Synthesis and evaluation of azalanstat analogues as heme oxygenase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.12.075

Several analogues based on the lead structure of azalanstat were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). A number of these compounds, which are structurally distinct from metalloporphyrin HO inhibitors, were found to be selec

Full text of DOI:10.1016/j.bmcl.2004.12.075

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1457–1461
Synthesis and evaluation of azalanstat analogues as
heme oxygenase inhibitors
Jason Z. Vlahakis,a Robert T. Kinobe,b Raymond J. Bowers,a James F. Brien,b
Kanji Nakatsub and Walter A. Szareka,*
aDepartments of Chemistry, Queenꢀs University, Kingston, Canada ON K7L 3N6
bPharmacology and Toxicology, Queenꢀs University, Kingston, Canada ON K7L 3N6
Received 22 November 2004; accepted 29 December 2004
Available online 22 January 2005
Abstract—Several analogues based on the lead structure of azalanstat were synthesized and evaluated as novel inhibitors of heme
oxygenase (HO). A number of these compounds, which are structurally distinct from metalloporphyrin HO inhibitors, were found
to be selective for the HO-1 isozyme (stress induced), and had substantially less inhibitory activity on HO-2, the constitutive
isozyme.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
to be a member of the glucocorticoid-regulated family of
enzymes and is upregulated by glucocorticoids. HO-3 is
the least known of the isozymes3 as its catalytic activity
is low, and its physiological function is unknown.
Carbon monoxide (CO) is formed in mammals primarily
through the action of heme oxygenase (HO) on the sub-
strate heme.1 Approximately 85% of the CO produced
by humans under normal physiological conditions is de-
rived from heme,2 and CO is increasingly becoming rec-
ognized as a cellular regulator with actions in brain,
blood vessels, and immune systems. HO activity is not
attributable to a single enzyme as there are in fact three
isozymes. HO-1 is also known as HSP-32 (molecular
weight ꢀ32 kD) and is a stress protein induced by a
number of stimuli including heat shock, heavy metals,
heme, and reactive oxygen species. HO-2 has a molecu-
lar weight of 36.5 kD and is constitutive; it is considered
Much of our current information on the role of CO and
HO has been garnered by judicious use of drugs, espe-
cially HO inhibitors. Almost all of the studies to date
have exploited HO inhibitors of the metalloporphyrin
class, such as tin protoporphyrin (SnPP).4,5 Because of
the close structural similarity between heme and the
metalloporphyrin HO inhibitors, a problem arises with
respect to specificity. Heme plays a key role in a number
of biologically relevant molecules, where it may be in-
volved in the active site of an enzyme or function as a regu-
latory prosthetic group. Examples of the former would
be cytochromes P-450 and nitric oxide synthase (NOS),
and an example of the latter would be soluble guanylyl
cyclase (sGC). Although the metalloporphyrins have
been subjected to criticism because of their ability to in-
hibit enzymes other than HO,6,7 it has been argued that
they are useful if they are used cautiously with great
attention to concentration.8 Our laboratory has found
that chromium mesoporphyrin was one of the more use-
ful drugs in broken cell preparations as a 10 lM concen-
tration inhibited HO by >90%, with little or no effect on
sGC or NOS.9 Nevertheless, the range of drug concen-
trations in which selectivity is expressed is limited, a fea-
ture which proscribes the utility of the metalloporphyrins
in distinguishing amongst a number of enzymes.10 Thus,
Keywords: Heme oxygenase; Selective inhibitors; Azalanstat; Carbon
monoxide; Heme.
*
Corresponding author. Tel.: +1 613 533 2643; fax: +1 613 533
6532; e-mail addresses: nakatsuk@post.queensu.ca; szarekw@chem.
queensu.ca
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.075

1458
J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1457–1461
we have embarked on a program to design HO inhibitors
that are not based on the porphyrin nucleus with the
objective of creating more-selective HO inhibitors. One
of our leads, azalanstat, was designed originally at Syn-
tex as an inhibitor of mammalian lanosterol 14a-demeth-
ylase (14-DM),11 and was discovered recently to show
activity against HO.2 Thus, using azalanstat (1) as the
lead compound and systematically modifying its struc-
ture, we have synthesized a series of novel drugs, several
of which are selective HO-1 inhibitors.
2,2-dimethyl-1,3-dioxolane, respectively, according to
the published tosylation procedure.13 The diastereo-
meric tosylates 5, 6, 7, and 8 were prepared from com-
pound 2 by acid-catalyzed transacetalation in toluene
using the corresponding tosylate 3 or 4, according to
the procedure reported by Walker et al.,14 as shown in
Scheme 1. In each case, the diastereomeric product mix-
ture was resolved by column chromatography on silica
gel using EtOAc as eluent. Azalanstat (1) and its ana-
logues (9–19) were synthesized by a nucleophilic dis-
placement reaction on the appropriate tosylate (5, 6, 7,
or 8) using the appropriately substituted aminothiophe-
nol in acetone/K2CO3 in a manner similar to that re-
ported by Walker et al.,14 and isolated/characterized as
the hydrochloride or dihydrochloride salts.15,16
2. Synthesis
4-(4-Chlorophenyl)-1-(1H-imidazol-1-yl)-2-butanone (2)
was prepared starting from 1-chloro-4-(chloromethyl)-
benzene.12
(4S)-(+)-4-p-Toluenesulfonyloxymethyl-
2,2-dimethyl-1,3-dioxolane (3) and
(4R)-(ꢁ)-4-
3. Biological evaluation
p-toluenesulfonyloxymethyl-2,2-dimethyl-1,3-dioxolane
(4) were prepared from (4R)-(ꢁ)-4-hydroxymethyl-2,2-
dimethyl-1,3-dioxolane and (4S)-(+)-4-hydroxymethyl-
Azalanstat (1) and its structural analogues were
screened for evidence of their ability to inhibit HO using
Scheme 1. Reagents and conditions: (a) p-TsOHÆH2O, n-butanol, toluene, reflux; (b) 2- or 3- or 4-aminothiophenol, K2CO3, acetone, reflux.

J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1457–1461
1459
an in vitro assay for HO in which heme was presented to
the enzyme complexed with albumin.17 HO-1 was ob-
tained from rat spleen18 and HO-2 was obtained from
rat brain19,20 in the microsomal fractions prepared by
differential centrifugation. Several compounds showed
good potency as inhibitors of HO activity (Table 1).
The ratio of IC50 values measured for HO-2 and HO-1
for each compound is shown in the fourth column,
and is used as the index of selectivity for HO-1 com-
pared to HO-2.
The data obtained using this series of azalanstat ana-
logues revealed general inhibitory selectivity for HO-1
over HO-2; this selectivity appeared to be dependent
on the diastereomeric configuration of the core. Com-
pounds with the (2R,4S) configuration for the 1,3-dioxo-
lane ring (11, 12, and 13) were found to be effective
against both HO-1 and HO-2 activity, but compounds
11 and 12 showed high selectivity for HO-1. The
(2S,4S) configuration was also found to produce potent
analogues (9, 10, and 1) against HO-1, with compounds
9 and 10 showing high selectivity for HO-1; compound
10 was the most selective inhibitor studied with a selec-
tivity index of 35 (Fig. 1). Nevertheless, the marked dif-
ferences in three-dimensional structure amongst the
compounds with the (2R,4S)/(2S,4S) cores appeared to
impact only moderately the selectivity of these HO
inhibitors; this result is interpreted to mean that the flex-
ible side chains at the C2 and C4 positions of the 1,3-
dioxolane ring in these compounds are not exploiting
efficiently the three-dimensional space within the active
site of the enzyme. The compounds with the (2R,4R)
configuration (14, 15, and 16) were found to exhibit
the lowest potency with respect to both HO-1 and
HO-2 activity, with IC50 values > 100 lM in all cases ex-
cept for mild inhibition of HO-1 in the case of com-
pound 16.
Figure 1. Inhibition of HO-1 and HO-2 activity by compound 10.
Enzyme activity was determined by measuring the quantity of CO
produced in 15 min from 50 lM substrate (methemalbumin). All
values of activity (ordinate) are expressed as a percentage of the
control with no inhibitor present. The values on the abscissa represent
the log of the inhibitor concentration. Solid triangles (m), HO-1
(spleen microsomes) and open squares (h), HO-2 (brain microsomes).
Concentrations that inhibited activity by 50% (IC50) were determined
by nonlinear regression using GRAPHPAD PRISM version 3.
evaluate the compounds having all four diastereomeric
configurations. For 14-DM inhibition, the (2R,4S) iso-
mer (13) was the least effective of the four diastereomers
(1, 13, 16, and 19); this difference in sensitivity to the
present diastereomers suggests that the synthesis of even
more-selective drugs is possible.
No striking pattern in selectivity was observed with re-
spect to the position of the amino functionality; never-
theless, the 4-amino compounds (1, 13, 16, and 19) all
appeared to have higher potency against HO-2 activity
than their 2- or 3-amino analogues, an aspect which
leads to lower selectivity for HO-1 in three of the four
cases (1, 13, and 19). In fact, compound 19 had the low-
est measurable selectivity index and could be taken as
the only member of this series with slight selectivity to-
wards HO-2 inhibition. Since the analogues in the
(2S,4R) series (17, 18, and 19) and the (2R,4R) series
(14, 15, and 16) proved to have low potency and/or
selectivity, further modifications to these structures to
create highly potent inhibitors selective for HO-1 should
focus on the (2R,4S) and (2S,4S) core configurations.
In contrast to the inhibition observed with 14-DM using
azalanstat analogues and other related antifungal
agents,11 it is the (2R,4S) stereoisomer (13) not the
(2S,4S) stereoisomer (1) that is most potent toward
HO. This initial result compelled us to synthesize and
Table 1. Inhibitory potency and selectivity of azalanstat (1) and
various analogues against HO-1 and HO-2 activity
Compound
IC50 (lM)
rat spleen
(HO-1)
IC50 (lM)
rat brain
(HO-2)
Selectivity index
IC50 (HO-2)/IC50
(HO-1)
4. Conclusion
1
6
5
1
1
28 18
4.7
13.6
35.0
25.2
20.0
9.6
We have synthesized several compounds (9, 10, 11, and
12) that express good selectivity as inhibitors of HO-1
relative to HO-2. As HO-1 is the isozyme that has at-
tracted the most research interest to date, these drugs
are anticipated to become useful tools in elucidating
the physiological roles of HO-1 in mammalian and other
biological systems.
9
68
35
5
6
3
9
3
10
11
12
13
14
15
16
17
18
19
1.0 0.2
2.5 0.1
1.6 0.7
0.52 0.03
>100
63
32
5
>100
>100
>100
>100
—
—
>100
40
>100
4
8
>2.5
—
2
9
34 17
15
8.5
0.6
Acknowledgements
27
4
This work was supported by a grant-in-aid from the
Canadian Institutes of Health Research, MOP 64305.
Data represent mean IC50 values standard deviation of replicate
experiments.

1460
J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1457–1461
137.9, 140.3, 141.3; HRMS (ES) [M+H]+ Calcd for
References and notes
C22H25ClN3O2S: 430.1350. Found: 430.1362. Anal. Calcd
for C22H26Cl3N3O2S: C, 52.54; H, 5.21; N, 8.36. Found:
C, 52.28; H, 5.80; N, 8.27.
1. Maines, M. D. Ann. Rev. Pharmacol. Toxicol. 1997, 37,
517.
16. Characterization of the new compounds synthesized
following the procedure in Note 15 as outlined in Scheme
1:
2. Vreman, H. J.; Wong, R. J.; Stevenson, D. K. In Carbon
Monoxide and Cardiovascular Function; Wang, R., Ed.;
CRC: Boca Raton, 2002; Chapter 15, p 273.
1 Dihydrochloride: beige solid in 73% yield from 5: mp 72–
3. McCoubrey, W. K., Jr.; Huang, T. J.; Maines, M. D. Eur.
J. Biochem. 1997, 247, 725.
4. Johnson, R. A.; Colombari, E.; Colombari, D. S.; Lavesa,
M.; Talman, W. T.; Nasjletti, A. Hypertension 1997, 30,
962.
5. Tulis, D. A.; Durante, W.; Peyton, K. J.; Evans, A. J.;
Schafer, A. I. Atherosclerosis 2001, 155, 113.
6. Luo, D.; Vincent, S. R. Eur. J. Pharmacol. 1994, 267,
263.
7. Meffert, M. K.; Haley, J. E.; Schumann, E. M.; Schulman,
H.; Madison, D. V. Neuron 1994, 13, 1225.
8. Zakhary, R.; Poss, K. D.; Jaffrey, S. R.; Ferris, C. D.;
Tonegawa, S.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 14848.
9. Appleton, S. D.; Chretien, M. L.; McLaughlin, B. E.;
Vreman, H. J.; Stevenson, D. K.; Brien, J. F.; Nakatsu,
K.; Maurice, D. H.; Marks, G. S. Drug Metab. Dispos.
1999, 27, 1214.
10. Vreman, H. J.; Cipkala, D. A.; Stevenson, D. K. Can. J.
Physiol. Pharmacol. 1996, 74, 278.
23
180 °C (decomp); Rf = 0.19 (EtOAc); ½aŠ +11.7 (c 0.70,
D
CH3OH); 1H NMR (400 MHz, D2O): d 2.01–2.09 (m, 2H),
2.68–2.74 (m, 2H), 2.95 (dd, J = 14.2, 4.6 Hz, 1H), 3.23–
3.27 (m, 1H), 3.99–4.03 (m, 1H), 4.25–4.30 (m, 1H), 4.43–
4.52 (m, 1H), 7.20 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.4 Hz,
2H), 7.36 (d, J = 8.4 Hz, 2H), 7.43–7.46 (m, 4H), 8.69 (s,
1H); 13C NMR (100 MHz, D2O): d 28.6, 35.3, 36.8, 53.2,
69.4, 75.6, 109.1, 119.4, 123.6, 123.9, 128.7, 129.7, 130.1,
130.7, 131.4, 135.9, 136.1, 140.1; HRMS (ES) [M+H]+
Calcd for C22H25ClN3O2S: 430.1350. Found: 430.1356.
Anal. Calcd for C22H26Cl3N3O2S: C, 52.54; H, 5.21; N,
8.36. Found: C, 51.89; H, 5.61; N, 7.94.
9 Dihydrochloride: beige solid in 99% yield from 5; mp 80–
22
175 °C (decomp); Rf = 0.19 (EtOAc); ½aŠ +28.7 (c 0.77,
D
D2O); 1H NMR (500 MHz, D2O): d 2.03–2.13 (m, 2H),
2.56–2.61 (m, 1H), 2.73–2.76 (m, 2H), 2.82–2.85 (m, 1H),
3.15–3.18 (m, 1H), 4.13–4.15 (m, 1H), 4.26–4.34 (m, 1H),
4.48 (s, 2H), 7.22–7.30 (m, 4H), 7.35–7.40 (m, 3H), 7.41–
7.46 (m, 1H), 7.48 (s, 1H), 7.56–7.60 (m, 1H), 8.63 (s, 1H);
13C NMR (100 MHz, D2O): d 28.6, 36.8, 36.9, 53.2, 69.6,
76.4, 109.1, 119.4, 121.8, 123.9, 125.3, 127.0, 128.7, 130.1,
130.4, 131.5, 135.5, 136.0, 136.9, 140.1; HRMS (ES)
[M+H]+ Calcd for C22H25ClN3O2S: 430.1350. Found:
430.1359. Anal. Calcd for C22H26Cl3N3O2S: C, 52.54; H,
5.21; N, 8.36. Found: C, 52.76; H, 5.24; N, 7.81.
11. Walker, K. A. M.; Kertesz, D. J.; Rotstein, D. M.;
Swinney, D. C.; Berry, P. W.; So, O.-Y.; Webb, A. S.;
Watson, D. M.; Mak, A. Y.; Burton, P. M.; Mills-Dunlap,
B.; Chiou, M. Y.; Tokes, L. G.; Kurz, L. J.; Kern, J. R.;
Chan, K. W.; Salari, A.; Mendizabal, G. R. J. Med. Chem.
1993, 36, 2235.
11 Dihydrochloride: off-white solid in 72% yield from 6: mp
12. Walker, K. A. M.; Braemer, A. C.; Hitt, S.; Jones, R. E.;
Matthews, T. R. J. Med. Chem. 1978, 21, 840.
13. Baldwin, J. J.; Raab, A. W.; Mensler, K.; Arison, B. H.;
McClure, D. E. J. Org. Chem. 1978, 43, 4876.
14. Walker, K. A. M.; Burton, P. M.; Swinney, D. C.
European Patent EP 0 492 474 B1, 1997.
22
125–165 °C (decomp); Rf = 0.11 (EtOAc); ½aŠ +5.6 (c
2.15, D2O); 1H NMR (400 MHz, D2O): dD 1.88 (t,
J = 8.0 Hz, 2H), 2.56 (t, J = 8.0 Hz, 2H), 2.92–3.06 (m,
2H), 3.63 (t, J = 8.0 Hz, 1H), 3.76–3.82 (m, 1H), 4.03 (t,
J = 7.4 Hz, 1H), 7.03 (d, J = 8.0 Hz, 2H), 7.21–7.39 (m,
5H), 7.43 (s, 2H), 7.55 (d, J = 7.6 Hz, 1H), 8.73 (s, 1H);
13C NMR (100 MHz, D2O): d 28.3, 37.3, 37.5, 53.7, 69.9,
77.1, 109.2, 119.8, 122.0, 123.6, 125.9, 127.3, 128.8, 130.2,
130.3, 131.5, 135.2, 136.0, 136.3, 140.1; HRMS (ES)
[M+H]+ Calcd for C22H25ClN3O2S: 430.1350. Found:
430.1336. Anal. Calcd for C22H26Cl3N3O2S: C, 52.54; H,
5.21; N, 8.36. Found: C, 52.92; H, 5.19; N, 8.18.
15. Representative procedure for the displacement of tosylates
using thiophenol-containing nucleophiles:
(2S,4S)-2-[2-(4-Chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)me-
thyl]-4-[{(3-aminophenyl)thio}methyl]-1,3-dioxolane dihydro-
chloride (10). Under a nitrogen atmosphere, a mixture of 5
(200 mg, 0.42 mmol), 3-aminothiophenol (113 mg,
0.90 mmol, 2.1 equiv) and K2CO3 (116 mg, 0.84 mmol,
4 equiv) in acetone (8 mL) was heated at reflux tempera-
ture with stirring for 5 h. The solids were removed by
filtration, and washed with hot acetone and then with hot
EtOAc. The filtrate was concentrated, and the residue
(Rf ffi 0.2 in EtOAc) purified by flash chromatography on
silica gel (EtOAc) to give 180 mg (0.42 mmol, 100%) of the
free base as a pale yellow oil. To a solution of the oil in hot
2-propanol (2 mL) was added a solution of 37% aqueous
HCl (82 mg, 0.84 mmol, 2 equiv) in 2-propanol (1 mL).
The mixture was concentrated and dried under high
vacuum. The residue was dissolved in hot 2-propanol
(5 mL), the solution cooled in the freezer, and then Et2O
was slowly added. The solid was removed by filtration and
washed with Et2O. High-vacuum drying left 103 mg (53%)
12 Hydrochloride: yellowish solid in 81% yield from 6: mp
22
165–166 °C; Rf = 0.09 (EtOAc); ½aŠ ꢁ14.1 (c 2.41,
D
CD3OD); 1H NMR (400 MHz, CD3OD): d 1.97 (t,
J = 8.4 Hz, 2H), 2.67–2.81 (m, 2H), 3.01 (dd, J = 14.0,
6.8 Hz, 1H), 3.16 (dd, J = 14.0, 5.2 Hz, 1H), 3.56–3.63 (m,
1H), 3.68 (t, J = 8.2 Hz, 1H), 4.02 (dd, J = 8.2, 5.8 Hz,
1H), 4.42 (s, 2H), 6.59–6.62 (m, 1H), 6.67–6.70 (m, 1H),
6.75 (t, J = 1.8 Hz, 1H), 7.05 (t, J = 8.0 Hz, 1H), 7.18 (d,
J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.47 (t,
J = 1.4 Hz, 1H), 7.56 (t, J = 1.6 Hz, 1H), 8.83 (s, 1H);
13C NMR (100 MHz, CD3OD): d 29.8, 36.4, 39.2, 54.7,
70.9, 77.9, 109.9, 115.3, 117.7, 120.7, 120.9, 124.9, 130.8,
131.0, 132.8, 137.1, 137.8, 141.4, 148.6; HRMS (ES)
[M+H]+ Calcd for C22H25ClN3O2S: 430.1350. Found:
430.1358. Anal. Calcd for C22H25Cl2N3O2S: C, 56.65; H,
5.40; N, 9.01. Found: C, 56.53; H, 5.21; N, 8.68.
of 10 as a beige solid: mp 189–200 °C (decomp); Rf =
22
0.10 (EtOAc); ½aŠ +9.1 (c 1.76, CD3OD); 1H NMR
D
(400 MHz, CD3OD):
13 Hydrochloride: beige solid in 41% yield from 6: mp 161–
d 1.96–2.10 (m, 2H), 2.75 (t,
23
163 °C; Rf = 0.06 (EtOAc); ½aŠ ꢁ11.6 (c 1.90, CD3OD);
J = 8.4 Hz, 2H), 2.92–3.03 (m, 2H), 3.39 (t, J = 7.8 Hz,
1H), 4.18 (dd, J = 8.4, 6.4 Hz, 1H), 4.39–4.46 (m, 1H),
4.50–4.63 (m, 2H), 7.19–7.30 (m, 5H), 7.40–7.51 (m, 3H),
7.58–7.62 (m, 2H), 8.95 (s, 1H); 13C NMR (100 MHz,
CD3OD): d 30.0, 36.3, 38.5, 54.1, 70.5, 76.8, 110.0, 120.4,
121.6, 123.8, 125.3, 129.6, 129.8, 131.0, 131.8, 132.9, 133.2,
D
1H NMR (400 MHz, CD3OD): d 1.92–1.99 (m, 2H), 2.66–
2.78 (m, 2H), 2.82 (dd, J = 13.6, 7.2 Hz, 1H), 3.00 (dd,
J = 13.6, 5.2 Hz, 1H), 3.51–3.56 (m, 1H), 3.60 (t,
J = 8.2 Hz, 1H), 4.00 (dd, J = 8.2, 5.8 Hz, 1H), 4.39 (s,
2H), 6.68 (d, J = 6.4 Hz, 2H), 7.17 (d, J = 2.0 Hz, 2H),

J. Z. Vlahakis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1457–1461
1461
7.19 (d, J = 2.0 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.46 (t,
J = 1.4 Hz, 1H), 7.53 (t, J = 1.4 Hz, 1H), 8.79 (s, 1H); 13C
18 Hydrochloride: beige solid in 95% yield from 8: mp 163–
21
164 °C; Rf = 0.09 (EtOAc); ½aŠ +12.5 (c 2.08, CD3OD);
D
NMR (100 MHz, CD3OD):
d
29.8, 39.3, 38.2 (in
1H NMR (400 MHz, CD3OD): d 1.97 (t, J = 8.6 Hz, 2H),
2.68–2.81 (m, 2H), 3.01 (dd, J = 13.8, 6.6 Hz, 1H), 3.15
(dd, J = 14.0, 5.2 Hz, 1H), 3.58–3.65 (m, 1H), 3.68 (t,
J = 8.2 Hz, 1H), 4.02 (dd, J = 8.0, 5.6 Hz, 1H), 4.42 (s,
2H), 6.61 (ddd, J = 8.0, 2.4, 0.8 Hz, 1H), 6.69 (ddd,
J = 8.0, 1.7, 0.9 Hz, 1H), 6.76 (t, J = 1.8 Hz, 1H), 7.05 (t,
J = 8.0 Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.26 (d,
J = 8.4 Hz, 2H), 7.46 (t, J = 1.4 Hz, 1H), 7.54 (t,
J = 1.6 Hz, 1H), 8.82 (s, 1H); 13C NMR (100 MHz,
CD3OD): d 29.8, 36.4, 39.3, 54.7, 71.0, 78.0, 109.9,
115.2, 117.7, 120.7, 121.0, 124.9, 129.5, 130.8, 131.0,
132.8, 137.1, 137.8, 141.4, 148.9; HRMS (ES) [M+H]+
Calcd for C22H25ClN3O2S: 430.1350. Found: 430.1335.
Anal. Calcd for C22H25Cl2N3O2S: C, 56.65; H, 5.40; N,
9.01. Found: C, 56.82; H, 5.31; N, 8.89.
DMSO-d6), 54.7, 71.2, 78.3, 109.8, 117.3, 120.9, 123.0,
124.9, 129.5, 131.0, 132.8, 135.2, 137.8, 141.4, 148.2;
HRMS (ES) [M+H]+ Calcd for C22H25ClN3O2S:
430.1350.
Found:
430.1361. Anal.
Calcd for
C22H25Cl2N3O2S: C, 56.65; H, 5.40; N, 9.01. Found: C,
57.00; H, 5.19; N, 9.17.
14 Dihydrochloride: white solid in 87% yield from 7: mp
22
90–160 °C (decomp); Rf = 0.14 (EtOAc); ½aŠ ꢁ27.0 (c
D
2.15, D2O); 1H NMR (400 MHz, D2O): d 1.85–1.97 (m,
2H), 2.52 (dd, J = 14.0, 7.6 Hz, 1H), 2.60 (t, J = 7.8 Hz,
2H), 2.75 (dd, J = 14.0, 4.4 Hz, 1H), 3.07 (t, J = 8.0 Hz,
1H), 3.99 (t, J = 7.2 Hz, 1H), 4.12–4.18 (m, 1H), 4.37 (s,
2H), 7.11 (d, J = 8.0 Hz, 2H), 7.19–7.25 (m, 4H), 7.29 (d,
J = 8.0 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.42 (s, 1H), 7.49
(d, J = 7.6 Hz, 1H), 8.57 (s, 1H); 13C NMR (100 MHz,
D2O): d 28.6, 36.9, 37.0, 53.2, 69.7, 76.4, 109.1, 119.5,
122.0, 123.8, 125.5, 127.1, 128.7, 130.1, 130.4, 131.5, 135.6,
136.0, 136.5, 140.1; HRMS (ES) [M+H]+ Calcd for
C22H25ClN3O2S: 430.1350. Found: 430.1336. Anal. Calcd
for C22H26Cl3N3O2S: C, 52.54; H, 5.21; N, 8.36. Found:
C, 53.04; H, 5.16; N, 8.11.
19 Hydrochloride: white solid in 62% yield from 8:
21
mp 163–164 °C; Rf = 0.06 (EtOAc); ½aŠ +11.7 (c 2.23,
D
CD3OD); 1H NMR (400 MHz, CD3OD): d 1.95 (t,
J = 8.4 Hz, 2H), 2.66–2.76 (m, 2H), 2.82 (dd, J = 13.8,
7.0 Hz, 1H), 3.00 (dd, J = 13.8, 5.4 Hz, 1H), 3.50–3.57 (m,
1H), 3.60 (t, J = 8.2 Hz, 1H), 4.01 (dd, J = 7.8, 5.8 Hz,
1H), 4.40 (s, 2H), 6.70 (d, J = 8.4 Hz, 2H), 7.17 (d,
J = 8.4 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 7.26 (d,
J = 8.0 Hz, 2H), 7.47 (s, 1H), 7.53 (s, 1H), 8.81 (s, 1H);
13C NMR (100 MHz, CD3OD): d 29.8, 39.2, 38.2 (in
DMSO-d6), 54.7, 71.2, 78.3, 109.8, 116.5, 117.3, 121.0,
123.0, 124.9, 129.5, 131.0, 132.8, 135.2, 137.8, 141.4, 148.2;
HRMS (ES) [M+H]+ Calcd for C22H25ClN3O2S:
15 Dihydrochloride: beige solid in 87% yield from 7: mp
22
191–200 °C (decomp); Rf = 0.11 (EtOAc); ½aŠ ꢁ9.5 (c
1.68, CD3OD); 1H NMR (400 MHz, CD3OD): dD1.93–2.06
(m, 2H), 2.75 (t, J = 8.4 Hz, 2H), 2.93–3.03 (m, 2H), 3.39
(t, J = 7.8 Hz, 1H), 4.18 (dd, J = 8.0, 6.4 Hz, 1H), 4.39–
4.46 (m, 1H), 4.57 (q, J = 15.2 Hz, 2H), 7.19–7.29 (m, 5H),
7.43 (d, J = 8.0 Hz, 2H), 7.49 (t, J = 8.0 Hz, 1H), 7.56–7.62
(m, 2H), 8.95 (s, 1H); 13C NMR (100 MHz, CD3OD): d
30.0, 36.3, 38.5, 54.1, 70.5, 76.8, 110.0, 120.4, 121.7, 123.9,
125.3, 129.5, 129.9, 131.0, 131.8, 132.9, 133.0, 137.9, 140.3,
141.3; HRMS (ES) [M+H]+ Calcd for C22H25ClN3O2S:
430.1350. Found:
430.1334. Anal.
Calcd
for
C22H25Cl2N3O2S: C, 56.65; H, 5.40; N, 9.01. Found: C,
56.85; H, 5.26; N, 8.86.
17. In vitro HO activity assay: HO activity in rat spleen and
brain microsomal fractions was determined by the quanti-
tation of CO formed from the degradation of methem-
albumin (heme complexed with albumin).21,22 Spleen and
brain (Sprague–Dawley rats) microsomal fractions were
prepared according to the procedure outlined by Appleton
et al.9 Protein concentration of microsomal fractions was
determined by a modification of the Biuret method.22
Incubations for HO activity analysis were done under
conditions for which the rate of CO formation (pmol
CO minꢁ1 mg proteinꢁ1) was linear with respect to time
and microsomal protein concentration. Briefly, reaction
mixtures (150 lL) consisting of 100 mM phosphate buffer
(pH 7.4), 50 lM methemalbumin and 1 mg/mL protein
were pre-incubated with the inhibitors at final concentra-
tions 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 mM 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
18. Xia, Z. W.; Cui, W. J.; Zhang, X. H.; Shen, Q. X.; Wang,
J.; Li, Y. Z.; Chen, S. N.; Yu, S. C. World J. Gastroenterol.
2002, 6, 1123.
19. Trakshel, G. M.; Kutty, R. K.; Maines, M. D. Arch.
Biochem. Biophys. 1988, 260, 732.
20. Maines, M. D. FASEB J. 1988, 10, 2557.
21. Vreman, H. J.; Stevenson, D. K. Anal. Biochem. 1988, 168,
31.
22. Cook, M. N.; Nakatsu, K.; Marks, G. S.; McLaughlin, B.
E.; Vreman, H. J.; Stevenson, D. K. Can. J. Physiol.
Pharmacol. 1995, 73, 515.
430.1350.
Found:
430.1348. Anal.
Calcd for
C22H26Cl3N3O2S: C, 52.54; H, 5.21; N, 8.36. Found: C,
52.61; H, 4.95; N, 8.22.
16 Dihydrochloride: white solid in 80% yield from 7: mp
22
75–185 °C (decomp); Rf = 0.06 (EtOAc); ½aŠ ꢁ11.3 (c
D
1.95, CD3OD); 1H NMR (400 MHz, CD3OD): d 1.93–2.06
(m, 2H), 2.74 (t, J = 8.4 Hz, 2H), 2.89 (dd, J = 13.6,
6.0 Hz, 1H), 2.97 (dd, J = 13.8, 5.8 Hz, 1H), 3.35 (dd,
J = 8.4, 7.6 Hz, 1H), 4.16 (dd, J = 8.4, 6.4 Hz, 1H), 4.34–
4.41 (m, 1H), 4.56 (q, J = 13.3 Hz, 2H), 7.21 (d,
J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.37 (d,
J = 8.8 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.57–7.61 (m,
2H), 8.95 (s, 1H); 13C NMR (100 MHz, CD3OD): d 30.0,
36.5, 38.5, 54.1, 70.5, 76.9, 110.0, 120.3, 124.8, 125.4,
129.6, 130.5, 131.0, 131.4, 132.9, 137.9, 138.6, 141.3;
HRMS (ES) [M+H]+ Calcd for C22H25ClN3O2S:
430.1350.
Found:
430.1335. Anal.
Calcd for
C22H26Cl3N3O2S: C, 52.54; H, 5.21; N, 8.36. Found: C,
52.86; H, 5.17; N, 8.11.
17 Hydrochloride: beige solid in 99% yield from 8: mp 78–
21
110 °C (decomp); Rf = 0.14 (EtOAc); ½aŠ ꢁ3.4 (c 1.74,
D
D2O); 1H NMR (400 MHz, D2O): d 1.77 (t, J = 8.0 Hz,
2H), 2.50 (t, J = 8.0 Hz, 2H), 2.71 (d, J = 5.6 Hz, 2H), 3.42
(t, J = 8.0 Hz, 1H), 3.52–3.59 (m, 1H), 3.88 (t, J = 7.0 Hz,
1H), 4.24 (s, 2H), 6.69 (t, J = 7.4 Hz, 1H), 6.87 (d,
J = 8.0 Hz, 1H), 6.98 (d, J = 8.4 Hz, 2H), 7.09–7.14 (m,
3H), 7.20 (d, J = 7.6 Hz, 1H), 7.27 (s, 1H), 7.36 (s, 1H),
8.61 (s, 1H); 13C NMR (100 MHz, D2O): d 28.5, 37.0,
37.5, 53.7, 69.9, 76.8, 108.9, 117.3, 118.8, 119.8, 120.7,
123.4, 128.7, 130.2, 130.5, 131.5, 135.6, 135.9, 140.1, 146.3;
HRMS (ES) [M+H]+ Calcd for C22H25ClN3O2S:
430.1350. Found: 430.1337.