI) calcd for C₂₄H₂₁N₃O: 367.1685 (M⁺); found:

367.1687.

1-(3-Methylthio-1H-1,2,4-triazol-1-yl)-4-phenyl-2-butanone (31) and

1-(5-methylthio-1H-1,2,4-triazol-1-yl)-4-phenyl-2-butanone hydrochlo-

ride (35). Column chromatography (silica gel, diethyl ether) gave

firstly the free base of compound 35 as a yellowish oil with R_f

0.57 (diethyl ether), which was transformed into the hydrochloride

by treatment with 37% HCl in 2-propanol followed by precipitation

with diethyl ether; white crystals (152 mg, 17%), mp 154–155 ꢀC

(dec.). ¹H NMR (400 MHz, CD₃OD): d 2.75 (s, 3H), 2.91–3.01 (m,

4H), 5.35 (s, 2H), 7.15–7.31 (m, 5H), 8.69 (s, 1H). ¹³C NMR

(100 MHz, CD₃OD): d 16.6, 30.2, 42.2, 59.4, 127.3, 129.4, 129.6,

141.7, 146.9, 155.7, 201.6. HRMS (ESI) calcd for C₁₃H₁₆N₃OS:

262.1014 [M+H]⁺; found: 262.1018. Compound 31 was isolated by

further elution with diethyl ether as a white solid (369 mg, 47%),

1-(5-Methylthio-1H-tetrazol-1-yl)-4-phenyl-2-butanone (38) and 1-(5-

methylthio-2H-tetrazol-2-yl)-4-phenyl-2-butanone (41). Column chro-

matography (silica gel, ethyl acetate–hexanes 1:2 v ⁄ v) gave firstly

compound 41 as a yellowish solid (441 mg, 56%), mp 116–117 ꢀC,

1

R_f0.62 (ethyl acetate–hexanes 1:2 v ⁄ v). H NMR (400 MHz, CDCl₃):

d 2.67 (s, 3H), 2.78 (t, J = 7.4 Hz, 2H), 2.95 (t, J = 7.4 Hz, 2H), 5.32

(s, 2H), 7.13–7.33 (m, 5H). ¹³C NMR (100 MHz, CDCl₃): d 14.5, 29.2,

41.6, 60.7, 126.7, 128.4, 128.8, 139.9, 166.1, 199.3. HRMS (EI) calcd

for C₁₂H₁₄N₄OS: 262.0888 (M⁺); found: 262.0880. Further elution

with ethyl acetate–hexanes (1:2 v ⁄ v) gave compound 38 as an off-

white solid (236 mg, 30%), mp 80–81 ꢀC, R_f0.24 (ethyl acetate–

1

mp 77–78 ꢀC, R_f0.39 (diethyl ether). H NMR (400 MHz, CDCl₃): d

1

2.57 (s, 3H), 2.78 (t, J = 7.4 Hz, 2H), 2.94 (t, J = 7.4 Hz, 2H), 4.83

(s, 2H), 7.12–7.24 (3H), 7.27–7.33 (2H), 7.97 (1H). ¹³C NMR

(100 MHz, CDCl₃): d 14.6, 29.5, 41.6, 57.9, 126.7, 128.5, 128.8,

140.1, 145.5, 162.6, 201.3. HRMS (EI) calcd for C₁₃H₁₅N₃OS:

261.0936 (M⁺); found: 261.0929.

hexanes 1:2 v ⁄ v). H NMR (400 MHz, CDCl₃): d 2.77 (s, 3H), 2.83 (t,

J = 7.4 Hz, 2H), 2.97 (t, J = 7.4 Hz, 2H), 5.00 (s, 2H), 7.14–7.34 (m,

5H). ¹³C NMR (100 MHz, CDCl₃): d 15.8, 29.4, 41.8, 55.2, 126.8,

128.4, 128.9, 139.8, 155.9, 198.9. HRMS (EI) calcd for C₁₂H₁₄N₄OS:

262.0888 (M⁺); found: 262.0880.

1-(3-Nitro-1H-1,2,4-triazol-1-yl)-4-phenyl-2-butanone (32). The solid

isolated after the processing was recrystallized twice from 2-propa-

nol to give cream-colored crystals (445 mg, 57%), mp 100–101 ꢀC.

¹H NMR (400 MHz, d₆-DMSO): d 2.85 (t, J = 7.4 Hz, 2H), 2.95 (t,

J = 7.6 Hz, 2H), 5.52 (s, 2H), 7.16–7.25 (m, 3H), 7.26–7.32 (m, 2H),

8.75 (s, 1H). ¹³C NMR (100 MHz, d₆-DMSO): d 28.3, 40.6, 58.6,

126.0, 128.2, 128.4, 140.6, 148.0, 162.0, 201.5. HRMS (EI) calcd for

C₁₂H₁₂N₄O₃: 260.0909 (M⁺); found: 260.0907.

Ethyl (1-(2-oxo-4-phenylbutyl)-1H-tetrazol-5-yl)acetate (39) and ethyl

(2-(2-oxo-4-phenylbutyl)-2H-tetrazol-5-yl)acetate (42). Column chro-

matography (silica gel, ethyl acetate–hexanes 1:2 v ⁄ v) gave firstly

compound 42 as a yellowish oil (354 mg, 39%), R_f0.40 (ethyl ace-

tate–hexanes 1:2 v ⁄ v). ¹H NMR (400 MHz, CDCl₃): d 1.25 (t,

J = 7.2 Hz, 3H), 2.76 (t, J = 7.6 Hz, 2H), 2.95 (t, J = 7.6 Hz, 2H),

4.00 (s, 2H), 4.19 (q, J = 7.2 Hz, 2H), 5.38 (s, 2H), 7.12–7.32 (m,

5H). ¹³C NMR (100 MHz, CDCl₃): d 14.2, 29.2, 32.0, 41.6, 60.6,

61.8, 126.7, 128.4, 128.8, 139.9, 160.9, 168.2, 199.4. HRMS (EI)

calcd for C₁₅H₁₈N₄O₃: 302.1379 (M⁺); found: 302.1367. Further elu-

tion with ethyl acetate–hexanes (1:2 v ⁄ v) gave compound 39 as a

1-(3-Phenyl-1H-1,2,4-triazol-1-yl)-4-phenyl-2-butanone (33). The crude

material isolated after the processing was passed through a silica

72

Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

yellowish solid (444 mg, 49%), mp 39–40 ꢀC, R_f0.21 (ethyl ace-

tate–hexanes 1:2 v ⁄ v). ¹H NMR (400 MHz, CDCl₃): d 1.26 (t,

J = 7.2 Hz, 3H), 2.85–2.93 (m, 2H), 2.93–3.02 (m, 2H), 3.91 (s, 2H),

4.14 (q, J = 7.2 Hz, 2H), 5.30 (s, 2H), 7.14–7.34 (m, 5H). ¹³C NMR

(100 MHz, CDCl₃): d 14.1, 29.5, 30.1, 41.6, 56.3, 62.5, 126.8, 128.4,

128.9, 139.8, 150.2, 167.1, 199.9. HRMS (EI) calcd for C₁₅H₁₈N₄O₃:

302.1379 (M⁺); found: 302.1367.

NMR (400 MHz, CDCl₃): d 2.89 (t, J = 6.8 Hz, 2H), 2.98 (t,

J = 7.2 Hz, 2H), 3.29 (s, 3H), 5.38 (s, 2H), 7.16–7.25 (m, 3H), 7.27–

7.33 (m, 2H), 8.03 (s, 1H). ¹³C NMR (100 MHz, CDCl₃): d 29.2, 41.5,

43.1, 59.7, 126.7, 128.4, 128.8, 140.0, 151.1, 151.7, 200.6. HRMS

(EI) calcd for C₁₃H₁₅N₃O₃S: 293.0834 (M⁺); found: 293.0843.

1-(5-Methylsulfonyl-1H-tetrazol-1-yl)-4-phenyl-2-butanone (47). White

solid (132 mg, 75%), mp 130–131 ꢀC (2-propanol). ¹H NMR

(400 MHz, CDCl₃): d 2.90–3.04 (m, 4H), 3.40 (s, 3H), 5.51 (s, 2H),

7.15–7.35 (m, 5H). ¹³C NMR (100 MHz, CDCl₃): d 29.3, 41.7, 44.0,

57.6, 126.8, 128.4, 128.9, 139.6, 154.1, 199.0. HRMS (EI) calcd for

C₁₂H₁₄N₄O₃S: 294.0787 (M⁺); found: 294.0782.

4-Phenyl-1-(5-phenyl-1H-tetrazol-1-yl)-2-butanone (40) and 4-phenyl-

1-(5-phenyl-2H-tetrazol-2-yl)-2-butanone (43). Column chromatogra-

phy (silica gel, ethyl acetate–hexanes 1:2 v ⁄ v) gave firstly com-

pound 43 as an off-white solid (658 mg, 75%), mp 109–110 ꢀC, R_f

1

0.63 (ethyl acetate–hexanes 1:2 v ⁄ v). H NMR (400 MHz, CDCl₃): d

2.81 (t, J = 7.4 Hz, 2H), 2.97 (t, J = 7.4 Hz, 2H), 5.42 (s, 2H), 7.13–

7.33 (m, 5H), 7.45–7.54 (m, 3H), 8.11–8.19 (m, 2H). ¹³C NMR

(100 MHz, CDCl₃): d 29.3, 41.6, 60.7, 126.7, 127.1, 127.2, 128.5,

128.8, 129.1, 130.7, 139.9, 165.8, 199.6. HRMS (EI) calcd for

C₁₇H₁₆N₄O: 292.1324 (M⁺); found: 292.1312. Further elution with

ethyl acetate–hexanes (1:2 v ⁄ v) gave compound 40 as an off-white

solid (88 mg, 10%), mp 88–89 ꢀC, R_f0.29 (ethyl acetate–hexanes

1-(5-Methylsulfonyl-2H-tetrazol-2-yl)-4-phenyl-2-butanone (48). White

solid (111 mg, 63%), mp 113–114 ꢀC (2-propanol). ¹H NMR

(400 MHz, CDCl₃): d 2.87–3.02 (m, 4H), 3.37 (s, 3H), 5.52 (s, 2H),

7.15–7.34 (m, 5H). ¹³C NMR (100 MHz, CDCl₃): d 29.3, 41.9, 43.3,

61.4, 126.8, 128.4, 128.9, 139.6, 165.8, 197.7. HRMS (EI) calcd for

C₁₂H₁₄N₄O₃S: 294.0787 (M⁺); found: 294.0785.

1

1:2 v ⁄ v). H NMR (400 MHz, CDCl₃): d 2.86 (t, J = 7.2 Hz, 2H), 2.95

(t, J = 7.2 Hz, 2H), 5.17 (s, 2H), 7.11–7.16 (m, 2H), 7.19–7.31 (m,

3H), 7.43–7.49 (m, 4H), 7.51–7.58 (m, 1H). ¹³C NMR (100 MHz,

CDCl₃): d 29.3, 41.9, 56.0, 123.5, 126.7, 128.4, 128.6, 128.9, 129.5,

131.6, 139.8, 155.5, 199.8. HRMS (EI) calcd for C₁₇H₁₆N₄O:

292.1324 (M⁺); found: 292.1325.

General procedure for the ketalization of 1-

azolyl-4-phenyl-2-butanones using ethylene

glycol

A mixture of 1-azolyl-4-phenyl-2-butanone (2 mmol), ethylene glycol

(1.24 g, 20 mmol), p-toluenesulfonic acid monohydrate (380 mg,

2 mmol), and toluene (30 mL) was heated at reflux temperature

while water was being removed as an azeotrope using a Dean–

Stark trap for 1 h. The trap was emptied, fresh toluene (15 mL)

was added to the reaction mixture and heating at reflux tempera-

ture continued for another hour, and then the trap was emptied

again. This sequence was repeated every 2 h until the reaction

time amounted to 10 h. The solvent was removed under reduced

pressure, and the residue was dissolved in ethyl acetate (30 mL);

the organic solution was sequentially extracted with a saturated

aqueous solution of NaHCO₃(3 · 50 mL), washed with water, dried

over anhydrous Na₂SO₄, and evaporated under reduced pressure to

give a residue that was purified as described.

General procedure for the oxidation of 1-

(methylthio-azolyl)-4-phenyl-2-butanones

A mixture of 1-(methylthio-azolyl)-4-phenyl-2-butanone (0.6 mmol)

and m-chloroperbenzoic acid (295 mg, 1.71 mmol, 75% purity) in

chloroform (10 mL) was stirred at room temperature for 24 h. The

mixture was diluted with dichloromethane (10 mL), and sequentially

extracted with

a

saturated aqueous solution of NaHCO₃

(3 · 30 mL), washed with water, and dried over anhydrous Na₂SO₄.

The solvent was removed to give a residue that was recrystallized.

1-(2-Methylsulfonyl-1H-imidazol-1-yl)-4-phenyl-2-butanone (44). White

solid (126 mg, 72%), mp 110–111 ꢀC (2-propanol). ¹H NMR

(400 MHz, CDCl₃): d 2.88 (t, J = 7.2 Hz, 2H), 2.99 (t, J = 7.4 Hz,

2H), 3.21 (s, 3H), 5.07 (s, 2H), 6.81 (d, J = 1.2 Hz, 1H), 7.17–7.25

(m, 4H), 7.28–7.34 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): d 29.5,

41.4, 43.1, 56.9, 125.3, 126.6, 128.5, 128.8, 129.7, 140.2, 142.5,

201.7. HRMS (ESI) calcd for C₁₄H₁₇N₂O₃S: 293.0960 [M+H]⁺; found:

293.0962.

1-((2-(2-Phenylethyl)-1,3-dioxolan-2-yl)methyl)-1H-1,2,4-triazole hydro-

chloride (50). The crude material was dissolved in 2-propanol

(5 mL) and transformed into the hydrochloride by the treatment of

the solution with 37% HCl (400 mg). After several hours, the sol-

vent was removed using a pipette, and the residue was washed

with diethyl ether (2 · 10 mL) and recrystallized from 2-propanol–

diethyl ether to give a white solid (455 mg, 77%), mp 157–159 ꢀC.

¹H NMR (400 MHz, CD₃OD): d 1.93–2.02 (m, 2H), 2.72–2.82 (m, 2H),

3.81–3.90 (m, 2H), 3.99–4.09 (m, 2H), 4.62 (s, 2H), 7.12–7.31 (m,

5H), 8.82 (s, 1H), 9.73 (s, 1H). ¹³C NMR (100 MHz, CD₃OD): d 30.4,

39.0, 56.6, 66.9, 108.8, 127.1, 129.3, 129.5, 142.7, 144.2, 145.2.

HRMS (ESI) calcd for C₁₄H₁₈N₃O₂: 260.1399 [M+H]⁺; found:

260.1404.

1-(3-Methylsulfonyl-1H-1,2,4-triazol-1-yl)-4-phenyl-2-butanone (45).

White crystals (143 mg, 81%), mp 104–105 ꢀC (dec.) (2-propanol).

¹H NMR (400 MHz, CDCl₃): d 2.88 (t, J = 7.2 Hz, 2H), 2.96 (t,

J = 7.0 Hz, 2H), 3.24 (s, 3H), 5.05 (s, 2H), 7.15–7.25 (m, 3H), 7.27–

7.33 (m, 2H), 8.21 (s, 1H). ¹³C NMR (100 MHz, CDCl₃): d 29.5, 41.9,

42.5, 58.6, 126.8, 128.5, 128.9, 139.9, 146.8, 162.3, 199.8. HRMS

(EI) calcd for C₁₃H₁₅N₃O₃S: 293.0834 (M⁺); found: 293.0830.

1-((2-(2-Phenylethyl)-1,3-dioxolan-2-yl)methyl)-1H-1,2,3-triazole hydro-

chloride (51). The crude material was dissolved in 2-propanol

(5 mL) and transformed into the hydrochloride by the treatment of

the solution with 37% HCl (400 mg). The mixture was kept at room

1-(5-Methylsulfonyl-1H-1,2,4-triazol-1-yl)-4-phenyl-2-butanone (46).

White crystals (97 mg, 55%), mp 102–103 ꢀC (dec.) (ethanol). ¹H

Chem Biol Drug Des 2010; 75: 68–90

73

Roman et al.

temperature overnight, the solvent was removed under reduced

pressure, and the residue was triturated with diethyl ether until it

crystallized. Recrystallization from 2-propanol afforded a white solid

(396 mg, 67%), mp 135–137 ꢀC. ¹H NMR (400 MHz, CD₃OD): d

1.89–1.97 (m, 2H), 2.72–2.80 (m, 2H), 3.70–3.80 (m, 2H), 3.96–4.05

(m, 2H), 4.78 (s, 2H), 7.13–7.21 (m, 3H), 7.22–7.29 (m, 2H), 8.24 (s,

1H), 8.37 (s, 1H). ¹³C NMR (100 MHz, CD₃OD): d 30.4, 39.0, 57.3,

66.9, 109.0, 127.1, 129.3, 129.5, 130.8, 131.2, 142.6. HRMS (ESI)

calcd for C₁₄H₁₈N₃O₂: 260.1399 [M+H]⁺; found: 260.1394.

NMR (400 MHz, CDCl₃): d 1.81–1.92 (m, 2H), 2.56–2.65 (m, 2H),

3.55–3.65 (m, 2H), 3.85–3.95 (m, 4H), 7.08–7.30 (m, 8H), 7.38–7.52

(m, 7H), 7.78 (s, 1H). ¹³C NMR (100 MHz, CDCl₃): d 29.4, 38.7, 49.9,

66.0, 109.0, 126.1, 126.4, 126.7, 128.2, 128.3, 128.6, 128.8, 129.0,

129.5, 130.8, 131.6, 134.7, 138.2, 141.4. HRMS (EI) calcd for

C₂₇H₂₆N₂O₂: 410.1994 (M⁺); found: 410.2004.

General procedure for the reduction of 1-azolyl-

4-phenyl-2-butanones using NaBH₄

2-((2-(2-Phenylethyl)-1,3-dioxolan-2-yl)methyl)-2H-1,2,3-triazole (52).

The crude material was chromatographed (silica gel, hexanes–ethyl

acetate 2:1 v ⁄ v), and the combined fractions (R_fꢀ0.4) were evapo-

rated to give a residue that was recrystallized from 2-propanol–

hexanes to give a white solid (321 mg, 62%), mp 57–58 ꢀC, R_f0.44

(hexanes–ethyl acetate 2:1 v ⁄ v). ¹H NMR (400 MHz, CDCl₃): d

1.91–1.98 (m, 2H), 2.73–2.80 (m, 2H), 3.82–3.92 (m, 2H), 3.97–4.06

(m, 2H), 4.59 (s, 2H), 7.13–7.20 (m, 3H), 7.24–7.30 (m, 2H), 7.65 (s,

2H). ¹³C NMR (100 MHz, CDCl₃): d 29.3, 37.9, 59.1, 65.7, 108.8,

126.0, 128.4, 128.5, 134.7, 141.7. HRMS (ESI) calcd for C₁₄H₁₈N₃O₂:

260.1399 [M+H]⁺; found: 260.1402.

To a solution of 1-azolyl-4-phenyl-2-butanones (1 mmol) in methanol

(15 mL) was added gradually sodium borohydride (114 mg, 3 mmol),

and the mixture was stirred at room temperature for 3 h. The sol-

vent was removed under reduced pressure, the residue was parti-

tioned between ethyl acetate (20 mL) and water (30 mL), and the

aqueous layer was further extracted with ethyl acetate (20 mL). The

combined organic phases were sequentially washed with water

(40 mL) and brine, dried over anhydrous Na₂SO₄, and then the sol-

vent was removed under reduced pressure to give the pure azole-

alcohols.

(€)-4-Phenyl-1-(1H-1,2,4-triazol-1-yl)-2-butanol hydrochloride (58). The

free base was converted into the hydrochloride by treatment with a

twofold-equimolar amount of 37% HCl in 2-propanol. Dilution with

diethyl ether afforded a white solid (193 mg, 76%), mp 125–

1-((2-(2-Phenylethyl)-1,3-dioxolan-2-yl)methyl)-1H-tetrazole (53). The

crude material was recrystallized from a small amount of 2-propanol

to give beige crystals (224 mg, 43%), mp 96–97 ꢀC. ¹H NMR

(400 MHz, CD₃OD): d 1.86–1.96 (m, 2H), 2.70–2.81 (m, 2H), 3.64–3.74

(m, 2H), 3.93–4.03 (m, 2H), 4.71 (s, 2H), 7.12–7.22 (m, 3H), 7.23–7.31

(m, 2H), 9.17 (s, 1H). ¹³C NMR (100 MHz, CD₃OD): d 30.4, 39.1, 53.5,

66.8, 109.0, 127.0, 129.3, 129.5, 142.7, 145.9. HRMS (ESI) calcd for

C₁₃H₁₇N₄O₂: 261.1352 [M+H]⁺; found: 261.1340.

1

126 ꢀC. H NMR (400 MHz, CD₃OD): d 1.73–1.94 (m, 2H), 2.68–2.90

(m, 2H), 3.95–4.04 (m, 1H), 4.37 (dd, J = 8.4 and 14.0 Hz, 1H), 4.51

(dd, J = 3.2 and 14.0 Hz, 1H), 7.14–7.31 (m, 5H), 8.90 (s, 1H), 9.82

(s, 1H). ¹³C NMR (100 MHz, CD₃OD): d 32.5, 37.3, 58.8, 69.1, 127.0,

129.5, 142.8, 143.1, 144.6. HRMS (ESI) calcd for C₁₂H₁₆N₃O:

218.1293 [M+H]⁺; found: 218.1283.

2-((2-(2-Phenylethyl)-1,3-dioxolan-2-yl)methyl)-2H-tetrazole (54). The

crude material was recrystallized from a small amount of 2-propanol

to give tan crystals (219 mg, 42%), mp 73–74 ꢀC. ¹H NMR (400 MHz,

CD₃OD): d 1.94–2.02 (m, 2H), 2.72–2.80 (m, 2H), 3.78–3.89 (m, 2H),

3.94–4.05 (m, 2H), 4.86 (s, 2H), 7.12–7.20 (m, 3H), 7.22–7.28 (m, 2H),

8.72 (s, 1H). ¹³C NMR (100 MHz, CD₃OD): d 30.2, 39.3, 57.6, 66.7,

109.1, 126.9, 129.3, 129.5, 142.9, 153.9. HRMS (ESI) calcd for

C₁₃H₁₆N₄NaO₂: 283.1171 [M+Na]⁺; found: 283.1184.

(€)-4-Phenyl-1-(1H-1,2,3-triazol-1-yl)-2-butanol hydrochloride (59). The

free base was converted into the hydrochloride by treatment with a

twofold-equimolar amount of 37% HCl in 2-propanol. Dilution with

diethyl ether afforded a white solid (218 mg, 86%), mp 84–85 ꢀC.

¹H NMR (400 MHz, CD₃OD): d 1.70–1.95 (m, 2H), 2.67–2.79 (m, 1H),

2.79–2.91 (m, 1H), 3.98–4.07 (m, 1H), 4.57 (dd, J = 8.4 and 13.6 Hz,

1H), 4.76 (dd, J = 3.2 and 14.0 Hz, 1H), 7.13–7.31 (m, 5H), 8.52 (t,

J = 1.6 Hz, 1H), 8.59 (t, J = 1.6 Hz, 1H). ¹³C NMR (100 MHz,

CD₃OD): d 32.5, 37.3, 59.6, 70.0, 127.1, 129.4, 129.5, 130.5, 130.7,

142.7. HRMS (ESI) calcd for C₁₂H₁₆N₃O: 218.1293 [M+H]⁺; found:

218.1283.

1-((2-Phenylethyl-1,3-dioxolan-2-yl)methyl)-4-phenyl-1H-imidazole hyd-

rochloride (55). The crude material was recrystallized from a small

volume of 2-propanol to give the pure free base, which was con-

verted into the hydrochloride by treatment with a twofold-equimolar

amount of 37% HCl in 2-propanol. Dilution with hexanes afforded an

(€)-4-Phenyl-1-(1H-tetrazol-1-yl)-2-butanol (60). White solid (209 mg,

1

off-white solid (349 mg, 47%), mp 190–191 ꢀC. H NMR (400 MHz,

96%), mp 79–80 ꢀC. H NMR (400 MHz, CD₃OD): d 1.65–1.89 (m,

CD₃OD): d 1.97–2.09 (m, 2H), 2.72–2.84 (m, 2H), 3.67–3.79 (m, 2H),

3.97–4.08 (m, 2H), 4.50 (s, 2H), 7.13–7.33 (m, 5H), 7.45–7.60 (m, 3H),

7.68–7.79 (m, 2H), 7.96 (d, J = 1.6 Hz, 1H), 9.06 (d, J = 1.6 Hz, 1H).

¹³C NMR (100 MHz, CD₃OD): d 30.5, 39.1, 54.9, 67.0, 109.1, 120.7,

126.8, 127.1, 127.6, 129.3, 129.6, 130.6, 131.1, 135.0, 138.2, 142.7.

HRMS (ESI) calcd for C₂₁H₂₃N₂O₂: 335.1760 [M+H]⁺; found:

335.1747.

2H), 2.64–2.90 (m, 2H), 3.88–4.00 (m, 1H), 4.42 (dd, J = 8.0 and

14.0 Hz, 1H), 4.59 (dd, J = 3.2 and 14.0 Hz, 1H), 7.12–7.32 (m, 5H),

9.13 (s, 1H). ¹³C NMR (100 MHz, CD₃OD): d 32.6, 37.4, 55.0, 69.8,

127.0, 129.5, 142.9, 145.4. HRMS (EI) calcd for C₁₁H₁₄N₄O:

218.1168 (M⁺); found: 218.1163.

(€)-4-Phenyl-1-(2H-tetrazol-2-yl)-2-butanol (61). White solid (212 mg,

1

97%), mp 65–66 ꢀC. H NMR (400 MHz, CD₃OD): d 1.70–1.92 (m,

1-((2-Phenylethyl-1,3-dioxolan-2-yl)methyl)-4,5-diphenyl-1H-imidazole

(56). Column chromatography of the crude material (silica gel,

ethyl acetate–hexanes 2:1 v ⁄ v) afforded an off-white solid (508 mg,

2H), 2.64–2.91 (m, 2H), 4.08–4.18 (m, 1H), 4.63–4.75 (m, 2H), 7.12–

7.30 (m, 5H), 8.69 (s, 1H). ¹³C NMR (100 MHz, CD₃OD): d 32.5,

37.4, 59.7, 70.0, 127.0, 129.4, 129.5, 142.9, 153.9. HRMS (EI) calcd

for C₁₁H₁₄N₄O: 218.1168 (M⁺); found: 218.1166.

1

62%), mp 139–140 ꢀC, R_f0.68 (ethyl acetate–hexanes 2:1 v ⁄ v). H

74

Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

(€)-4-Phenyl-1-(4-phenyl-1H-imidazol-1-yl)-2-butanol hydrochloride (62).

The free base was recrystallized from 2-propanol and then con-

verted into the hydrochloride in the usual manner to yield a white

Francisco). Bacterial expression and purification of hHO-1 were

based on previously published protocols (23,25,26). The formation of

the heme–HO-1 complex was performed as previously described

(27). Protein concentration of the heme–HO-1 complex was deter-

mined by absorbance [e₄₀₅= 140 ⁄ (mM cm)]. Purity was assessed by

measurement of the R_zratio (A₄₀₅⁄ A₂₈₀> 2.1) and by SDS-PAGE

analysis.

1

solid (164 mg, 50%), mp 155–156 ꢀC. H NMR (400 MHz, CD₃OD):

d 1.71–1.96 (m, 2H), 2.68–2.81 (m, 1H), 2.82–2.94 (m, 1H), 3.90–

3.99 (m, 1H), 4.15 (dd, J = 8.4 and 14.0 Hz, 1H), 4.39 (dd, J = 2.4

and 14.0 Hz, 1H), 7.14–7.32 (m, 5H), 7.45–7.57 (m, 3H), 7.69–7.76

(m, 2H), 7.98 (d, J = 1.6 Hz, 1H), 9.02 (d, J = 1.6 Hz, 1H). ¹³C NMR

(100 MHz, CD₃OD): d 32.6, 37.4, 56.6, 70.1, 119.7, 126.7, 127.1,

127.8, 129.5, 130.6, 131.0, 135.3, 137.3, 142.9. HRMS (ESI) calcd

for C₁₉H₂₁N₂O: 293.1654 [M+H]⁺; found: 293.1664.

Crystallization

Crystallization of 7 in complex with heme–HO-1 was similar to that

previously described for another HO-1 inhibitor, namely 1-(adaman-

tan-1-yl)-2-(1H-imidazol-1-yl)ethanone (27). The heme–hHO-1 com-

plex (412 lM in 20 mM potassium phosphate) was mixed with 7 at

a molar ratio of 1:3 and incubated for 1–1.5 h at room temperature.

Binding was confirmed using spectral analysis to confirm a shift in

the native Soret peak (from 404 to 409 nm) prior to setting up crys-

tallization plates. Sitting-drop vapor diffusion trials were performed

at room temperature using a reservoir solution containing 100 m^M

HEPES (pH 7.5), 2.00–2.28 M ammonium sulfate, and 0.8–1.1% 1,6-

hexanediol.

(€)-1-(4,5-Diphenyl-1H-imidazol-1-yl)-4-phenyl-2-butanol (63). The

resultant residue after methanol had been removed from the reac-

tion mixture was stirred with water to give a residue that was

removed by filtration, washed thoroughly with water, and recrystal-

lized from 2-propanol to yield a white solid (306 mg, 83%), mp

1

177–178 ꢀC. H NMR (400 MHz, CDCl₃): d 1.49–1.65 (m, 1H), 1.65–

1.80 (m, 1H), 2.53–2.67 (m, 1H), 2.79–2.92 (m, 1H), 3.45–3.67 (m,

3H), 6.68 (d, J = 7.2 Hz, 2H), 7.12–7.38 (m, 13 H), 7.67 (s, 1H). ¹³C

NMR (100 MHz, CDCl₃): d 32.0, 36.2, 52.1, 69.5, 126.0, 126.5,

126.8, 128.3, 128.4, 128.6, 128.7, 128.8, 130.2, 131.4, 134.5, 137.1,

137.8, 142.0. HRMS (EI) calcd for C₂₅H₂₄N₂O: 368.1889 (M⁺); found:

368.1878.

Data collection and structure determination

X-ray diffraction measurements were performed at the F1 beamline

of the Cornell High Energy Synchrotron Source. A cryoprotectant

comprised of 100 m^MHEPES (pH 7.5), 2.32 M ammonium sulfate,

0.9% 1,6-hexanediol, and 20% (v ⁄ v) glycerol was used for data

collection. Crystals were subsequently flash-cooled in a stream of

nitrogen at 100 K. Data were processed using HKL2000 (28). The

structure of the protein–inhibitor complex was solved by molecular

replacement using Phaser. The structure of heme–hHO-1 in complex

with inhibitor 64 (PDB# 3CZY) in which both the inhibitor and the

heme had been omitted was the initial probe. An initial template of

7 was generated using CS Chem3D Ultra (Version 6.0, Copyright

2000; CambridgeSoft.com) and the Dundee PRODRG2 Server (29).

Following initial refinement with Refmac5 in the CCP4 suite, the

structures, first of heme and then of compound 7, were inserted

manually and subsequently refined using iterative cycles of Xfit and

Refmac5 in the CCP4 suite (30). Standard parameters for heme

were used as described in its library entry in the program during

refinement, with no additional restraints on planarity and bond

lengths and angles. No restraints other than the defaults generated

by the CCP4 suite have been applied to the ligand 7 during

refinement. Structural alignments were performed using CCP4 (30).

Electrostatic surface potentials were calculated and all images were

prepared using PyMOL (31).

Biologic evaluation of the in vitro HO activity

Heme oxygenase activity in rat spleen and brain microsomal frac-

tions was determined by the quantitation of CO formed from the

degradation of methemalbumin (heme complexed with albumin)

(21,22). Spleen and brain (Sprague–Dawley rats) microsomal frac-

tions were prepared according to the procedure outlined by Apple-

ton et al. (8). Protein concentration of microsomal fractions was

determined by a modification of the Biuret method (22). Incubations

for HO activity analysis were performed under conditions for which

the rate of CO formation [pmol CO ⁄ (min mg protein)] was linear

with respect to time and microsomal protein concentration. Briefly,

reaction mixtures (150 lL) consisting of 100 m^Mphosphate buffer

(pH 7.4), 50 lM methemalbumin, and 1 mg ⁄ mL protein were pre-

incubated with the inhibitors at final concentrations ranging from

0.1 to 100 lM for 10 min at 37 ꢀC. Reactions were initiated by

adding NADPH at a final concentration of 1 m^M, and incubations

were performed for an additional 15 min at 37 ꢀC. Reactions were

stopped by instantly freezing the reaction mixture on dry ice, and

CO formation was monitored by gas chromatography according to

the method described by Vreman and Stevenson (21). The data from

all of the experiments were analyzed and processed as reported in

an earlier article (15).

Molecular modeling of compounds in the

inhibitor-binding site of hHO-1

X-ray crystallography study

Initial templates of the various inhibitors were generated as

described earlier for compound 7. Virtual docking simulation was

performed using A^UTODOCK (Version 3.0) (32). The macromolecule

used for docking was the A chain of heme-conjugated hHO-1 in

complex with inhibitor 7, in which compound 7 had been omitted;

the sulfate ion and 1,6-hexanediol molecule were also removed

prior to docking.

Expression and puriﬁcation of human heme

oxygenase-1 (hHO-1)

For crystallization purposes, a truncated, soluble version of hHO-1

that contains 233 amino acids (hHO1-t233) was used. The hHO1-

t233 ⁄ pBace expression plasmid (23,24) was a generous gift from

Drs. Paul Ortiz de Montellano and John Evans (University of San

Chem Biol Drug Des 2010; 75: 68–90

75

Roman et al.

were assigned using correlation spectroscopy Nuclear Overhauser

effect spectroscopy (NDESY) and Heteronuclear Multiple Bond

Coherence (HMBC) technique.

Results and Discussion

Chemistry

Starting from commercially available benzylacetone (3), the key

intermediate 1-bromo-4-phenylbutan-2-one (4) was prepared by bro-

mination in methanol rather than the usual halogenated solvents

with a view of minimizing the formation of the favored isomeric

3-bromoketone (33). Subsequent alkylation of NH-azoles with 4 in

DMF in the presence of anhydrous K₂CO₃afforded the desired 1-az-

olyl-4-phenylbutan-2-ones (Scheme 1). The N-alkylation of pyrazole

and imidazole yielded only 4-phenyl-1-(1H-pyrazol-1-yl)-2-butanone

(5) and 1-(1H-imidazol-1-yl)-4-phenylbutan-2-one (6), respectively. As

expected, 1,2,4-1H-triazole produced almost exclusively 4-phenyl-1-

(1,2,4-1H-triazol-1-yl)-2-butanone (7); because the formation of the

product arising from the N ⁴-alkylation is thermodynamically sup-

pressed (34), only small amounts (below 5%) of the isomeric 4-phe-

nyl-1-(1,2,4-1H-triazol-4-yl)-2-butanone could be identified in the

crude mixtures by NMR spectroscopy.

Under the same experimental conditions, 1H-benzimidazole led to

the corresponding N ¹-alkylated derivative 12, whereas 1H-benzo-

triazole yielded a mixture of 1-(1H-benzotriazole-1-yl)-4-phenylbutan-

2-one (13) and 1-(2H-benzotriazole-2-yl)-4-phenylbutan-2-one (14) in

a relative ratio of 7:3 (Scheme 2). The correct structures for these

N-alkylated benzotriazoles were assigned based on their different

¹H NMR spectral profile for the protons in the benzene ring of the

heterocyclic fused system (a combination of doublets and multiplets

for the unsymmetrical N-alkylated benzotriazole 13 as opposed to

two sets of distinctive doublet of doublets for the symmetrical

N-alkylated benzotriazole 14).

A single type of N-alkylation product, namely the 1,2-disubstituted

imidazole as in 15–19 and the 1,4,5-trisubstituted imidazole as in

20–23, was produced in the case of 2-substituted imidazoles and

imidazoles 4,5-disubstituted with identical substituents, respectively,

owing to their symmetry (Scheme 3). On the other hand, 4(5)-substi-

tuted imidazoles led to mixed results upon alkylation using bromok-

etone 4. In some cases, only the 1,4-disubstituted imidazole could

be evidenced in the crude reaction mixtures, whereas both 1,4- and

1,5-disubstituted were identified and isolated in other cases

(Scheme 3). It seems that the steric hindrance caused by the bulki-

ness of a substituent rather than the substituent's electronic effects

is responsible for the regioselective N-alkylation of 4(5)-phenyl-1H-

imidazole and 4(5)-nitro-1H-imidazole to give exclusively compounds

24 and 26, respectively. 4(5)-Bromo-1H-imidazole yielded predo-

minantly 1-(4-bromo-1H-imidazol-1-yl)-4-phenylbutan-2-one (25),

together with the isomeric 1-(5-bromo-1H-imidazol-1-yl)-4-phenyl-

butan-2-one (28) representing approximately 10% of the crude

The tautomerism of 1,2,3-1H-triazole and 1H-tetrazole in each case

led to mixtures of regioisomeric azole-ketones that were separable

by means of column chromatography. The major component in the

crude reaction mixture was the N ¹-alkylation product in both

instances. The NMR spectroscopic analysis of the crude reaction

mixture showed that the ratio of regioisomer 8 to regioisomer 9 in

the mixture was approximately 4:3 in the case of 1,2,3-1H-triazole,

and about 3:2 for regioisomers 10 and 11 resulting from the

N-alkylation of 1H-tetrazole. The structural assignment of the sepa-

rated regioisomers 8 and 9 was possible owing to their different

¹H NMR spectra (two singlets each corresponding in intensity to

one magnetically non-equivalent triazole proton in 8; one singlet

corresponding in intensity to two magnetically equivalent triazole

protons in 9). The correct structures for regioisomers 10 and 11

Scheme 1: N-Alkylation of various azoles using 1-bromo-4-phenyl-2-butanone (4). (a) azole, K₂CO₃, N,N-dimethylformamide, rt, 2 h.

76 Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

Scheme 2: Benzimidazole- and benzotriazole-containing heme oxygenase inhibitors. (a) benzo-fused azole, K₂CO₃, N,N-dimethylformamide,

rt, 2 h.

Scheme 3: Preparation of ring-substituted imidazoles as heme oxygenase inhibitors. (a) substituted imidazole, K₂CO₃, N,N-dimethylforma-

mide, rt, 2 h.

reaction mixture, as estimated by NMR spectroscopy. On the other

hand, the N-alkylation of methyl 1H-imidazole-4(5)-carboxylate using

bromoketone 4 led to a mixture in which the 1,4-disubstituted

isomer 27 was present to the extent of approximately 60%,

whereas the 1,5-disubstituted isomer 29 accounted for the rest.

Correlation spectroscopy (NOESY) was employed again was for the

correct structural assignments of these 1,4- and 1,5-disubstituted

imidazoles.

separated from the minor component, the 1,5-disubstituted triazole,

only by a combination of column chromatography and repeated

recrystallization. 3(5)-Nitro-1H-1,2,4-triazole yielded only the steri-

cally less-hindered 1,3-disubstituted regioisomer 32. The correlation

between the proton at position 5 in the triazole ring and the pro-

tons in the methylene group adjacent to the triazole served to

assign correctly the structure for the 1,3-disubstituted triazoles 30–

33, whereas its lack was diagnostic in the structural assignment of

1,5-disubstituted triazoles 34–35. Two triazoles having identical

substituents at positions 3 and 5 were also N-alkylated with bro-

moketone 4 to yield 4-phenyl-1-(3,5-disubstituted-1,2,4-1H-triazol-1-

yl)-2-butanones 36 and 37 (Scheme 4).

The library was extended to incorporate products arising from the

N-alkylation of variously 3(5)-substituted 1H-1,2,4-triazoles with bro-

moketone 4, a process that led in some cases to mixtures of 4-phe-

nyl-1-(3-substituted-1,2,4-1H-triazol-1-yl)-2-butanones (30 and 31)

and 4-phenyl-1-(5-substituted-1,2,4-1H-triazol-1-yl)-2-butanones (34

and 35) and that were resolvable by column chromatography

(Scheme 4); the former regioisomer was the major component in

each case. 3(5)-Phenyl-1H-1,2,4-triazole gave a mixture from which

the major component, the 1,3-disubstituted triazole 33, could be

Under the same conditions, the reaction between bromoketone 4

and a series of 5-substituted-1H-tetrazoles has in each case led to

mixtures of 4-phenyl-1-(5-substituted-1H-tetrazol-1-yl)-2-butanones

38–40 and 4-phenyl-1-(5-substituted-2H-tetrazol-2-yl)-2-butanones

41–43. The latter regioisomer was the major component in the

Chem Biol Drug Des 2010; 75: 68–90

77

Roman et al.

Scheme 4: Synthesis of 1,2,4-triazole- and tetrazole-ketones substituted in the heteroaromatic ring. (a) azole, K₂CO₃, N,N-dimethylforma-

mide, rt, 2 h.

crude reaction mixtures derived from 5-phenyl-1H-tetrazole (ratio

approximately 1:7) and from 5-(methylthio)-1H-tetrazole (ratio

approximately 1:2), presumably on the account of better access of

the alkylating agent to the sterically less-hindered nitrogen atom at

position 2. Interestingly, the ratio of the regioisomers in the crude

reaction mixture resulting from the N-alkylation of ethyl 1H-tetraz-

ole-5-carboxylate with bromoketone 4 was approximately 1:1; in

this case, it was the small size of the methylene group directly

attached to the tetrazole ring that granted equal access to both N ¹

and N ²atoms in the tetrazole. Correlation spectroscopy (NOESY)

was used to distinguish the N ¹- from the N ²-alkylated tetrazoles.

Inhibition of heme oxygenase-1 and heme

oxygenase-2

A preliminary investigation of imidazole and several other azoles as

inhibitors of HOs proved that the NH-azoles themselves were not

active toward these enzymes (data not shown). On the other hand,

all of the imidazole-based inhibitors developed by our group feature

a substituent at N ¹. As a previous report (15) from our group

showed that all of the compounds in a series of 1-(1H-imidazol-1-

yl)-4-(substituted phenyl)-2-butanones were good inhibitors of HOs,

it has been rationalized that a 4-phenyl-2-oxo-1-butyl substituent at

position 1 of the azole would be a suitable appendage for the

design of a series of azole-ketones as potential inhibitors of HOs.

The X-ray structure of co-crystals of two structurally different imid-

azole-based inhibitors and human (27) and rat (35) HO-1 that have

become recently available further confirm that these compounds

inhibit HO-1 by a common binding mode. Regardless of the nature

of the appendage at N ¹of imidazole, these inhibitors coordinate

with heme iron using the lone pair of electrons at N ³, while the

appendage's contribution appears to be in the anchoring of the inhi-

bitor within a hydrophobic pocket on the distal side of the enzyme's

binding pocket. Therefore, any azole having an N ³atom relative to

the appendage was contemplated as a potential replacement for

imidazole. Because of the large number of known five-membered

rings having two or more heteroatoms and the difficulty of synthe-

sizing azoles having the appendage attached to a carbon atom,

azoles containing only nitrogen have been considered for this study.

A first group of inhibitors that have been designed comprises

pyrazole, imidazole, 1,2,3- and 1,2,4-triazole, tetrazole, as well as

benzimidazole and benzotriazole, all being N-alkylated with the

selected appendage. Although pyrazole does not fit the criteria as

The oxidation of the available methylthio-substituted 1-(azolyl)-4-

phenyl-2-butanones 17, 31, 35, 38, and 41 using m-chloroper-

benzoic acid (Scheme 5) afforded new additions to the series of

N ¹-alkylated 2-substituted imidazoles, 3- and 5-substituted triazoles,

and 4- and 5-substituted tetrazoles. The use of an excess of oxidiz-

ing reagent ensured the direct conversion of these thioethers into

sulfones 44–48.

A few selected azole-ketones have been converted into the corre-

sponding azole-dioxolanes 49–56 upon heating with ethylene gly-

col in the presence of p-toluenesulfonic acid in toluene at reflux

temperature (Scheme 6). The reaction conditions established in the

previous report (15) were improved as it was found out that the

use of a tenfold excess of ethylene glycol and the extension of the

heating at reflux temperature to 10 h converts approximately 95%

of the azole-ketones into azole-dioxolanes. Reduction of the same

azole-ketones using NaBH₄in methanol at room temperature

yielded the azole-alcohols 57–63 (Scheme 6).

78

Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

Scheme 5: Oxidation of the

thioether function to sulfone in

azole-ketones. (a) m-chloroperbenzoic

acid, chloroform, rt, 24 h.

Scheme 6: Derivatization of the

carbonyl function in azole-ketones.

(a) ethylene glycol, p-toluenesulfonic

acid, toluene, reflux, 8 h; (B) NaBH₄,

methanol, rt, 3 h.

it does not have an N ³atom relative to the appendage, it has been

included in this group solely for the purpose of supporting the

hypothesis by providing an example of an azole-based non-inhibitor

of HOs. As expected, compound 5 is completely devoid of activity

toward both isozymes (Table 1), and, although it could bind poten-

tially to heme iron by using the lone pair of electrons at N ², the

formation of the complex with the enzyme is prevented by the prox-

imity of the bulky appendage to heme's porphyrin macrocycle. This

rationale is also true for the symmetrical 1,2,3-triazole 9 that does

not have a nitrogen atom at position 3 relative to the appendage

and shows no activity in the CO assay. The replacement of the car-

bon atom at position 2 of imidazole in the fairly potent inhibitor 6

by a nitrogen atom, as in the other isomer of the N ¹-alkylated

1,2,3-triazole 8, leads to a very weak inhibitor of HO-1. The

Chem Biol Drug Des 2010; 75: 68–90

79

Roman et al.

Table 1: Inhibitory potency and

selectivity of the candidate com-

pounds against heme oxygenases

Selectivity index

IC₅₀HO-2/

IC₅₀HO-1 (rat)

IC₅₀(µM) HO-1 IC₅₀(µM) HO-2

Compound

Heterocycle (Het)

(rat spleen)

(rat brain)

O

Het

N

5

6^a

7

>> 100

-

N

4

2

11

18

5

1

2.8

7.2

N

2.5 0.4

89

N

8

9

1

> 100

> 1.1

-

N

>> 100

N

10

11

2.6

1

30

4

11.5

N

9.6 0.2

>> 100

> 100

> 10.4

N

12

13

>> 100

-

N

>> 100

N

14

15

>> 100

-

N

H₃C

N

C₆H₅

N

16

17

18

>> 100

-

H₃CS

N

C₂H₅OOC

N

C₂H₅OOC

N

19

20

>> 100

-

N

Cl

80

Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

Table 1: (Continued)

Selectivity index

IC HO-2/

IC₅₀(μM) HO-1 IC₅₀(μM) HO-2

IC⁵₅⁰₀HO-1 (rat)

(rat spleen)

(rat brain)

Compound

21

Heterocycle (Het)

N

40

2

> 100

> 2.5

C₆H₅

N

22

>> 100

-

COOCH₃

H₃COOC

N

23

24

>> 100

> 100

-

CN

NC

N

32

2

> 3.1

C₆H₅

N

25

26

>> 100

-

N

Br

N

NO₂

27

28

>> 100

> 100

-

N

COOCH₃

N

22

2

> 4.5

N

Br

N

29

69 19

> 100

> 1.4

N

H₃COOC

N

30

31

>> 100

-

COOCH₃

SCH₃

N

32

33

>> 100

90 33

-

NO₂

N

9

2

> 10

N

C₆H₅

Chem Biol Drug Des 2010; 75: 68–90

81

Roman et al.

Table 1: (Continued)

Selectivity index

IC₅₀HO-2/

IC₅₀(µM) HO-1 IC₅₀(µM) HO-2

IC₅₀HO-1 (rat)

Compound

34

Heterocycle (Het)

H₃COOC

(rat spleen)

(rat brain)

>> 100

-

N

H₃CS

N

35

>> 100

-

N

Br

36

37

-

N

Br

C₆H₅

N

>> 100

N

C₆H₅

N

38

39

40

>> 100

-

N

H₃CS

N

C₂H₅OOCH₂C

N

C₆H₅

N

41

42

43

44

>> 100

> 100

>> 100

> 100

-

N

SCH₃

N

CH₂COOCH

3

N

C₆H₅

H₃CO₂S

N

>> 100

N

82

Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

Table 1: (Continued)

Selectivity index

IC₅₀HO-2/

IC₅₀HO-1 (rat)

IC₅₀(µM) HO-1 IC₅₀(µM) HO-2

Compound

45

Heterocycle (Het) (rat spleen)

(rat brain)

N

>> 100

-

N

SO₂CH₃

H₃CO₂S

N

46

N

>> 100

N

47

48

>> 100

-

H₃CO₂S

N

SO₂CH₃

O

Het

N

49^a

50

0.7 0.4

>> 100

> 100

> 143

> 7.6

N

13

2

N

51

> 100

-

N

52

53

54

>> 100

> 100

-

N

39

72

5

1

> 2.6

> 1.4

N

55

56

> 100

>> 100

> 100

-

C₆H₅

N

C₆H₅

OH

Het

Chem Biol Drug Des 2010; 75: 68–90

83

Roman et al.

Table 1: (Continued)

Selectivity index

IC₅₀HO-2/

IC₅₀(µM) HO-1 IC₅₀(µM) HO-2

IC₅₀HO-1 (rat)

Compound

Heterocycle (Het) (rat spleen)

(rat brain)

N

57^a

58

6

1

16

8

2.6

5.3

N

10.2 0.2

54 12

> 100

N

59

60

61

44

56

1

6

> 2.3

> 1.8

-

N

> 100

N

> 100

N

62

63

15

1

> 100

> 6.7

-

C₆H₅

N

> 100

C₆H₅

HO-1, heme oxygenase-1; HO-2, heme oxygenase-2.

^aValues obtained from Roman et al. (15).

retention of the two carbon atoms adjacent to N ³in the imidazole-

based inhibitor 6 and the formal replacement of its C ⁵by a nitro-

gen atom, as in 1,2,4-triazole-containing compound 7, result in the

production of a very effective inhibitor of HO-1, with a selectivity

index toward this isozyme superior to that of the imidazole-based

inhibitor 6. At the highest concentration used in this study

(100 lM), compound 7 decreased the control activity of HO-1 by

approximately 85%, whereas the loss of the control activity of HO-

2 at the same concentration was approximately 70% (Figure S1 in

Supporting Information). To discern the contributions of various

structural features of these azole inhibitors to binding, the structure

of the imidazole variant 6 was superimposed on the structure of

the inhibitor 7 in complex with hHO-1. As stated in the X-ray crys-

tallography section, the additional nitrogen atom in the 1,2,4-triaz-

ole group results in the formation of a potential, weak hydrogen

bond with the backbone amide group of Gly143, which would not

be present with the imidazole variant. This may explain the slightly

increased potency of inhibitor 7 compared to that of compound 6.

Furthermore, calculation of the Mꢁlliken charge [Gaussian03;

Method: B3LYP ⁄ 6-31 +G(d,p)] for the coordinating nitrogen of the

triazole ring in 7 gave a value of )0.464, which is very similar to

the corresponding value for the imidazole variant, namely )0.446

(Figure 2). In contrast, the 1,2,3-triazole variant 8 has a significantly

higher IC₅₀value than does compound 7 (Table 1). For the 1,2,3-

triazole ring, the Mꢁlliken charge of the coordinating nitrogen

decreases to )0.281 with the N ²atom gaining a charge of only

)0.075 as opposed to the corresponding charge of 0.219 corre-

sponding to the C⁵atom of the 1,2,4-triazole-based inhibitor 7.

Thus, the resultant delocalized electron system in the 1,2,3-triazole

Figure 2: Calculated Mꢁlliken charges for selected atom in

inhibitors 6, 7, 8, 10 and 11.

ring causes a decrease in the electronegativity of the coordinating

nitrogen atom, a feature that potentially weakens the coordination

with the heme iron. Moreover, the superimposition of the structure

84

Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

of the 1,2,3-triazole variant 8 on the structure of the inhibitor 7 in

complex with hHO-1 showed that the N ²atom of compound 8 is

located only 3.05 ꢀ away from the ketone group of Gly139, a situa-

tion that may contribute to repulsive forces, as opposed to the

other variants having carbon atoms at the same position. Docking

experiments using A^UTODOCK were also performed with inhibitors 6,

7, and 8. Interestingly, the convergence of docking conformations

for the triazole variants 7 and 8 appeared to be less than that of

the imidazole-containing inhibitor 6; this aspect may be a result of

the delocalization of electrons in these compounds.

ture of the inhibitor 7 in complex with hHO-1, and the distance

between the nitrogen atoms N ²and N ³and the amide group of

Gly 143 was determined to be 3.61 and 3.88 ꢀ, respectively. Given

the flexibility of the distal helix of hHO-1, it is possible for these

nitrogen atoms to form hydrogen bonds with the amide group of

Gly143 and further stabilize the binding of this compound, as in the

case of inhibitor 7. In the structure of compound 11, the N ¹atom

with a Mꢁlliken charge of )0.324 has replaced formally the C⁵

atom in the tetrazole ring of inhibitor 10. For compound 11, coordi-

nation of the heme iron by N ⁴and the putative repulsive forces

between N ³and the ketone moiety of Gly139 of hHO-1, in a man-

ner similar to that proposed for compound 8, may result in the ring

structure being positioned such that N ¹in compound 11 is in a

position similar to that of N ²of compound 7, and may form a

hydrogen bond with the amide group of Gly143. While the greater

electronegativity at this position would result potentially in a stron-

ger hydrogen bond then that of compound 10, the electronegativity

of N ³facing the proximal pocket may contribute a slight repulsive

force given the proximity to the ketone group of Gly139, as the cor-

responding position in compound 10 is held by a carbon atom.

The N ¹-alkylated tetrazole 10 is another example of a good inhibi-

tor of HO-1 having an improved selectivity toward HO-1 over that

of the imidazole-based inhibitor 6 (Table 1). The loss of the control

activity is approximately 85% for HO-1 and about 60% for HO-2 at

a concentration of 100 lM (Figure S1 in Supporting Information). Its

isomer the N ²-alkylated tetrazole 11 is a somewhat weaker inhibi-

tor than 10 toward both isozymes, but still fairly potent with a

maximum inhibition at 100 lM of 80% for HO-1 and 45% for HO-2.

It should be noted that compound 10 could bind potentially to

heme iron through both N ³and N ⁴and that one of the atoms

adjacent to N ⁴is a carbon atom, whereas the only possibility of

compound 11 for binding to heme iron is presented by N ⁴, which

is adjacent to one carbon and one nitrogen atom. An examination

of the structures of inhibitor 11 and of the 1,2,4-triazole-based

compound 7 shows that they are identical with respect to the rela-

tive position of the nitrogen atoms and that the only difference is

the modification of one of the carbon atoms adjacent to the coordi-

nating nitrogen in 7 into a nitrogen atom. We hypothesize that the

presence of two carbon atoms adjacent to the nitrogen atom that

binds to heme iron is important, and that a third heteroatom in the

azole ring is best tolerated when it occupies the position neighbor-

ing N ¹and farthest away from the nitrogen atom that binds to

heme iron in HO. Therefore, an 1H-1,4,2-diazaphosphole alkylated

with the appropriate appendage at either N ¹or P², or 5-substituted

thiazoles and -oxazoles could be projected as good inhibitors of HO

based on azoles containing other heteroatoms beside nitrogen (Fig-

ure 3). Calculation of Mꢁlliken charges for compounds 10 and 11

(Figure 2) supports this hypothesis. The electronegativity of the

putative coordinating nitrogen atom (N ⁴in both cases) is )0.295

for compound 10 and )0.298 for compound 11, and both values

are lower than the value of the electronegativity for the coordinat-

ing nitrogen in either compound 6 or compound 7. The structure of

the tetrazole-based compound 10 was superimposed on the struc-

Finally, the benzo-fused imidazole and triazoles 12–14 were inactive

toward HOs, indicating that a fused-ring system containing an active

azole is too bulky to fit into the active site of these enzymes.

The most active azole-ketones toward HO-1 in the first group are the

1,2,4-triazole- and 1-tetrazolyl-based ketones 7 and 10, respectively,

followed closely by the imidazole-derived ketone 6; the 2-tetrazolyl-

based ketone 11 ranked fourth, with a fourfold higher IC₅₀value rela-

tive to that of its isomer 10. Therefore, imidazole, 1,2,4-triazole, and

tetrazole have been selected as the most promising scaffolds for the

development of a second series of compounds that aims at the explo-

ration of the relationship between the substitution in these azoles

and the inhibitory action toward HOs. First, a series of potential inhib-

itors has been designed that was based mostly on commercially avail-

able imidazoles bearing in one case small or bulky, and in the second

case either electron-withdrawing or electron-releasing groups. The

evaluation of the activity toward HOs showed that none of the com-

pounds 15–19 featuring an imidazole substituted at position 2 was

active. In the series of the 4(5)-substituted imidazoles, the compounds

24 and 28 containing a 4-phenylimidazole and a 5-bromoimidazole

scaffold, respectively, were weak inhibitors of HO-1, whereas com-

pound 29 having a 5-ethoxycarbonylimidazol-1-yl moiety was very

weak. Interestingly, the isomers of compounds 28 and 29 having the

same substituents at position 4 of imidazole were inactive toward

HO-1. Unfortunately, the comparison between the activities of inhi-

bitors 24 and its 5-subtituted isomer was not possible as only the

former was produced by alkylation of 4-phenylimidazole using bro-

moketone 4. Furthermore, in the subgroup comprising the inhibitors

20–23 based on identically 4,5-disubstituted imidazoles, the phenyl

substitution led to inhibitor 21 that was even weaker than compound

24 having only one phenyl moiety.

The triazole-based candidates were designed with the view to

include essentially the same choice of substituents. Regardless of

the position (either 3 or 5) and the nature of the substituent, these

compounds were inactive, with the exception of the inhibitor 33,

having a phenyl moiety at position 3 of the triazole scaffold, which

Figure 3: Azoles containing other heteroatoms besides nitrogen

that could act as pharmacophores in inhibitors of heme oxygenases.

Chem Biol Drug Des 2010; 75: 68–90

85

Roman et al.

is a fairly potent inhibitor of HO-1 and has an improved selectivity

index over the standard imidazole-containing inhibitor 6. Again, the

activities of 33 and that of its isomer having a phenyl moiety at

position 5 could not be compared as the latter was not available

through the synthetic route. It is noteworthy that, unlike the activity

noticed in the imidazole-based series, neither the inhibitor having a

3,5-diphenyltriazole scaffold 37 nor the compounds 30 and 34 hav-

ing an ester function at either position 3 or 5 of the triazole moiety

inhibited HO-1.

by imidazole-based and triazole- or tetrazole-based compounds, the

most promising candidates in the azole-ketone series were converted

into the corresponding azole-dioxolanes and azole-alcohols. From the

structure–activity relationship standpoint, a general remark is that

both the activities of azole-dioxolanes and the activities of azole-

alcohols are significantly lower than the activities of the parent

azole-ketones. In the cases of weak azole-ketone inhibitors such as

21 and 24, the drop in activity toward HO-1 was so considerable

that the corresponding dioxolanes 56 and 55 and alcohol 63 did

not even inhibit activity by 50% at a concentration of 100 lM. There

is one exception, namely the 4-phenylimidazole-based alcohol 62,

whose activity toward HO-1 is comparable to the activity of the

1,2,4-triazole-based alcohol 58. In the azole-dioxolane series, the

enhancement of selectivity toward HO-1 attributed previously to

the presence of the dioxolane moiety is confirmed; however, the

most active inhibitor remains the imidazole-containing compound 49,

with a 20-times weaker 1,2,4-triazole-derived dioxolane 50 trailing

(Figure S2 in Supporting Information). Although the dioxolane 53

derived from tetrazole-ketone 10 did not inhibit HO-2 even at a con-

centration of 10 lM (the loss of control activity of the enzyme is vir-

tually nil at this concentration), the high IC₅₀value for HO-1 makes

this inhibitor inadequate for further development (Figure S2 in Sup-

porting Information). The same trend is followed in the azole-alcohol

series, although the gap between the activities of the imidazole- and

1,2,4-triazole-based alcohols 57 and 58, respectively, was narrower.

The investigation of a structure–activity relationship toward HOs

was extended to substituted tetrazoles appended either at position

1 or at position 2 and substituted with a range of small or bulky,

electron-withdrawing or electron-releasing substituents. Although no

compound in this series was active, the N ²-alkylated compound 43

derived from 5-phenyltetrazole inhibited HO-1 to a certain degree;

however, the IC₅₀value was slightly higher than the 100-lM thresh-

old (at this concentration, the loss of the control activity was 40–

45%). Its isomer 40 was in this case available through synthesis,

and it was found to be inactive (loss of the control activity was

<10% at 100 lM). This result was not unexpected, as the inhibitor

11 having the unsubstituted tetrazole as pharmacophore is less

active than the imidazole- or the1,2,4-triazole-based analogs 6 and

7, respectively. Furthermore, it seems that the hydrophobic phenyl

substituent is tolerated on an azole, and a comparison between the

activity for the pair of imidazole-based inhibitors 16 (inactive) and

24 (active) and the pair of tetrazole-based inhibitors 40 (inactive)

and 43 (active) suggests that this substituent needs to be in a cer-

tain position relative to the nitrogen atom binding to heme iron.

One possible explanation is that the phenyl ring in the active pair

of compounds hinders the binding of the pharmacophore to a lesser

degree than the same substituent does in the inactive pair of com-

pounds. Comparison of these derivatives to that of the crystal struc-

ture of 7 bound to hHO-1 provided some insights to these

observations. In compound 24, the phenyl group at position 4 of

the imidazole ring would be situated toward the opening of the

heme pocket, in a position similar to that of the 1,6-hexanediol

molecule. In contrast, the 2-phenyl-substituted isomer 16 has this

bulky group positioned toward the less-flexible proximal helix,

resulting in steric hindrance.

Crystal structure of hHO-1 bound to the 1,2,4-

triazole-based inhibitor 7

There are two available X-ray crystallographic studies of imidazole-

based inhibitors 1-(adamantan-1-yl)-2-(1H-imidazol-1-yl)ethanone

(64) and 1-((2-(4-chlorophenethyl)-1,3-dioxolan-2-yl)methyl)-1H-imid-

azole (65) (Figure 4) bound to human HO-1 (27) and rat HO-1 (rHO-

1) (35), respectively. The discovery of inhibitors of HO-1 that contain

azoles other than imidazole as pharmacophore has prompted further

structural studies of complexes formed between human HO-1 and

these novel compounds. Among the azole-based inhibitors reported

in this article, the 1,2,4-triazole-based compound 7 has emerged as

a valuable candidate for crystallographic studies with a view of

further optimizing inhibitor design owing to its potent inhibition of

HO-1 and good solubility in water.

A previous report from our group (15) suggested that an oxygen-con-

taining function on the four-carbon atom linker between imidazole

and the remote, substituted phenyl ring is required for a good inhibi-

tory activity toward HOs in a series of 2-oxy-substituted 1-(1H-imi-

dazol-1-yl)-4-phenylbutanes, and confirmed that imidazole-dioxolanes

are a class of selective inhibitors of HO-1. Moreover, the activity of

imidazole-dioxolanes and imidazole-alcohols toward HO-1 was gener-

ally similar to the inhibition of HO-1 noticed for the corresponding

imidazole-ketones. With a view of comparing the inhibition of HOs

The crystal structure of hHO-1 in complex with 7 was solved to a

resolution of 2.20 ꢀ (Table S1 in Supporting Information). The two

molecules in the asymmetric unit (A and B) were found to be virtu-

ally identical with a Ca root mean square deviation of 0.169 ꢀ

(upon alignment of all the atoms). The flexible distal helix had a

maximal displacement of 0.478 ꢀ at Gly144. Given the closeness in

structure of the two molecules in the asymmetric unit, further

discussion will focus on the A chain unless otherwise stated.

Figure 4: Heme oxygenase inhib-

itors employed for complexing with

heme oxygenase-1 for X-ray crystal-

lographic studies.

86

Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

The overall structure of the hHO-1 complex with 7 was very similar

to that of the native heme-conjugated hHO-1 (36), being mostly

a-helical with the heme sandwiched between the proximal and dis-

tal helices (Figure 5) with a Ca RMSD of 0.69 and 0.42 ꢀ with the

closed (A chain) and open (B chain) forms, respectively. Additional

alignments of the structure with that of the complex of 64 with

hHO-1 or with the structure of the complex of 65 with rat hHO-1

(35) gave RMSD values of 0.50 and 0.71 ꢀ, respectively. Just as in

the case of complexes of 65 and 64 with HO-1, co-crystallization

of 7 with hHO afforded a complex in which the inhibitor binds to

the distal side of the heme to coordinate the iron atom through the

electrons at N ⁴, thus preventing the oxidation of iron by disrupting

a critical hydrogen-bond network. In addition, inhibitor 7 interferes

with oxygen binding to the heme iron, without competing with

heme for binding to HO as do the metalloporphyrin-based inhibitors.

The X-ray structure contained some electron density that did not

correspond to heme, the inhibitor, or water; the electron density

was assigned to a sulfate ion and a molecule of 1,6-hexanediol,

both of which were present in the crystallization medium. The 1,6-

hexanediol molecule is situated at the opening of the heme ⁄ inhibi-

tor-binding pocket and is involved in a hydrogen bond with Gln38

(3.21 ꢀ). This hydrogen bond is not present in the B molecule. In

addition, the 1,6-hexanediol molecule can form hydrophobic con-

tacts with C³of 7, with two points of potential contact, namely at

distances of 3.76 and 3.88 ꢀ.

The inhibitor 7 is bound to the distal side of the heme in a manner

similar to the binding of 64 to hHO-1 or the binding of 65 to rHO-

1, with the triazole ring coordinating the heme iron through N ⁴at

a distance of 2.2 ꢀ and the rest of the molecule extending into a

distal hydrophobic pocket (Figure 6). Residues within the van der

Waals distance of the inhibitor (i.e., 4 ꢀ) include Met34, Phe37,

Val50, Arg136, Gly139, Asp140, Gly143, Leu147, Phe214, heme, 1,6-

hexanediol, and two water molecules. There is a potential hydrogen

bond between the N ²of triazole and the Gly143 amide group

(3.61 ꢀ). This potential point of stabilization is lost in the cases of

complexes of 64 and 65 with HO-1 as these imidazole-based inhib-

itors have a carbon atom at that position instead. The carbonyl

function of the inhibitor 7 putatively forms a second hydrogen bond

with a water molecule in the active site (3.32 ꢀ). This water mole-

cule appears to be involved in the hydrogen-bonding network of the

critical Asp140 residue as it is also hydrogen-bonded to the amino

group of Arg136 (3.13 ꢀ), and another water molecule (2.74 ꢀ),

with potential weak bonds with the carboxyl group of Asp140

(3.84 ꢀ). The second water molecule is involved further in hydrogen

bonds with the amino group (3.08 ꢀ) and carbonyl group (3.19 ꢀ) of

Arg136, and with the amide (3.26 ꢀ) and carboxyl groups (2.64 ꢀ)

of Asp140. This second water molecule is involved also in a hydro-

gen bond with the carbonyl group of Thr135 (2.81 ꢀ). The phenyl

moiety of the inhibitor is bound within the hydrophobic pocket that

extends back toward the distal side of the heme-binding pocket.

Figure 5: Ribbon diagram of the inhibitor-binding site of heme-conjugated human heme oxygenase-1(hHO-1) in complex with 7 at 2.20 ꢀ

resolution. Heme (orange) and 7 (yellow) are depicted as stick models. An omit map contoured at 3.0 r is superimposed. The heme iron is

coordinated by the nitrogens of the imidazole and triazole rings of His25 and 7, respectively. Residues within van der Waals distance of the

inhibitor are indicated (black), as are the two water molecules involved in inhibitor stabilization. The 1,6-hexanediol residue situated in the

opening of the distal pocket is indicated in purple, and has been made opaque for clarity of inhibitor binding. Potential hydrogen bonds con-

tributing to inhibitor stabilization are indicated as is that between 1,6-hexanediol and Gln38. A break in the proximal helix of hHO-1 (Asn30–

Gln32) upon binding of 7 is highlighted (olive green).

Chem Biol Drug Des 2010; 75: 68–90

87

Roman et al.

in 7 is not parallel to the 4-chlorophenyl moiety in 65, the former

being skewed by approximately 45ꢀ. Upon closer inspection, the

4-chlorophenyl moiety in 65 is in contact with four aromatic groups

from Phe33, Phe37, Phe214, and Phe167, albeit Phe33 and Phe37

are in contact with the chlorine atom; as expected, the unsubstitut-

ed phenyl moiety of 7 is in contact only with Phe37 and Phe214.

Thus, aromatic stacking interactions, while not perfectly aligned,

may also be a contributing factor to the binding of these phenyl

substituents in the hydrophobic pocket. In addition to the aromatic

residues in amino acids lining the hydrophobic pocket and shown to

be within the van der Waals distance of the respective inhibitors,

the phenyl rings in Phe47 and Phe166 may contribute also to the

anchoring of the inhibitors through such interactions. Thus, the

potential contribution of p–p stacking interactions, in addition to

the general hydrophobic interactions, may explain the observed

activity of inhibitors having a phenyl, and particularly a halogen-

substituted phenyl group in this region of the molecule, in compari-

son to the lack of activity of inhibitors deficient in such moieties.

Figure 6: Electrostatic surface potentials of the complex of inhibi-

tor 7 with human heme oxygenase-1 illustrating the hydrophobic

pocket into which the hydrophobic phenyl moiety fits. Blue and red

colors indicate positive and negative electrostatic potentials, respec-

tively, as calculated using PyMOL (31). The 1,6-hexanediol molecule

present in the crystal structure has been omitted for clarity.

The comparison of the inhibitor-bound structure with the closed

form (A molecule) of the native holoenzyme revealed that the flexi-

ble distal helix has shifted to open up the distal pocket to accom-

modate the inhibitor 7, similarly to the changes noticed with the

binding of 65 to the rat rHO-1 as well as with the binding of 64

to hHO-1. The distal helix appears to move back more than what is

seen with the 65–rat hHO-1 complex, but not as much as is

required to accommodate the bulky adamantanyl group of 64. A

helical break, which is seen to accommodate the inhibitor 64, is

not seen with compounds 7 or 65. Thus, the flexibility of the distal

helix allows it to freely shift to expand the distal hydrophobic

pocket to the extent required to accommodate the inhibitor that is

being bound. As for the changes in the proximal helix, the kink in

the proximal helix of the native closed structure of hHO-1 becomes

a 'break' from Asn30 to Glu32 upon binding of 7 or 64. In contrast,

the 65–rat hHO-1 structure maintains a kink, albeit slightly less

pronounced (35). Close inspection of the position of the heme in

these structures revealed that the a-meso carbon is shifted back by

0.62 and 0.82 ꢀ upon binding of 7 and 64, respectively, relative to

the native structure, while upon binding to 65, the shift is only

0.42 ꢀ. The break in the proximal helix may result to accommodate

the greater, albeit small, shifts in heme position with 7 and 64.

The fact that such small shifts resulted in these breaks suggests

that the proximal helix of hHO-1exhibits a more limited flexibility

than that seen with the distal helix.

Residues lining this hydrophobic pocket that may contribute to

hydrophobic interactions include Met34, Phe37, Val50, Leu147 and

Phe214.

Although inhibitors 7 and 65 share a common general structure as

they both feature a phenyl moiety and the azole pharmacophore

separated by a four-carbon linker having an oxygen-containing func-

tion, these compounds differ in terms of the substitution of the

phenyl moiety, the nature of the azole, and the type of the oxygen-

containing function on the linker. Despite these structural dissimilar-

ities, a comparison of the complex of inhibitor 7 to hHO-1 with that

of compound 65 to rat HO-1 reveals that the two compounds bind

in a very analogous manner, and that the two molecules are almost

superimposable (data not shown). However, because compound 65

is an imidazole-based structure, there is no hydrogen bond between

this ring structure and the Gly143 amide group, as opposed to the

case of inhibitor 7. Furthermore, in the structure of the complex of

inhibitor 65 with rHO-1, a water molecule that is hydrogen-bonded

to Thr135 is weakly bonded also to the oxygen atom of the dioxo-

lane group, whereas in the structure of the complex of inhibitor 7

with hHO-1 the carbonyl function on the linker is involved in a

hydrogen-bonding network consisting of a two water molecules that

are ultimately hydrogen-bonded with the carbonyl of Thr135. It

should be noted that, despite the presence of a similar carbonyl

function on the two-carbon linker in the structure of inhibitor 64,

no such stabilization of this function can be observed in the struc-

ture of the complex of 64 with hHO-1. In this latter case, the bind-

ing pocket is more open to accommodate the bulky adamantanyl

moiety of 64, a feature that may account for the unrestricted

movement of water molecules into and out of the binding pocket

(data not shown).

Conclusions and Future Directions

For the first time, 1,2,4-triazole- and 1H-tetrazole-based inhibitors

have been shown to be potent inhibitors of HO-1, with an improved

selectivity toward the stress-induced isozyme over the constitutive

HO-2 isozyme when compared to the corresponding imidazole-based

analogs. The hypothesis that a nitrogen atom at position 3 relative

to the appendage is required for the binding to heme iron was

confirmed by a structure–activity relationship study. Several other

inhibitors that featured either an imidazole pharmacophore having

a hydrophobic substituent (phenyl, bromine, ethoxycarbonyl) at

position 4 or 5 or a phenyl-substituted 1H-1,2,4-triazole or 1H-tet-

Both phenyl moieties furthest from the azole pharmacophore in

inhibitors 7 and 65 fit into the distal hydrophobic pocket and

appear to be stabilized by hydrophobic interactions from residues

within this pocket. Interestingly, the orientation of the phenyl group

88

Chem Biol Drug Des 2010; 75: 68–90

Azole-Based Heme Oxygenase Inhibitors

razole pharmacophore exhibited activity toward HO-1 in various

degrees; especially, a phenyl group appears to be tolerated as a

substituent of these active azoles, although it decreases the inhibi-

tory activity. Surprisingly, the dioxolanes and the alcohols derived

from the corresponding active 1,2,4-triazole- and 1H-tetrazole-based

ketones were less potent than their corresponding imidazole-con-

taining analogs. In all of the cases, the azole-dioxolanes selectively

inhibited HO-1 over HO-2. These 1,2,4-triazole- and 1H-tetrazole-

based compounds could be developed further into more-potent HO

inhibitors that do not interfere with the activity of other hemoen-

zymes, a hypothesis that was supported by preliminary inhibition

experiments with several cytochrome P450 enzymes. The X-ray crys-

tallographic investigation of the first example of hHO-1 in complex

with an inhibitor based on 1H-1,2,4-triazole showed that imidazole-

and 1,2,4-triazole-containing inhibitors of HO-1 share a common

mode of binding. The study aids the design of improved inhibitors

for HOs, advances our general understanding of the CO ⁄ heme oxy-

genase system, and assists the transition to future therapeutic

applications.

9. Walker K.A.M., Kertesz D.J., Rotstein D.M., Swinney D.C., Berry

P.W., So O.-Y., Webb A.S. et al. (1993) Selective inhibition of

mammalian lanosterol 14a-demethylase: a possible strategy for

cholesterol lowering. J Med Chem;36:2235–2237.

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aran L., Johnson R.M. (1998) Identification of nonporphyrin

inhibitors of heme oxygenase-1. Abstr Soc Neurosci;24:2058.

11. Vreman H.J., Wong R.J., Stevenson D.K., Clark J.D. (2002) Aza-

lanstat: a novel heme oxygenase inhibitor. 2nd International

Conference on Heme Oxygenase (HO ⁄ CO) and Cellular Stress

Response, Catania, Italy.

12. Vlahakis J.Z., Kinobe R.T., Bowers R.J., Brien J.F., Nakatsu K.,

Szarek W.A. (2005) Synthesis and evaluation of azalanstat ana-

logues as heme oxygenase inhibitors. Bioorg Med Chem

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13. Vlahakis J.Z., Hum M., Rahman M.N., Jia Z., Nakatsu K., Szarek

W.A. (2009) Synthesis and evaluation of imidazole-dioxolane

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14. Vlahakis J.Z., Kinobe R.T., Bowers R.J., Brien J.F., Nakatsu K.,

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Nakatsu K., Szarek W.A. (2007) Heme oxygenase inhibition by

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Acknowledgments

This research was supported by a Canadian Institutes of Health

Research Grant-in-Aid (MOP 64305). The award of a postdoctoral

fellowship to Dr. Gheorghe Roman by the Canadian Institutes of

Health Research Gasotransmitter Research Training (GREAT) Program

is gratefully acknowledged. The authors thank also Ms. Tracy Gif-

ford and Mr. Brian E. McLaughlin for the assistance with the bio-

logic evaluations.

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Figure S1: Inhibition of heme oxygenase-1 (solid triangles,

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The values on the abscissa represent the decimal logarithm of the

inhibitor concentration.

Figure S2: Inhibition of heme oxygenase-1 (solid triangles,

)

and heme oxygenase-2 (open squares, h) by compound 50 (graph

A) and compound 53 (graph B). All values of activity (ordinate) are

expressed as a percentage of the control with no inhibitor present.

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Please note: Wiley-Blackwell is not responsible for the content or

functionality of any supporting materials supplied by the authors.

Any queries (other than missing material) should be directed to the

corresponding author for the article.

90

Chem Biol Drug Des 2010; 75: 68–90

Article Doi

Heme oxygenase inhibition by 2-Oxy-substituted 1-Azolyl-4-phenylbutanes: Effect of variation of the azole moiety. X-ray crystal structure of human heme oxygenase-1 in complex with 4-phenyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone

DOI: 10.1111/j.1747-0285.2009.00909.x

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Article abstract of DOI:10.1111/j.1747-0285.2009.00909.x

Full text of DOI:10.1111/j.1747-0285.2009.00909.x

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