Synthesis of sulfaphenazole derivatives and their use as inhibitors and tools for comparing the active sites of human liver cytochromes P450 of the 2C subfamily

Article abstract of DOI:10.1021/jm010861y

Twenty-three new derivatives of sulfaphenazole (SPA) were synthesized to further explore the topology of the active sites of human liver cytochromes P450 of the 2C subfamily and to find new selective inhibitors of these cytochromes. These compounds are derived from SPA by replacement of the NH₂ and H (of the SO₂NH function) substituents of SPA with various R₁ and R₂ groups, respectively. Their inhibitory effects were studied on recombinant CYP 2C8, 2C9, 2C18, and 2C19 expressed in yeast. High affinities for CYP 2C9 (IC₅₀ < 1 μM) were only observed for SPA derivatives having the SO₂NH function and a relatively small R₁ substituent (R₁ = NH₂, CH₃). Any increase in the size of R₁ led to a moderate decrease of the affinity, and the N-alkylation of the SO₂NH function of SPA to a greater decrease of this affinity. The same structural changes led to opposite effects on molecular recognition by CYP 2C8 and 2C18, which generally exhibited similar behaviors. Thus, contrary to CYP 2C9, CYP 2C8 and 2C18 generally prefer neutral compounds with relatively large R₁ and R₂ substituents. CYP 2C19 showed an even lower affinity for anionic compounds than CYP 2C8 and 2C18. However, as CYP 2C8 and 2C18, CYP 2C19 showed a much better affinity for neutral compounds derived from N-alkylation of SPA and for anionic compounds bearing a larger R₁ substituent. One of the new compounds (R₁ = methyl, R₂ = propyl) inhibited all human CYP 2Cs with IC₅₀ values between 10 and 20 μM, while another one (R₁ = allyl, R₂ = methyl) inhibited all CYP 2Cs except CYP 2C9, and a third one (R₁ = R₂ = methyl) inhibited all CYP 2Cs except CYP 2C8. Only 2 compounds of the 25 tested derivatives were highly selective toward one human CYP 2C; these are SPA and compound 1 (R₁ = CH₃, R₂ = H), which acted as selective CYP 2C9 inhibitors. However, some SPA derivatives selectively inhibited CYP 2C8 and 2C18. Since CYP 2C18 is hardly detectable in human liver, these derivatives could be interesting molecules to selectively inhibit CYP 2C8 in human liver microsomes. Thus, compound 11 (R₁ = NH₂, R₂ = (CH₂)₂CH(CH₃)₂) appears to be particularly interesting for that purpose as its IC₅₀ value for CYP 2C8 is low (3 μM) and 20-fold smaller than those found for CYP 2C9 and 2C19.

Full text of DOI:10.1021/jm010861y

3622
J . Med. Chem. 2001, 44, 3622-3631
Syn th esis of Su lfa p h en a zole Der iva tives a n d Th eir Use a s In h ibitor s a n d Tools
for Com p a r in g th e Active Sites of Hu m a n Liver Cytoch r om es P 450 of th e 2C
Su bfa m ily
Nguyeˆt-Thanh Ha-Duong, Sylvie Dijols, Cristina Marques-Soares, Claire Minoletti, Patrick M. Dansette, and
Daniel Mansuy*
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite´ Paris V,
45, rue des Saints Pe`res, 75270 Paris Cedex 06, France
Received February 26, 2001
Twenty-three new derivatives of sulfaphenazole (SPA) were synthesized to further explore the
topology of the active sites of human liver cytochromes P450 of the 2C subfamily and to find
new selective inhibitors of these cytochromes. These compounds are derived from SPA by
replacement of the NH2 and H (of the SO2NH function) substituents of SPA with various R1
and R2 groups, respectively. Their inhibitory effects were studied on recombinant CYP 2C8,
2C9, 2C18, and 2C19 expressed in yeast. High affinities for CYP 2C9 (IC50 < 1 µM) were only
observed for SPA derivatives having the SO2NH function and a relatively small R1 substituent
(R1 ) NH2, CH3). Any increase in the size of R1 led to a moderate decrease of the affinity, and
the N-alkylation of the SO2NH function of SPA to a greater decrease of this affinity. The same
structural changes led to opposite effects on molecular recognition by CYP 2C8 and 2C18, which
generally exhibited similar behaviors. Thus, contrary to CYP 2C9, CYP 2C8 and 2C18 generally
prefer neutral compounds with relatively large R1 and R2 substituents. CYP 2C19 showed an
even lower affinity for anionic compounds than CYP 2C8 and 2C18. However, as CYP 2C8 and
2C18, CYP 2C19 showed a much better affinity for neutral compounds derived from N-alkylation
of SPA and for anionic compounds bearing a larger R1 substituent. One of the new compounds
(R1 ) methyl, R2 ) propyl) inhibited all human CYP 2Cs with IC50 values between 10 and 20
µM, while another one (R1 ) allyl, R2 ) methyl) inhibited all CYP 2Cs except CYP 2C9, and
a third one (R1 ) R2 ) methyl) inhibited all CYP 2Cs except CYP 2C8. Only 2 compounds of
the 25 tested derivatives were highly selective toward one human CYP 2C; these are SPA and
compound 1 (R1 ) CH3, R2 ) H), which acted as selective CYP 2C9 inhibitors. However, some
SPA derivatives selectively inhibited CYP 2C8 and 2C18. Since CYP 2C18 is hardly detectable
in human liver, these derivatives could be interesting molecules to selectively inhibit CYP 2C8
in human liver microsomes. Thus, compound 11 (R1 ) NH2, R2 ) (CH2)2CH(CH3)2) appears to
be particularly interesting for that purpose as its IC50 value for CYP 2C8 is low (3 µM) and
20-fold smaller than those found for CYP 2C9 and 2C19.
In tr od u ction
including (S)-mephenytoin12 and omeprazole.13,14 By
contrast, very few substrates of CYP 2C18 are presently
known even though this enzyme has been described to
catalyze the oxidation of tienilic acid,15 diazepam,16 and
warfarin17 with a low efficiency. Recently, a selective,
efficiently oxidized substrate of CYP 2C18 has been
reported; it is an alcohol derived from tienilic acid.18
Cytochrome P450 dependent monooxygenases play a
key role in the oxidative metabolism of exogenous and
endogenous compounds. Most metabolic oxidations of
xenobiotics such as drugs are catalyzed by P450s of the
first three families (CYP 1, 2, and 3).1,2 In fact, P450s
of the 3A and 2C subfamilies are the major enzymes in
human liver.1-3 From the four members of the 2C
subfamily, only CYP 2C8, 2C9, and 2C19 are expressed
at a significant level in human liver and are involved
in the metabolism of many drugs.1-3 Although CYP 2C8,
2C9, and 2C19 exhibit more than 80% sequence identity,
they show very different substrate specificities.3 Good
substrates of CYP 2C9 most often are anionic or polar
compounds such as diclofenac,4 tienilic acid,5 or tol-
butamide.6 CYP 2C8 appears to be able to oxidize much
larger substrates such as paclitaxel,7,8 retinoic acid,9
benzo[a]pyrene,10 and cerivastatine.11 CYP 2C19 is
responsible for the oxidative metabolism of many drugs
To interpret or to predict various problems that may
occur with some drugs, such as drug-drug interactions
or consequences of genetic polymorphism, it is crucial
to determine which human liver P450 is involved in the
metabolism of a new drug. A classical approach is the
use of human liver microsomes in the presence of
specific inhibitors of the different human liver P450s.
For that purpose, it is necessary to possess highly
selective inhibitors for each human liver P450.
Sulfaphenazole (SPA) is a reasonably selective inhibi-
tor of CYP 2C9.2,3,19 Its potent (Ki ≈ 0.3 µM) and
selective inhibitory effects toward CYP 2C919 appear to
be due to two strong interactions, a π-stacking interac-
tion of its phenyl substituent with an aromatic residue
of the protein, and an ionic (or hydrogen-bonding)
* To whom correspondence should be addressed. Phone: 33 1
42 86 21 87. Fax: 33 1 42 86 83 87. E-mail: Daniel.Mansuy@
biomedicale.univ-paris5.fr.
10.1021/jm010861y CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/08/2001

Inhibition of P450 2Cs by Sulfaphenazole Derivatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3623
Sch em e 1
Sch em e 2
interaction between its SO2N- anionic site and a cat-
ionic (or hydrogen-bonding) residue of CYP 2C9 (ref 19;
N. T. Ha-Duong, C. Marques-Soares, S. Dijols, M. A.
Sari, P. M. Dansette, D. Mansuy, manuscript in prepa-
ration). To further explore the origins of the selectivity
of SPA as a CYP 2C9 inhibitor within the human 2C
subfamily, and to find new selective inhibitors for the
other members of the 2C subfamily, we have synthe-
sized a series of new derivatives of SPA and compared
their inhibitory effects toward recombinant CYP 2C8,
2C9, 2C18, and 2C19.
Ch em istr y
To further explore the topology of the active site not
only of CYP 2C9 but also of CYP 2C8, 2C18, and 2C19,
and to find new selective inhibitors of these human P450
2Cs, many SPA derivatives with various R1 and R2
substituents that replace the NH2 and H (of SO2NH)
groups of SPA were synthesized. Three series of SPA
derivatives have been synthesized.
The compounds of the first series, 1-5, derive from
SPA by replacement of its NH2 substituent with R1
groups of various sizes and polarities (R1 ) CH3, CH2-
CHdCH2, Br, m-NO2Ph, and m-NH2Ph). Compounds
1-3 were obtained from reaction of (2-phenyl-2H-
pyrazol-3-yl)amine with benzenesulfonyl chlorides bear-
ing the appropriate p-R1 substituent (Scheme 1). Com-
pounds 4 and 5 were derived from 2 and 3, respectively,
by classical procedures (Scheme 1).
function of the precursor with benzoic acid 3-bromopro-
pyl ester, followed by deprotection of the alcohol (and
amino, in the case of 14 and 15) function with NaOH.
Compound 16 was prepared by treatment of its bromo
precursor with allyltributyltin in DMF in the presence
of Pd(PPh3)4 under argon ((a) in Scheme 3). Reduction
of 19 into 17 was carried out using Na2S2O4 as reducing
agent20 ((b) in Scheme 3), since the classical method of
reduction of nitroaromatic molecules using H2 in the
presence of Ni or Pd led to longer reaction times and
lower yields.
Bioch em istr y
The inhibitory effects of compounds 1-24 toward
human liver CYP 2Cs were studied on microsomes of
yeast strain W(R)fur121 expressing each human P450
of the 2C subfamily. The assays used for studying P450
2C activities were based on the 5-hydroxylation of
2-aroylthiophenes, the 5-hydroxy-2-aroylthiophene me-
tabolites being easily detected by UV-vis spectroscopy
at 390 nm.22 Tienilic acid and 2-[2,3-dichloro-4-(2-
thenoyl)phenoxy]ethanol were used as substrates for
CYP 2C95 and CYP 2C8,18 respectively. 3-[2,3-Dichloro-
4-(2-thenoyl)phenoxy]propan-1-ol was used as a sub-
strate for measuring the activities of CYP 2C18 and
2C19.18 In this paper, our objective was not to study the
detailed mechanism of inhibition of CYP 2Cs by the
various SPA derivatives, but more to compare their
efficiency as inhibitors of CYP 2C8, 2C9, 2C18, and
2C19. Thus, determination of the IC50 values was done
by measuring the effects of the inhibitors on the activity
of each CYP 2C using a substrate concentration equal
Compounds of the second series, 6-13, simply derive
from SPA by N-alkylation of its sulfonamide function.
They were prepared by direct N-alkylation of acetyl-
amino-SPA using the appropriate alkyl halide and Na2-
CO3 in DMF, followed by deprotection of the acetylami-
no function (Scheme 2).
Finally, the compounds of the third series, 14-24,
bear both substituents R1 and R2 different from those
found in SPA. Some of them, 18 and 20-24, were
directly obtained by N-alkylation of the SO2NH function
of compounds 3, 4, and 1 with the appropriate alkyl
halide (Scheme 3). Such a direct N-alkylation of para-
substituted (2-phenyl-2H-pyrazol-3-yl)arenesulfonam-
ides (with R1 ) Br, m-NO2Ph, (CH2)2NHCOCH3, and
(CH2)3NHCOCH3) with 3-bromopropanol gave second-
ary products, not easily separated by column chroma-
tography. Thus compounds 14, 15, and 19 were pre-
pared in two steps, the N-alkylation of the sulfonamide

3624 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Ha-Duong et al.
Ta ble 2. Comparison of the Inhibitory Effects of Various
N-Alkylated SPA Derivatives (See Scheme 2) toward
Recombinant CYP 2C8, 2C9, 2C18, and 2C19 Expressed
in Yeast
Sch em e 3a
a
IC50 (µM)
compd
R2
CYP 2C8 CYP 2C9 CYP 2C18 CYP 2C19
SPA
6
7
8
9
H
CH3
130
50
13
8
30
4
0.6
100
60
15
7
8
3
>500b
200
225
250
130
60
CH2CH3
(CH2)2CH3
CH(CH3)2
(CH2)3CH3
(CH2)2CH-
(CH3)2
90
55
80
50
60
10
11
6
9
3
60
12
CH2Ph
15
55
7
45
a
IC50 values were measured from increasing concentrations of
potential inhibitors added to the assay incubation mixture includ-
ing yeast microsomes expressing CYP 2C in 0.1 M Tris buffer, pH
7.4, substrate, and an NADPH-generating system. The experi-
b
mental details are described in the Experimental Section. CYP
2C activity higher than 50% of control activity at the indicated
inhibitor concentration. Values are the mean from three experi-
ments.
the space occupied by R1 leads to a decrease of selectivity
toward CYP 2C9, as already with R1 ) allyl, the IC50
value for CYP 2C8 reaches 10 µM. Further increases of
the R1 size, as in compounds 3 and 5, lead to strong
inhibitory effects not only toward CYP 2C8 but also
toward CYP 2C18, and even to moderate effects toward
CYP 2C19. In fact compound 3 acts as a reasonable
inhibitor for all human CYP 2Cs, with IC50 values of 7,
4, 5, and 65 µM for CYP 2C8, 2C9, 2C18, and 2C19,
respectively.
a
Reagents: (a) Allyltributyltin, Pd[PPh3]4, in DMF, under
argon. (b) Na2S2O4, 60 °C, 2 h, under argon.
N-alkylation of the SO2NH function of SPA has a
dramatic effect on the inhibition of CYP 2C9 (Table 2),
as it leads to IC50 values 2 orders of magnitude larger
than that of SPA (50-100 µM instead of 0.6 µM). These
results are in agreement with previous reports indicat-
ing that an ionic (or hydrogen-bonding) interaction of
the SO2N- (or SO2NH) function of SPA with an amino
acid residue of the CYP 2C9 active site is very important
in SPA recognition by CYP 2C9.19,23 In parallel with this
spectacular decrease of the inhibitory effects toward
CYP 2C9, N-alkylation of SPA leads to a marked
increase of the inhibitory effects toward CYP 2C8 and
2C18. Thus, compounds 8, 10, and 11 (R2 ) (CH2)2CH3,
(CH2)3CH3, and (CH2)2CH(CH3)2 respectively) exhibit
IC50 values of 8, 4, and 3 µM toward CYP 2C8 and 8, 6,
and 9 µM toward CYP 2C18. In fact, CYP 2C8 and CYP
2C18 exhibit a similar behavior toward the N-alkylated
SPA derivatives 6-12, with a large 10-fold decrease of
the IC50 values on passing from SPA to 7 and a further
smaller decrease of IC50 on increasing the length of R2.
Compounds 6-12 are less efficient inhibitors of CYP
2C9 and 2C19, as they show IC50 values between 45 and
250 µM for those two enzymes. However, their inhibi-
tory potency also increases in parallel with the size of
R2. As a consequence, compounds 10-12 inhibit the four
human CYP 2Cs with IC50 values smaller than 60 µM.
Previous studies on tienilic acid derivatives have
shown that replacement of the anionic moiety of tienilic
acid, -OCH2COOH, with a neutral substituent contain-
ing an alcohol function, -O(CH2)3OH, led to a very good
selective substrate of CYP 2C18.18 On the basis of these
Ta ble 1. Comparison of the Inhibitory Effects of SPA
Derivatives Bearing Various R1 Substituents (See Scheme 1)
toward Recombinant CYP 2C8, 2C9, 2C18, and 2C19 Expressed
in Yeast
a
IC50 (µM)
compd
R1
CYP 2C8 CYP 2C9 CYP 2C18 CYP 2C19
SPA NH2
130
220
7
10
8
0.6
0.6
4
1
12
60
170
5
150
20
>500b
100
65
450
95
1
3
4
5
CH3
m-NO2Ph
CH2CHdCH2
m-NH2Ph
a
IC50 values were measured from increasing concentrations of
potential inhibitors added to the assay incubation mixture includ-
ing yeast microsomes expressing CYP 2C in 0.1 M Tris buffer, pH
7.4, substrate, and an NADPH-generating system. The experi-
b
mental details are described in the Experimental Section. CYP
2C activity higher than 50% of control activity at the indicated
inhibitor concentration. Values are the mean from three experi-
ments.
5,18
to KM
(see the Experimental Section). However, it is
noteworthy that the few compounds, such as SPA,19 1,
or 4 (data not shown), for which the type of inhibition
of CYP 2C9 was determined on the basis of Line-
weaver-Burk or Dixon plots were found to be competi-
tive inhibitors.
Table 1 illustrates the effects of replacing the NH2
substituent of SPA with R1 groups of different sizes and
polarities on the inhibition of CYP 2C activities. SPA
itself and its derivative bearing a relatively small
substituent (R1 ) CH3) are selective inhibitors of CYP
2C9 with IC50 values 2 orders of magnitude lower than
those observed for CYP 2C8, 2C18, and 2C19. Increasing
results, a series of SPA derivatives bearing an R2
)
-CH2(CH2)nOH chain, with n ) 1, 2, or 3, have been

Inhibition of P450 2Cs by Sulfaphenazole Derivatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3625
Ta ble 3. Comparison of the Inhibitory Effects of Various Polar SPA Derivatives (See Schemes 2 and 3) toward Recombinant CYP
2C8, 2C9, 2C18, and 2C19 Expressed in Yeast
a
IC50 (µM)
compd
R1
R2
CYP 2C8
CYP 2C9
CYP 2C18
CYP 2C19
13
14
15
16
17
18
19
20
NH2
(CH2)3OH
(CH2)3OH
(CH2)3OH
(CH2)3OH
(CH2)3OH
(CH2)2OH
(CH2)3OH
(CH2)4OH
22
>250b
>200b
35
>100b
10
30
65
30
18
3
9
8
4
150
10
15
10
40
165
200
>200b
(CH2)2NH2
(CH2)3NH2
CH2CHdCH2
m-NH2Ph
m-NO2Ph
50
80
12
20
28
15
13
>250b
>250b
>250b
m-NO2Ph
m-NO2Ph
a
IC50 values were measured from increasing concentrations of potential inhibitors added to the assay incubation mixture including
yeast microsomes expressing CYP 2C in 0.1 M Tris buffer, pH 7.4, substrate, and an NADPH-generating system. The experimental
b
details are described in the Experimental Section. CYP 2C activity higher than 50% of control activity at the indicated inhibitor
concentration. Values are the mean from three experiments.
Ta ble 4. Comparison of the Inhibitory Effects of Hydrophobic SPA Derivatives (See Scheme 3) toward Recombinant CYP 2C8, 2C9,
2C18, and 2C19 Expressed in Yeast
a
IC50 (µM)
compd
R1
R2
CH3
CH2Ph
CH3
CYP 2C8
CYP 2C9
CYP 2C18
CYP 2C19
21
22
23
24
CH2CHdCH2
CH2CHdCH2
CH3
20
30
180
15
>200b
>200b
10
10
125
12
20
20
20
10
CH3
(CH2)2CH3
20
20
a
IC50 values were measured from increasing concentrations of potential inhibitors added to the assay incubation mixture including
yeast microsomes expressing CYP 2C in 0.1 M Tris buffer, pH 7.4, substrate, and an NADPH-generating system. The experimental
b
details are described in the Experimental Section. CYP 2C activity higher than 50% of control activity, even with 200 µM potential
inhibitor. Values are the mean from three experiments.
synthesized as potential good inhibitors of CYP 2C18.
Table 3 shows the results obtained with such com-
pounds, 13-20. Some of these compounds such as 17-
20 are good inhibitors of CYP 2C18, with IC50 values
between 3 and 9 µM; however, they are not selective,
as all of them except 13 also inhibit CYP 2C9, and some
of them, 16 and 17, inhibit all human CYP 2Cs with
IC50 values between 3 and 80 µM. It is noteworthy that
compounds 14-16 are relatively good inhibitors of CYP
2C19, with IC50 values between 10 and 15 µM, which
have never been reached with compounds 1-13. Com-
pound 13 derives from SPA by N-alkylation of its SO2-
NH function and may be compared to compounds 6-12.
As expected, 13, 8, and 10, which bear R2 groups of
similar size (R2 ) CH2CH2CH2OH, CH2CH2CH3, and
CH2CH2CH2CH3, respectively), exhibit similar inhibi-
tory properties with a marked preference for CYP 2C8
and 2C18. Addition of 13 to yeast microsomes express-
ing CYP 2C8 leads to the appearance of a difference
visible spectrum characterized by a peak at 430 nm and
a trough at 405 nm. This difference spectrum is typical
of cytochrome P450 Fe(III) bound to a nitrogenous
ligand, and indicates that 13 binds to CYP 2C8 iron via
its NH2 function, with a Ks value for dissociation of the
CYP 2C8-13 complex of 2 ( 1 µM (data not shown).
Conversely, addition of 100 µM 13 to yeast microsomes
expressing CYP 2C9 does not lead to any significant
difference spectrum. These spectral results are in agree-
ment with the inhibitory properties of 13 toward CYP
2C8 (relatively low IC50 value of 22 µM, which would
correspond to a Ki value of 11 µM in the case of
competitive inhibition) and CYP 2C9 (IC50 > 100 µM)
(Table 3).
strong inhibitors of CYP 2C19, with IC50 values between
10 and 20 µM. Compound 24 acts as a good inhibitor
for all human CYP 2Cs with IC50 values around 15 µM,
whereas compound 21 inhibits all human CYP 2Cs with
IC50 values at the same level except CYP 2C9, and
compound 23 inhibits all human CYP 2Cs except CYP
2C8.
Discu ssion
The aforementioned results provide some data about
the structural factors that are important for molecules
to be recognized by each human CYP 2C.
High affinities (IC50 ≈ 1 µM) for CYP 2C9 were only
observed for SPA derivatives having the intact SO2NH
function and a relatively small R1 substituent (R1
)
NH2, CH3, or CH2CHdCH2). Any increase in the size
of R1 leads to a moderate decrease of the affinity (Table
1) and the N-alkylation of the SO2NH function of SPA
(Table 2) or of 1 (see 23 and 24 in Table 4) to a greater
decrease of the affinity in general (IC50 between 10 and
>200 µM).
The same structural changes have opposite effects on
molecular recognition by CYP 2C18, since an increase
in the size of R1 (compounds 3 and 5 of Table 1) or the
N-alkylation of SPA (Table 2) generally results in a
marked decrease of IC50 values from 60 to 5 or 3 µM (if
one excepts compound 4). Thus, contrary to CYP 2C9,
CYP 2C18 generally prefers neutral compounds with
relatively large R1 and R2 substituents. This conclusion
is in agreement with previous data on the interactions
of another series of compounds related to tienilic acid
with CYP 2C9 and 2C18.18
Finally, four compounds bearing two hydrophobic
alkyl substituents R1 and R2 were synthesized and
compared as inhibitors of human CYP 2Cs (Table 4).
Interestingly, these four hydrophobic compounds are
In a general manner, CYP 2C8 exhibits a behavior
toward these structural changes of SPA similar to that
of CYP 2C18. The only exceptions to this rule are
concerned with primary amines 14 and 15, and com-

3626 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Ha-Duong et al.
The abbreviations s, d, t, q, m, br s, and dd are used for singlet,
doublet, triplet, quadruplet, multiplet, broad singlet, and
doublet of doublets, respectively. Mass spectra (MS) were
obtained with chemical ionization (CI) using NH3 or CH4
(HRMS) on a Nermag R1010 apparatus. Elemental analyses
were carried out at the Centre Regional de Microanalyse,
Paris.
pounds 18-20. CYP 2C19 shows an even lower affinity
for anionic compounds than CYP 2C8 and 2C18 (see
SPA, 1, and 4). However, as CYP 2C8 and 2C18, it
exhibits a better affinity for neutral compounds derived
from N-alkylation of the SO2NH function of SPA (Table
2) or for anionic compounds bearing a larger R1 sub-
stituent (Table 1). Several SPA derivatives with R1
different from NH2 and R2 different from H lead to IC50
values as low as 10 µM.
Syn th esis of Su lfa p h en a zole Der iva tives. 4-Meth yl-N-
(2-p h en yl-2H-p yr a zol-3-yl)ben zen esu lfon a m id e, 1. 1 was
prepared as described previously:27 mp 142 °C (lit.27 mp 142-
143 °C).
Coming back to the search of new inhibitors for
human CYP 2Cs, we have found two molecules that
inhibit all human CYP 2Cs with IC50 values smaller
than 40 µM. These are compounds 17 (IC50 ) 13, 12, 3,
and 40 µM for CYP 2C8, 2C9, 2C18, and 2C19, respec-
tively) and 24 (IC50 ) 15, 20, 20, and 10 for CYP 2C8,
2C9, 2C18, and 2C19, respectively). Such molecules
might be useful to evaluate the contribution of the whole
human subfamily 2C in the oxidative metabolism of a
xenobiotic in various microsomes or tissues, provided
that they do not inhibit the CYPs of the other subfami-
lies. In that context, compound 21 might also be
interesting as it inhibits all human CYP 2Cs at a
reasonable level (IC50 < 20 µM) except CYP 2C9 (Table
4). In the same idea, compound 23 inhibits all human
CYP 2Cs with IC50 < 20 µM, except CYP 2C8, for which
an IC50 of 180 µM was found. Further experiments are
required to know whether compounds 17, 24, 21, and
23 do not act as inhibitors of human CYPs of subfamilies
different from 2C.
4-Br om o-N-(2-p h en yl-2H-p yr a zol-3-yl)ben zen esu lfon -
a m id e, 2. 2 was prepared by reaction of 2-phenyl-2H-pyrazol-
3-ylamine (1.26 mmol) with 4-bromobenzenesulfonyl chloride
(1.37 mmol) in the presence of pyridine. Recrystallization from
CH2Cl2 gave compound 2 (61% yield) as pale yellow crystals:
mp 185-186 °C; UV (MeOH) λ 235 (22); 1H NMR (CDCl3)
δ 7.55 (d, 1H, J ) 2), 7.53-7.43 (m, 4H), 7.37 (m, 3H), 7.11
(m, 2H), 6.55 (br s, 1H, NH), 6.27 (d, 1H, J ) 2); MS (CI, NH3)
m/z 378 ([M + H]+, 97), 163 (18). Anal. (C15H12BrN3O2S) C,
H, N.
3′-Nitr obip h en yl-4-su lfon ic Acid (2-P h en yl-2H-p yr a -
zol-3-yl)a m id e, 3. 3-Nitrobiphenyl (920 mg, 4.62 mmol) was
progressively added to chlorosulfonic acid (2 mL). The reaction
mixture was stirred for 1 h at room temperature, followed by
thin-layer chromatography (TLC) (SiO2, cyclohexane/CH2Cl2,
50/50). The solution was dripped into ice, extracted with CH2-
Cl2, and dried over MgSO4, and the solvent was evaporated.
The product was purified by column chromatography (SiO2,
cyclohexane/CH2Cl2, 50/50) and obtained in a 65% yield: 1H
NMR (CDCl3) δ 7.70 (t, 1H, J ) 8), 7.85 (d, 2H, J ) 8.6), 7.93
(d, 1H, J ) 8), 8.16 (d, 2H, J ) 8.6), 8.31 (d, 1H, J ) 8), 8.48
(s, 1H); IR (cm-1) 1348, 1164. The previous compound (870
mg, 2.92 mmol) was added to 2-phenyl-2H-pyrazol-3-ylamine
(465 mg, 2.92 mmol) in anhydrous pyridine (4.5 mL). After
the mixture was heated under reflux at 95 °C for 1 h, pyridine
was evaporated; the residue was dissolved in 1 N HCl,
extracted with CH2Cl2, and dried over MgSO4. Purification by
column chromatography (SiO2, CH2Cl2/EtOAc, 90/10), and two
recrystallizations from CH2Cl2 and Et2O, led to a crystalline
product in a 47% yield: mp 164-165 °C ; UV (MeOH) λ 253
Finally, when one looks for a selective inhibitor for
one of the human CYP 2Cs among the 24 compounds
that have been synthesized, one only finds one molecule
leading to an IC50 value for a given CYP 2C 2 orders of
magnitude smaller than the IC50 values for the three
other CYP 2Cs. This is compound 1, which acts as a
specific inhibitor of CYP 2C9, with IC50 values for CYP
2C8, 2C18, and 2C19 more than 200 times higher than
that for CYP 2C9. Compound 1 is even slightly more
selective toward CYP 2C9 than SPA itself. It is note-
worthy that some of the described molecules, such as
7-12, are much better inhibitors of CYP 2C8 and 2C18
than of CYP 2C9 and 2C19 (Table 2). If one takes into
account that CYP 2C18 is hardly detectable in human
liver microsomes,24,25 these molecules could be interest-
ing to selectively inhibit CYP 2C8 in these microsomes.
Compound 11 seems to be the most interesting molecule
for that purpose, as its IC50 value for CYP 2C8 is low (3
µM) and 20-fold lower than the IC50 values found for
CYP 2C9 and 2C19.
1
(37); H NMR (CDCl3) δ 6.30 (d, 1H, J ) 1.9), 6.70 (br s, 1H,
NH), 7.16 (m, 2H), 7.35 (m, 3H), 7.56 (d, 1H, J ) 1.9), 7.63 (d,
2H, J ) 8.6), 7.67 (t, 1H, J ) 8), 7.77 (d, 2H, J ) 8.6), 7.90 (d,
1H, J ) 8), 8.28 (d, 1H, J ) 8), 8.43 (s, 1H); MS (CI, NH3) m/z
421 ([M + H]+, 100), 391 (39), 163 (26). Anal. (C21H16N4O4S)
C, H, N.
4-Allyl-N-(2-p h en yl-2H-p yr a zol-3-yl)ben zen esu lfon a m -
id e, 4. Reaction of 2 (210 mg) with allyltributyltin (0.19 mL)
in DMF was performed in the presence of tetrakis(triphen-
ylphosphine)palladium(0) under argon at 110 °C for 7.5 h. Re-
crystallization from ether of the crude product obtained after
purification by column chromatography (SiO2, CH2Cl2/Et2O,
5%) led to 102 mg of compound 4 as pale yellow crystals: mp
1
115-116 °C; UV (MeOH) λ 229 (18); H NMR (CDCl3) δ 7.58
(d, 2H, J ) 8.3), 7.53 (d, 1H, J ) 1.9), 7.35 (m, 3H), 7.23 (d,
2H, )8.3), 7.07 (m, 2H), 6.47 (br s, 1H, NH), 6.25 (d, 1H, J )
1.9), 5.91 (m, 1H, Ha, J ) 6.7, J ab ) 10.1, J ab′ ) 16.9), 5.14 (m,
1H, Hb, J ba ) 10.1, J bb′ ) 1.6), 5.08 (m, 1H, Hb, J ba ) 16.9,
J bb′ ) 1.6), 3.43 (d, 2H, J ) 6.7); MS (CI, NH3) m/z 340 ([M +
H]+, 100), 357 (16), 163 (50). Anal. (C18H17N3O2S) C, H, N.
Exp er im en ta l Section
Ch em ica ls. All chemicals used were of the highest quality
commercially available. Tienilic acid was provided by Anphar-
Rolland (Chilly-Mazarin, France). 3-[2,3-Dichloro-4-(2-thenoyl)
phenoxy]propan-1-ol and 2-[2,3-dichloro-4-(2-thenoyl)phe-
noxy]ethanol were prepared by previously described proce-
dures.18,26 SPA was purchased from Sigma.
P h ysica l Mea su r em en ts. UV-vis spectra were recorded
on Kontron Uvikon 860 and 820 spectrophotometers equipped
with a diffusion sphere. UV characteristics of compounds 2-24
reported in the following are λmax (indicated as λ followed by
its value (nm) in the text) and ꢀ (mM-1·cm-1) (values in
parentheses). 1H NMR spectra were recorded at 27 °C on a
Bruker ARX-250 instrument; chemical shifts are reported
downfield from (CH3)4Si, and coupling constants are in hertz.
3′-Am in obip h en yl-4-su lfon ic Acid (2-P h en yl-2H-p yr a -
zol-3-yl)a m id e, 5. To a solution of compound 3 (91 mg, 0.22
mmol) dissolved in hot EtOH (10 mL) was added Pd black (7.5
mg). This solution was hydrogenated under H2 (1 atm) for 20
h. After filtration over Celite, the solvent was evaporated. The
product was purified by column chromatography (SiO2, CH2-
Cl2/EtOAc, 70/30). The solvent was evaporated, and the ob-
tained oil was dissolved in Et2O and then evaporated. Com-
pound 5 was obtained as a foam in a 65% yield: UV (MeOH)
1
λ 245 (29), 310 (4); H NMR (CDCl3) δ 7.67 (d, 2H, J ) 8.5),
7.56 (d, 2H, J ) 8.5), 7.55 (d, 1H, J ) 1.9), 7.33 (m, 3H), 7.25
(t, 1H, J ) 7.8), 7.10 (m, 2H), 6.94 (d, 1H, J ) 7.8), 6.85 (t,

Inhibition of P450 2Cs by Sulfaphenazole Derivatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3627
1H, J ) 1.7), 6.73 (dd, 1H, J ) 7.8, J ) 1.7), 6.5 (br s, 1H,
NH), 6.30 (d, 1H, J ) 1.9), 3.79 (br s, 2H, NH2); MS (CI, NH3)
m/z 391 ([M + H]+, 100), 163 (24). Anal. (C21H18N4O2S) C,
H, N.
1H), 4.16 (br s, 2H, NH2), 0.75 (br s, 3H), 0.58 (br s, 3H); MS
(CI, NH3) m/z 357 ([M + H]+, 100), 202 (82). Anal. (C18H20N4O2S)
C, H, N.
4-Am in o-N-bu tyl-N-(2-ph en yl-2H-pyr azol-3-yl)ben zen e-
su lfon a m id e, 10. 1-Bromobutane (0.1 mL, 0.93 mmol) was
added to a solution of 4-(acetylamino)-N-(2-phenyl-2H-pyrazol-
3-yl)benzenesulfonamide (150 mg, 0.42 mmol) in 1.5 mL of
anhydrous DMF containing 100 mg of Na2CO3. After 38 h at
50 °C, the solvent was evaporated and the residue dissolved
in CH2Cl2. The organic phase was washed with water and
dried over MgSO4. After purification by column chromatogra-
phy (SiO2, CH2Cl2/EtOAc, 70/30), 160 mg of N-acetyl-10 was
obtained in a 92% yield: 1H NMR (CDCl3) δ 7.66 (m, 4H),
7.62-7.59 (m, 3H), 7.53 (br s, 1H), 7.47-7.33 (m, 3H), 5.87 (d,
1H, J ) 2), 3.26 (t, 2H, J ) 7.2), 2.20 (s, 3H), 1.22 (m, 2H),
1.05 (m, 2H), 0.67 (t, 3H, J ) 7.2). The previous compound
(160 mg, 0.38 mmol) was heated under reflux in 3 mL of 3 N
NaOH and 6 mL of EtOH for 2 h. EtOH was evaporated, and
the solution was diluted in water and extracted with CH2Cl2.
The organic phase was dried over MgSO4. After recrystalliza-
tion from Et2O/cyclohexane, 10 was obtained (125 mg) in an
Gen er a l P r oced u r e for th e P r ep a r a tion of th e N-
Alk yla ted Com p ou n d s 6-13. 4-(Acetylamino)-N-(2-phenyl-
2H-pyrazol-3-yl)benzenesulfonamide was prepared as previ-
ously described.19 The appropriate alkyl halide (1.5-7.5 equiv)
was added to a solution of 4-(acetylamino)-N-(2-phenyl-2H-
pyrazol-3-yl)benzenesulfonamide (0.42 mmol) in 1.5 mL of
anhydrous DMF containing 100 mg of Na2CO3. After 1 h to 3
days at 20-80 °C, DMF was evaporated and the residue was
dissolved in CH2Cl2. The organic phase was washed with water
and dried over MgSO4. After purification by column chroma-
tography (SiO2), the intermediate acetylated compound was
heated under reflux in 3 mL of 3 N NaOH and 3-7 mL of
EtOH for 2-2.5 h. EtOH was evaporated, and the solution was
diluted with water and extracted with CH2Cl2. The organic
phase was dried over MgSO4, and the final product was
purified by crystallization.
4-Am in o-N-m et h yl-N-(2-p h en yl-2H -p yr a zol-3-yl)b en -
zen esu lfon a m id e, 6. 6 was prepared in an 84% yield using
the general procedure described above. Reaction with methyl
iodide (1.6 equiv) was done at 80 °C for 1 h. Product 6 was
purified by crystallization from CH2Cl2/Et2O: mp 210-211 °C;
UV (MeOH) λ 271 (22); 1H NMR (CDCl3) δ 7.61-7.56 (m, 3H),
7.47-7.30 (m, 5H), 6.63 (d, 2H, J ) 8.7), 5.94 (d, 1H, J ) 1.9),
4.17 (br s, 2H, NH2), 3.01 (s, 3H, CH3); MS (CI, NH3) m/z 329
([M + H]+, 100), 174 (16). Anal. (C16H16N4O2S) C, H, N.
1
89% yield: mp 111-112 °C; UV (MeOH) λ 271 (31); H NMR
(CDCl3) δ 7.67 (d, 2H, J ) 7.2), 7.59 (d, 1H, J ) 2), 7.49 (d,
2H, J ) 8.7), 6.65 (d, 2H, J ) 8.7), 5.91 (d, 1H, J ) 2), 4.16 (br
s, 2H, NH2), 3.24 (t, 2H, J ) 7.2), 1.18 (m, 2H), 1.05 (m, 2H),
0.68 (t, 3H, J ) 7.2); MS (CI, NH3) m/z 371 ([M + H]+, 100),
216 (92). Anal. (C19H22N4O2S) C, H, N.
4-Am in o-N-(3-m eth ylbu t-1-yl)-N-(2-p h en yl-2H-p yr a zol-
3-yl)ben zen esu lfon am ide, 11. 1-Bromo-3-methylbutane (0.11
mL, 0.92 mmol) was added to a solution of 4-(acetylamino)-
N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (150 mg, 0.42
mmol) in 1.5 mL of anhydrous DMF containing 100 mg of Na2-
CO3. After 30 h at 60 °C and 60 h at room temperature, the
solvent was evaporated and the residue dissolved in CH2Cl2.
The organic phase was washed with water and dried over
MgSO4. After purification by column chromatography (SiO2,
CH2Cl2/EtOAc, 70/30), 160 mg of N-acetyl-11 was obtained in
an 84% yield: 1H NMR (CDCl3) δ 9.01 (br s, 1H), 7.67-7.55
(m, 7H), 7.40-7.27 (m, 3H), 5.85 (d, 1H, J ) 2), 3.24 (t, 2H,
J ) 6.6), 2.06 (s, 3H), 1.20 (m, 1H), 1.04 (q, 2H, J ) 6.6), 0.63
(d, 6H, J ) 6.6). The previous compound (150 mg, 0.35 mmol)
was heated under reflux in 2.4 mL of 3 N NaOH and 3 mL of
EtOH for 2 h. EtOH was evaporated, and the solution was
diluted in water and extracted with CH2Cl2. The organic phase
was dried over MgSO4. 11 was recrystallized from Et2O/
cyclohexane, and obtained (118 mg) in an 88% yield: mp 143-
144 °C; UV (MeOH) λ 271 (27); 1H NMR (CDCl3) δ 7.66 (d,
2H, J ) 7.1), 7.59 (d, 1H, J ) 2), 7.49 (d, 2H, J ) 8.7), 6.65 (d,
2H, J ) 8.7), 5.92 (d, 1H, J ) 2), 4.16 (br s, 2H, NH2), 3.27 (t,
2H, J ) 6.6), 1.27 (m, 2H), 1.09 (q, 2H, J ) 6.6), 0.68 (d, 6H,
J ) 6.6); MS (CI, NH3) m/z 385 ([M + H]+, 100), 230 (61). Anal.
(C20H24N4O2S) C, H, N.
4-Am in o-N-eth yl-N-(2-ph en yl-2H-pyr azol-3-yl)ben zen e-
su lfon a m id e, 7. 7 was prepared in a 67% yield using the
general procedure described above. Reaction with ethyl bro-
mide (7.5 equiv) was performed at room temperature for 39
h. Product 7 was recrystallized from CH2Cl2/cyclohexane: mp
1
209-210 °C; UV (MeOH) λ 271 (29); H NMR (CDCl3) δ 7.66
(d, 2H, J ) 8.7), 7.60 (d, 1H, J ) 2), 7.50-7.32 (m, 5H), 6.64
(d, 2H, J ) 8.7), 5.93 (d, 1H, J ) 2), 4.15 (br s, 2H, NH2), 3.35
(q, 2H, J ) 7.2), 0.88 (t, 3H, J ) 7.2); MS (CI, NH3) m/z 343
([M + H]+, 100), 188 (26). Anal. (C17H18N4O2S) C, H, N.
4-Am in o-N-p r op yl-N-(2-p h en yl-2H -p yr a zol-3-yl)b en -
zen esu lfon a m id e, 8. The title compound was prepared in
an 81% yield using the general procedure described above.
Reaction with propyl iodide (2.2 equiv) was performed at 60
°C for 24 h. Product 8 (83 mg) was obtained after recrystal-
lization from CH2Cl2/Et2O: mp 169-170 °C; UV (MeOH) λ 271
(27); 1H NMR (CDCl3) δ 7.67 (d, 2H, J ) 7.1), 7.59 (d, 1H, J )
2), 7.50 (d, 2H, J ) 8.7), 7.47-7.35 (m, 3H), 6.65 (d, 2H, J )
8.7), 5.91 (d, 1H, J ) 2), 4.16 (br s, 2H, NH2), 3.21 (t, 2H, J )
7.4), 1.21 (m, 2H), 0.63 (t, 3H, J ) 7.4); MS (CI, NH3) m/z 357
([M + H]+, 100), 202 (18). Anal. (C18H20N4O2S·1/8H2O) C,
H, N.
4-Am in o-N-isop r op yl-N-(2-p h en yl-2H-p yr a zol-3-yl)ben -
zen esu lfon a m id e, 9. 2-Iodopropane (0.1 mL, 1 mmol) was
added to a solution of 4-(acetylamino)-N-(2-phenyl-2H-pyrazol-
3-yl)benzenesulfonamide (150 mg, 0.42 mmol) in 1.5 mL of
anhydrous DMF containing 100 mg of Na2CO3. After 24 h at
50 °C, the solvent was evaporated and the residue dissolved
in CH2Cl2. The organic phase was washed with water and
dried over MgSO4. After purification by column chromatogra-
phy (SiO2, CH2Cl2/EtOAc, 70/30), 141 mg of N-acetyl-9 was
obtained in an 84% yield: 1H NMR (CDCl3) δ 8.76 (br s, 1H,
NH), 7.73 (d, 2H, J ) 7), 7.68 (s, 4H), 7.64 (d, 1H, J ) 1.9),
7.43-7.30 (m, 3H), 5.96 (d, 1H, J ) 1.9), 4.18 (m, 1H, CH),
2.10 (s, 3H, CH3), 0.68 (br s, 3H, CH3), 0.58 (br s, 3H, CH3).
The previous compound (141 mg, 0.35 mmol) was heated under
reflux in 2.4 mL of 3 N NaOH and 7 mL of EtOH for 2 h. EtOH
was evaporated, and the solution was diluted with water and
extracted with CH2Cl2. The organic phase was dried over
MgSO4. Compound 9 was recrystallized from CH2Cl2/cyclo-
hexane, and obtained (99 mg) in a 79% yield: mp 223-224
°C; UV (MeOH) λ 271 (27); 1H NMR (CDCl3) δ 7.77 (d, 2H,
J ) 7.5), 7.64 (d, 1H, J ) 2), 7.59 (d, 2H, J ) 8.7), 7.47-7.32
(m, 3H), 6.67 (d, 2H, J ) 8.7), 5.99 (d, 1H, J ) 2), 4.20 (m,
4-Am in o-N-b en zyl-N-(2-p h en yl-2H -p yr a zol-3-yl)b en -
zen esu lfon a m ide, 12. Benzyl bromide (0.075 mL, 0.63 mmol)
was added to a solution of 4-(acetylamino)-N-(2-phenyl-2H-
pyrazol-3-yl)benzenesulfonamide (150 mg, 0.42 mmol) in 1.5
mL of anhydrous DMF containing 70 mg of Na2CO3. After 3.5
h at 80 °C, the solvent was evaporated and the residue
dissolved in CH2Cl2. The organic phase was washed with water
and dried over MgSO4. After purification by column chroma-
tography (SiO2, CH2Cl2/EtOAc 70/30), 160 mg of N-acetyl-12
was obtained in an 85% yield: 1H NMR (CDCl3) δ 7.70 (s, 4H),
7.53 (br s, 2H), 7.26 (m, 3H), 7.14 (m, 3H), 7.07 (t, 2H, J )
7.2), 6.83 (d, 2H, J ) 7.2), 5.87 (d, 1H, J ) 2), 4.38 (s, 2H,),
2.23 (s, 3H). The previous compound (140 mg, 0.31 mmol) was
heated under reflux in 3 mL of 3 N NaOH and 3 mL of EtOH
for 2 h. EtOH was evaporated, and the solution was diluted
in water and extracted with CH2Cl2. The organic phase was
dried over MgSO4. 12 was recrystallized from Et2O/cyclohex-
ane, and obtained (103 mg) in an 81% yield: mp 153-154 °C;
UV (MeOH) λ 272 (27); 1H NMR (CDCl3) δ 7.54-7.51 (m, 3H),
7.26-7.11 (m, 7H), 7.03 (t, 1H, J ) 7.3), 6.82 (d, 2H, J ) 7.3),
6.68 (d, 2H, J ) 8.7), 5.93 (d, 1H, J ) 1.9), 4.36 (s, 2H), 4.20

3628 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Ha-Duong et al.
(br s, 2H, NH2); MS (CI, NH3) m/z 405 ([M + H]+, 100), 250
MgSO4. After purification by column chromatography (SiO2,
CH2Cl2/MeOH, 20%, and then 1+% NH3(aq)), 65 mg of 14 was
obtained in a 36% yield: 1H NMR (CDCl3) δ 7.65 (d, 2H, J )
8.2) 7.59 (m, 2H), 7.55 (d, 1H, J ) 1.7), 7.41-7.35 (m, 3H),
7.30 (d, 2H, J ) 8.2), 5.84 (d, 1H, J ) 1.7), 3.37 (t, 2H, J )
6.1), 3.18 (t, 2H, J ) 6.1), 2.92 (t, 2H, J ) 6.6), 2.77 (t, 2H,
J ) 6.6), 1.34 (m, 2H). Anal. (C20H24N4O3S) C, H, N.
(71). Anal. (C22H20N4O2S) C, H, N.
4-Am in o-N-(3-h yd r oxyp r op yl)-N-(2-p h en yl-2H-p yr a zol-
3-yl)ben zen esu lfon a m id e, 13. 3-Bromopropanol (0.35 mL,
3.9 mmol) was added to a solution of 4-(acetylamino)-N-(2-
phenyl-2H-pyrazol-3-yl)benzenesulfonamide (210 mg, 0.59
mmol) in anhydrous DMF (1.5 mL) containing 70 mg of Na2-
CO3. After 16 h at 50 °C, DMF was evaporated and the residue
was dissolved in CH2Cl2. The organic phase was washed with
water and dried over MgSO4. Purification by column chroma-
tography (SiO2, CH2Cl2/EtOAc, 40/60, and 2% MeOH and then
4% MeOH) gave 146 mg of N-acetyl-13 in a 59% yield: mp
162-163 °C; 1H NMR (CDCl3) δ 2.22 (s, 3H), 1.38 (m, 2H),
3.41 (t, 2H, J ) 6), 3.28 (m, 2H), 5.88 (d, 1H, J ) 2), 7.48-
7.38 (m, 4H), 7.65-7.60 (m, 3H), 7.74-7.66 (m, 4H). The
previous compound (125 mg, 0.3 mmol), 3 mL of 2 N NaOH,
and 4 mL of EtOH were heated under reflux for 2.5 h. The
reaction mixture was cooled, water was added, and the solution
was extracted with CH2Cl2. The organic phase was dried over
MgSO4 and evaporated, and 13 was recrystallized from CH2-
4-(3-Am in op r op yl)-N-(3-h yd r oxyp r op yl)-N-(2-p h en yl-
2H-p yr a zol-3-yl)ben zen esu lfon a m id e, 15. A solution of
acetic anhydride (1.5 mL) in CH2Cl2 (3 mL) was added
dropwise at 0 °C to a solution of 3-phenylpropylamine (2 mL,
14 mmol) in CH2Cl2 (15 mL) in the presence of triethylamine
(2.2 mL). After 15 min, the reaction mixture was allowed to
warm to room temperature for the night. The solution was
washed with an aqueous HCl solution (pH 4) and extracted
with CH2Cl2. The organic phases were gathered, washed with
water until neutralization, and dried over MgSO4. The solvent
was evaporated, leading to 2.48 g of N-acetyl-3-phenylpropyl-
amine, as an oil, in a quantitative yield: 1H NMR (CDCl3) δ
7.30-7.14 (m, 5H), 5.46 (br s, 1H), 3.27 (q, 2H, J ) 7.2), 2.64
(t, 2H, J ) 7.2), 1.82 (m, 2H), 1.92 (s, 3H). N-Acetyl-3-
phenylpropylamine (2.17 g, 12 mmol) was then added dropwise
to 4 mL of chlorosulfonic acid contained in a 50 mL round-
bottomed flask fitted with a reflux condenser and a CaCl2 tube
and cooled in ice. The solution was stirred for 1 h at room
temperature and for 1.5 h at 70 °C. The mixture was then
poured onto ice, extracted with CH2Cl2, and dried over MgSO4.
Evaporation of the solvent led to 440 mg of crude 4-[3-
(acetylamino)propyl]benzenesulfonyl chloride (13% yield): 1H
NMR (CDCl3) δ 7.93 (d, 2H, J ) 8.1), 7.41 (d, 2H, J ) 8.1),
5.60 (br s, 1H), 3.31 (t, 2H, J ) 7.2), 2.76 (t, 2H, J ) 7.2), 1.87
(m, 2H), 1.97 (s, 3H). A 435 mg sample of this arylsulfonyl
chloride was then added to a solution of (2-phenyl-2H-pyrazol-
3-yl)amine (250 mg, 1.57 mmol) in 2.5 mL of anhydrous
pyridine. After 5 min at room temperature, the mixture was
heated at 95 °C for 1 h. Pyridine was evaporated, and the
residue was dissolved in 1 N NaOH, washed with CH2Cl2,
acidified with 1 N HCl, and extracted with CH2Cl2. The organic
phase was dried over MgSO4 and evaporated. After purification
by column chromatography (SiO2, EtOAc, and MeOH, 1%, and
then MeOH, 2%), 110 mg of 4-[3-(acetylamino)propyl]-N-(2-
phenyl-2H-pyrazol-3-yl)benzenesulfonamide was obtained in
a 17% yield : 1H NMR (CDCl3) δ 7.59 (d, 2H, J ) 8.4), 7.53 (s,
1H), 7.35 (m, 3H), 7.23 (m, 2H), 7.10 (m, 2H), 6.51 (br s, 1H),
6.23 (s, 1H), 5.45 (br s, 1H), 3.27 (q, 2H, J ) 7.2), 2.69 (t, 2H,
J ) 7.2), 1.95 (s, 3H), 1.82 (m, 2H). Benzoic acid 3-bromopropyl
ester (130 mg, 0.54 mmol) was then added dropwise to a
solution of this product (110 mg, 0.27 mmol) in 1 mL of
anhydrous DMF containing 34 mg of Na2CO3. After 20 h at
80 °C, the solvent was evaporated and the residue was
dissolved in CH2Cl2. The organic phase was washed with water
and dried over MgSO4. After purification by column chroma-
tography (SiO2, CH2Cl2, and MeOH, 2%), 93 mg of protected
15 was obtained in a 61% yield: 1H NMR (CDCl3) δ 7.92 (d,
2H, J ) 7.4),7.68-7.24 (m, 13H), 5.93 (s, 1H), 5.45 (br s, 1H,
NH), 3.97 (t, 2H, J ) 6.4), 3.45 (t, 2H, J ) 6.4), 3.29 (q, 2H,
J ) 7), 2.70 (t, 2H, J ) 7), 1.96 (s, 3H), 1.87 (m, 2H), 1.66 (m,
2H). The previous compound (93 mg, 0.16 mmol) was heated
under reflux in 2 mL of 5 N NaOH and 2 mL of EtOH for 24
h. After evaporation of EtOH, the solution diluted in water
was extracted with CH2Cl2. The organic phase was dried over
MgSO4. 15 was recrystallized from CH2Cl2, and obtained (34
mg) in a 49% yield: mp 133-134 °C; UV (MeOH) λ 233 (25);
1H NMR (CDCl3) δ 7.70-7.60 (m, 5H), 7.44-7.32 (m, 5H), 5.87
(d, 1H, J ) 1.9), 3.42 (t, 2H, J ) 6), 3.28 (t, 2H, J ) 6), 2.75
(m, 4H), 1.80 (m, 2H), 1.42 (m, 2H); MS (CI, NH3) m/z 415
([M + H]+, 86), 218 (100). Anal. (C21H26N4O3S·H2O) C, H, N.
1
Cl2 (79% yield): mp 170-171 °C; UV (MeOH) λ 271 (22); H
NMR (CDCl3) δ 1.36 (m, 2H), 1.66 (t, 1H, J ) 6), 3.26 (q, 2H,
J ) 6), 3.39 (t, 2H, J ) 6), 4.21 (br s, 2H, NH2), 5.91 (d, 1H,
J ) 2), 6.68 (d, 2H, J ) 8.7), 7.41 (m, 3H), 7.54 (d, 2H, J )
8.7), 7.60 (d, 1H, J ) 2), 7.67 (dd, 2H, J ) 8.2 and 1.5); MS
(CI, NH3) m/z 373 ([M + H]+, 100), 218 (18) 160 (17). Anal.
(C18H20N4O3S) C, H, N: calcd 58.05, found 57.60.
4-(2-Am in oeth yl)-N-(3-h ydr oxypr opyl)-N-(2-ph en yl-2H-
p yr a zol-3-yl)ben zen esu lfon a m id e, 14. In a 50 mL round-
bottomed flask, fitted with a reflux condenser and a CaCl2
tube, containing 3 mL of chlorosulfonic acid cooled in an ice
bath, was added dropwise N-acetyl-2-phenylethylamine (1.5
g, 9.2 mmol). The solution was stirred for 1 h at room
temperature, 1.5 h at 50 °C, and then 1.5 h at 70 °C, and the
reaction was followed by TLC (SiO2, CH2Cl2/EtOAc, 2/1). The
reaction mixture was poured onto ice, extracted with CH2Cl2,
and dried over MgSO4. The residue was dissolved in CH2Cl2.
An insoluble fraction was filtered. When cyclohexane was
added to the solution, a white precipitate was formed, leading
to 1.78 g of 4-[2-(acetylamino)ethyl]benzenesulfonyl chloride
in a 74% yield: 1H NMR (CDCl3) δ 7.97 (d, 2H, J ) 8.4), 7.43
(d, 2H, J ) 8.4), 5.48 (br s, 1H), 3.53 (q, 2H, J ) 7), 2.95 (t,
2H, J ) 7), 1.95 (s, 3H). This product (800 mg, 3 mmol) was
added to a solution of 636 mg of (2-phenyl)-2H-pyrazol-3-
ylamine (4 mmol) in 6 mL of anhydrous pyridine. After 5 min
at room temperature, the reaction mixture was heated at 95
°C for 2 h. Pyridine was evaporated, and the residue was
dissolved in CH2Cl2, with a little volume of MeOH, and washed
with 1 N HCl. The aqueous phase was extracted with CH2Cl2
and the organic phase dried over MgSO4 and evaporated.
Purification by column chromatography (SiO2, CH2Cl2/MeOH,
5%) and recrystallization from MeOH/acetone led to 0.3 g of
4-(2-(acetylamino)ethyl]-N-(2-phenyl-2H-pyrazol-3-yl)benzene-
1
sulfonamide in a 26% yield: mp 174-175 °C; H NMR (CD3-
SOCD3) δ 10.32 (s, 1H), 7.90 (m, 1H),7.54 (d, 1H, J ) 1.7),
7.57 (d, 2H, J ) 8.2) 7.44 (m, 5H), 7.36 (d, 2H, J ) 8.2), 5.80
(d, 1H, J ) 1.7), 3.28 (m, 2H), 2.77 (t, 2H, J ) 7), 1.78 (s, 3H).
Benzoic acid 3-bromopropyl ester (334 mg, 1.37 mmol) was
added to a solution of 4-[2-(acetylamino)ethyl]-N-(2-phenyl-
2H-pyrazol-3-yl)benzenesulfonamide (200 mg, 0.52 mmol) in
2 mL of anhydrous DMF containing 66 mg of Na2CO3. After
24 h at 80 °C, the solvent was evaporated and the residue was
dissolved in CH2Cl2. The organic phase was washed with water
and dried over MgSO4. After purification by column chroma-
tography (SiO2, CH2Cl2, and then MeOH, 2%), 244 mg of
protected 14 was obtained in an 86% yield: 1H NMR (CDCl3)
δ 7.92 (d, 2H, J ) 7.5), 7.68 (d, 2H, J ) 8.4), 7.65-7.61 (m,
3H), 7.56 (t, 1H, J ) 7.5), 7.48-7.39 (m, 5H), 7.28 (d, 2H, J )
8.4), 5.99 (d, 1H, J ) 1.9), 5.64 (m, 1H), 3.93 (t, 2H, J ) 6.1),
3.55-3.44 (m, 4H), 2.88 (t, 2H, J ) 6.9), 1.94 (s, 3H), 1.68 (m,
2H). This compound (244 mg, 0.45 mmol) was heated under
reflux in 5 mL of NaOH (5 N) and 5 mL of EtOH for 24 h.
After evaporation of EtOH, the solution was diluted with water
and extracted with CH2Cl2. The organic phase was dried over
4-Allyl-N-(3-h yd r oxyp r op yl)-N-(2-p h en yl-2H-p yr a zol-
3-yl)ben zen esu lfon a m id e, 16. Benzoic acid 3-bromopropyl
ester (515 mg, 2.12 mmol) was added to a solution of 4-bromo-
N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide, 2 (400 mg,
1.06 mmol), in 4 mL of anhydrous DMF containing 500 mg of
Na2CO3. After 16 h at 80 °C, DMF was evaporated and the
residue was dissolved in CH2Cl2. The organic phase was

Inhibition of P450 2Cs by Sulfaphenazole Derivatives
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3629
washed with water and dried over MgSO4. After purification
by column chromatography (SiO2, CH2Cl2, and then Et2O, 3%),
440 mg of the expected benzoate was obtained in a 77% yield:
mp 123-124 °C; 1H NMR (CDCl3) δ 1.73 (m, 2H), 3.48 (t, 2H,
J ) 7.2), 4.02 (t, 2H, J ) 6), 5.96 (d, 1H, J ) 2), 7.46-7.41 (m,
5H), 7.62-7.57 (m, 7H), 7.63 (d, 1H, J ) 2), 7.93 (d, 2H, J )
8). The previous compound (400 mg, 0.74 mmol) was boiled in
10 mL of EtOH and 5 mL of 2 N NaOH for 2 h. After
evaporation of EtOH, dilution in water, and extraction with
CH2Cl2, 250 mg of product was obtained (78% yield): 1H NMR
(CDCl3) δ 1.44 (m, 2H), 3.32 (q, 2H, J ) 6), 3.45 (t, 2H, J ) 6),
5.89 (d, 1H, J ) 2), 7.68-7.59 (m, 7H), 7.44 (m, 3H). A solution
containing 240 mg (0.55 mmol) of this compound, 43 mg of
Pd[PPh3]4, and 6 mL of anhydrous DMF was deoxygenated
under argon for 0.5 h. A 0.22 mL sample of allyltributyltin
was then added dropwise with a syringe. The reaction mixture
was stirred under argon at room temperature for 0.5 h and
then at 110 °C for 16 h. The solution was cooled, and the
solvent was evaporated under vacuum. After purification by
column chromatography (SiO2, CH2Cl2, and then Et2O, 10%),
and recrystallization from Et2O, 190 mg of compound 16 was
obtained (86% yield): mp 114-115 °C; UV (MeOH) λ 234 (23);
1H NMR (CDCl3) δ 7.70 (d, 2H, J ) 8.3), 7.65-7.60 (m, 2H),
7.60 (d, 1H, J ) 2), 7.48-7.38 (m, 3H), 7.35 (d, 2H, J ) 8.3),
5.96 (m, 1H), 5.87 (d, 1H, J ) 2), 5.13 (m, 2H), 3.48 (d, 2H,
J ) 6.7), 3.42 (m, 2H), 3.29 (q, 2H, J ) 6), 1.39 (m, 2H); MS
(CI, NH3) m/z 398 ([M + H]+, 100), 218 (76). Anal. (C21H23N3O3S)
C, H, N.
an 88% yield: 1H NMR (CDCl3) δ 1.74 (m, 2H), 3.53 (t, 2H,
J ) 6.6), 4.02 (t, 2H, J ) 6.6), 6.02 (d, 1H, J ) 1.8), 7.37-7.48
(m, 5H), 7.54 (t, 1H, J ) 7.3), 7.62-7.72 (m, 6H), 7.86 (d, 2H,
J ) 8.5), 7.93, (m, 3H), 8.29 (d, 1H, J ) 8.2), 8.44 (s, 1H). This
compound (215 mg, 0.37 mmol) was dissolved in EtOH (10 mL)
and added to a solution of 148 mg of NaOH (10 equiv) in 5 mL
of water. The mixture was heated under reflux. At the end of
the reaction, EtOH was evaporated, the mixture was diluted
with H2O, extracted with CH2Cl2, and dried over MgSO4, and
the solvents were evaporated. Compound 19 was obtained as
a resin in an 85% yield: UV (MeOH) λ 259 (49); 1H NMR
(CDCl3) δ 1.46 (m, 2H), 3.34 (m, 2H), 3.51 (t, 2H, J ) 6.6),
5.94 (d, 1H, J ) 1.8), 7.35-7.50 (m, 3H), 7.63-7.67 (m, 3H),
7.69 (t, 1H, J ) 8), 7.77 (d, 2H, J ) 8.4), 7.89 (d, 2H, J ) 8.4),
7.95 (d, 1H, J ) 8), 8.29 (d, 1H, J ) 8), 8.49 (s, 1H); MS (CI,
NH3) m/z 479 ([M + H]+, 100), 449 (22), 218 (60). Anal.
(C24H22N4O5S) C, H, N.
3′-Nitr obip h en yl-4-su lfon ic Acid (4-Hyd r oxybu tyl)(2-
p h en yl-2H-p yr a zol-3-yl)a m id e, 20. Compound 3 (120 mg,
0.29 mmol) was added to 1 mL of DMF containing 90 mg of
Na2CO3, 0.1 mL of 4-chlorobutanol (1 mmol), and 130 mg of
NaI (3 equiv). The solution was stirred at 80 °C. After 1 day,
the solvent was evaporated and the residue was diluted in
water and extracted with CH2Cl2. The organic phase was dried
over MgSO4. After purification by column chromatography
(SiO2, CH2Cl2/EtOAc, 20%) and then on preparative TLC
plates (SiO2), 34 mg of 20 was obtained as a white-green
powder in a 24% yield: mp 145-146 °C; UV (MeOH) λ 259
(38); 1H NMR (CDCl3) δ 8.49 (s, 1H), 8.29 (d, 1H, J ) 8), 7.95
(d, 1H, J ) 8), 7.86 (d, 2H, J ) 8.5), 7.75 (d, 2H, J ) 8.5),
7.71-7.64 (m, 3H), 7.63 (d, 1H, J ) 1.9), 7.48-7.38 (m, 3H),
5.94 (d, 1H, J ) 1.9), 3.41 (m, 4H), 1.33 (m, 4H), 1.06 (br s,
1H); MS (CI, NH3) m/z 493 ([M + H]+, 73), 463 (41), 232 (100).
Anal. (C25H24N4O5S) C, H, N.
4-Allyl-N-m eth yl-N-(2-ph en yl-2H-pyr azol-3-yl)ben zen e-
su lfon a m id e, 21. Methyl iodide (0.01 mL, 0.16 mmol) was
added to a solution of 4-allyl-N-(2-phenyl-2H-pyrazol-3-yl)-
benzenesulfonamide, 4 (34 mg, 0.1 mmol), in 0.3 mL of
anhydrous DMF containing Na2CO3 (12 mg, 0.11 mmol). After
45 min at 80 °C, DMF was evaporated and the residue was
diluted with water and extracted with CH2Cl2. The organic
phase was dried over MgSO4. After purification by column
chromatography (SiO2, CH2Cl2, and then Et2O, 2%), 35 mg of
21 was obtained in a quantitative yield as an oil, which
crystallized after a few days: mp 85-86 °C; 1H NMR (CDCl3)
δ 7.61 (d, 2H, J ) 8.3), 7.59-7.53 (m, 2H), 7.57 (d, 1H, J ) 2),
7.48-7.34 (m, 3H), 7.31 (d, 2H, J ) 8.3), 5.94 (m, 1H), 5.88 (d,
1H, J ) 2), 5.14 (m, 1H), 5.11 (m, 1H), 3.46 (d, 2H, J ) 6.7),
3.04 (s, 3H); HRMS (CI, CH4) (M + H)+ calcd for C19H20O2N3S
354.1276, found 354.1274.
4-Allyl-N-ben zyl-N-(2-p h en yl-2H-p yr a zol-3-yl)ben zen e-
su lfon a m id e, 22. Benzyl bromide (0.013 mL, 0.11 mmol) was
added to a solution of 4-allyl-N-(2-phenyl-2H-pyrazol-3-yl)-
benzenesulfonamide, 4 (24.6 mg, 0.073 mmol), in 0.3 mL of
anhydrous DMF containing Na2CO3 (10 mg, 0.1 mmol). After
2 h at 80 °C, DMF was evaporated and the residue was
dissolved in water and extracted with CH2Cl2. The organic
phase was dried over MgSO4. After purification by column
chromatography (SiO2, CH2Cl2/Et2O, 1%), 26 mg of 22 was
obtained as an oil, which crystallized after a few days in an
83% yield: mp 94-95 °C; UV (MeOH) λ 235; 1H NMR (CDCl3)
δ 7.70 (d, 2H, J ) 8.3), 7.53 (d, 1H, J ) 1.9), 7.35 (d, 2H, J )
8.3), 7.30-7.10 (m, 6H), 7.04 (t, 2H, J ) 7.3), 6,82 (d, 2H, J )
7.3), 5.97 (m, 1H), 5.86 (d, 1H, J ) 1.9), 5.18-5.09 (m, 2H),
4.39 (s, 2H), 3.50 (d, 2H, J ) 6.6); HRMS (CI, CH4) (M + H)+
calcd for C25H24O2N3S 430.1589, found 430.1592.
3′-Am in obip h en yl-4-su lfon ic Acid (3-Hyd r oxyp r op yl)-
(2-p h en yl-2H-p yr a zol-3-yl)a m id e, 17. Compound 19 (195
mg, 0.41 mmol) was dissolved in a minimum volume of
acetone, and then a half-volume of water was added. The
reaction mixture was deoxygenated under argon. A 1.42 g
sample of Na2S2O4 was added, and the solution was heated at
60 °C under argon for 2 h. The reaction was followed by TLC
(SiO2, CH2Cl2/EtOAc, 90/10). The mixture was diluted with
water, extracted with EtOAc, and dried over MgSO4, and the
solvents were evaporated. Compound 17 was purified on
preparative TLC plates (SiO2, 1 mm, CH2Cl2/MeOH, 90/10),
and obtained as a resin in a 38% yield : UV (MeOH) λ 250
1
(30); H NMR (CDCl3) δ 1.43 (m, 2H), 3.32 (m, 2H), 3.50 (m,
2H), 3.80 (s, 2H, NH2), 5.93 (s, 1H), 6.75 (d, 1H, J ) 7.7), 6.91
(s, 1H), 7.00 (d, 1H, J ) 7.7), 7.26 (t, 1H, J ) 7.7), 7.30-7.50
(m, 3H), 7.62-7.66 (m, 3H), 7.69 (d, 2H, J ) 8.5), 7.80 (d, 2H,
J ) 8.5); MS (CI, NH3) m/z 449 ([M + H]+, 100), 218 (48). Anal.
(C24H24N4O3S) C, H, N.
3′-Nitr obip h en yl-4-su lfon ic Acid (2-Hyd r oxyeth yl)(2-
p h en yl-2H-p yr a zol-3-yl)a m id e, 18. Compound 3 (120 mg,
0.29 mmol) was added to 1 mL of DMF containing 90 mg of
Na2CO3 and 0.06 mL of 2-chloroethanol (0.89 mmol). The
solution was stirred at 80 °C. After 1 day, the reaction was
not finished. A 3 equiv sample of NaI was then added, and
the reaction was heated for 24 h at 80 °C. The solvent was
evaporated, and the residue was diluted with water and
extracted with CH2Cl2. The organic phase was dried over Na2-
SO4. After purification by column chromatography (SiO2, CH2-
Cl2/EtOAc, 10%), 60 mg of an oil was obtained, which crys-
tallized slowly, leading to 18 in a 45% yield: mp 168-169 °C;
1
UV (MeOH) λ 259 (42); H NMR (CDCl3) δ 8.49 (s, 1H), 8.29
(d, 1H, J ) 8), 7.94 (d, 1H, J ) 8), 7.89 (d, 2H, J ) 8.5), 7.76
(d, 2H, J ) 8.5), 7.73-7.63 (m, 4H), 7.42-7.54 (m, 3H), 5.96
(d, 1H, J ) 1.9), 3.49 (m, 4H), 1.33 (t, 1H, J ) 5.8); MS (CI,
NH3) m/z 465 ([M + H]+, 67), 204 (100). Anal. (C23H20N4O5S)
C, H, N.
3′-Nit r obip h en yl-4-su lfon ic Acid (3-Hyd r oxyp r op yl)-
(2-p h en yl-2H-p yr a zol-3-yl)a m id e, 19. To a solution of com-
pound 3 (180 mg, 0.43 mmol) in DMF (2 mL) was added
benzoic acid 3-bromopropyl ester (208 mg, 0.86 mmol), in the
presence of Na2CO3 (55 mg, 0.52 mmol). The solution was
stirred at 80 °C for 1 day. After evaporation of DMF, the
residue was dissolved in water and CH2Cl2, then extracted
with CH2Cl2, and dried over MgSO4, and the solvents were
evaporated. The benzoate of 19 was purified by column
chromatography (SiO2, CH2Cl2/EtOAc, 90/10), and obtained in
4-Met h yl-N-m et h yl-N-(2-p h en yl-2H -p yr a zol-3-yl)b en -
zen esu lfon a m id e, 23. Methyl iodide (0.022 mL, 0.35 mmol)
was added to a solution of 4-methyl-N-(2-phenyl-2H-pyrazol-
3-yl)benzenesulfonamide (75 mg, 0.24 mmol) in 0.5 mL of
anhydrous DMF containing 40 mg of Na2CO3. After 3 h at 70
°C, DMF was evaporated and the residue was dissolved in CH2-
Cl2. The organic phase was washed with water and dried over
MgSO4. After purification by column chromatography (SiO2,

3630 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
CH2Cl2/Et2O, 3%), 76 mg of 23 was obtained in a 97% yield:
Ha-Duong et al.
was plotted versus the inhibitor concentration. IC50 values
were estimated by curve fit analyses.
1
mp 121-122 °C; UV (MeOH) λ 232 (22); H NMR (CDCl3) δ
2.44 (s, 3H), 3.03 (s, 3H), 5.88 (d, 1H, J ) 2), 7.29 (d, 2H, J )
8.1), 7.39 (t, 1H, J ) 7.2), 7.44 (t, 2H, J ) 7.2), 7.60-7.56 (m,
5H); MS (CI, NH3) m/z 328 ([M + H]+, 100), 174 (36). Anal.
(C17H17N3O2S) C, H, N.
Stu d y of Su bstr a te Bin d in g to Yea st-Exp r essed CYP
2C9 by Differ en ce Visible Sp ectr oscop y. Difference visible
spectra produced by sulfaphenazole derivatives were recorded
at room temperature with a Kontron 820 spectrophotometer.
Yeast microsomes were suspended in 50 mM Tris, 1 mM
EDTA, pH 7.4, to obtain a P450 concentration of 0.13 µM. The
solution was equally divided between two 500 µL quartz
cuvettes (1 cm path length), and a baseline was recorded.
Aliquots (1-5 µL) of solutions containing the studied com-
pound were added to the sample cuvette, the same volume of
solvent being added to the reference cuvette. The difference
spectra were recorded between 380 and 520 nm.37
4-Met h yl-N-p r op yl-N-(2-p h en yl-2H -p yr a zol-3-yl)b en -
zen esu lfon a m id e, 24. 1-Iodopropane (0.03 mL, 0.3 mmol)
was added to a solution of 4-methyl-N-(2-phenyl-2H-pyrazol-
3-yl)benzenesulfonamide (45 mg, 0.14 mmol) in 0.5 mL of
anhydrous DMF containing 30 mg of Na2CO3. After 18 h at
60 °C, DMF was evaporated and the residue was dissolved in
CH2Cl2. The organic phase was washed with water and dried
over MgSO4. After purification by column chromatography
(SiO2, CH2Cl2), 45 mg of 24 was obtained (88% yield). Recrys-
tallization from ether/cyclohexane led to 32 mg of pure 24: mp
127-128 °C; UV (MeOH) λ 233 (20); 1H NMR (CDCl3) δ 7.66-
7.58 (m, 5H), 7.42 (m, 3H), 7.30 (d, 2H, J ) 8.1), 5.86 (d, 1H,
J ) 1.9), 3.22 (t, 2H, J ) 7.5), 2.44 (s, 3H), 1.24 (m, 2H), 0.64
(t, 3H, J ) 7.5); MS (CI, NH3) m/z 356 ([M + H]+, 100), 202
(41). Anal. (C19H21N3O2S) C, H, N.
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2C9, 2C18, and 2C19, respectively.
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Drug Metab. Dispos. 1996, 24, 1081-1087.
The microsomal P450 content was determined according to
the method of Omura and Sato.35 The protein content in
microsomal suspensions was determined by the Lowry proce-
dure36 using bovine serum albumin as the standard.
En zym e Activity Assa y. 5-Hyd r oxyla tion of 2-Ar oyl-
th iop h en es. Quantitation of 5-hydroxy-2-aroylthiophenes was
based on a spectrophotometric method22 adapted to yeast
microsomes expressing CYP 2C9 in the case of tienilic acid.5
Incubations for metabolic activity with yeast microsomes were
carried out at 28 °C, using glass tubes in a shaking bath. The
incubation mixtures contained the yeast microsomal suspen-
sion, providing 0.2, 0.2, 0.1, and 0.075 µM P450 for CYP 2C8,
2C9, 2C18, and 2C19, respectively, the substrate, and an
NADPH-generating system (1 mM NADP+, 10 mM glucose
6-phosphate, and 2 units of glucose 6-phosphate deshydroge-
nase/mL) diluted in 0.1 M Tris buffer, pH 7.4, containing 1
mM EDTA and 8% glycerol (final concentrations). Activity
assays were routinely initiated (t0 ) 0 min) by incorporation
of the NADPH-generating system into the incubation mixture
after 3 min of separate preincubation at 28 °C for temperature
equilibration. At t0 and regularly thereafter, aliquots (140 µL)
were taken and the reaction was quickly stopped by treatment
with 70 µL of a cold CH3CN/CH3COOH (10/1) mixture.
As previously reported,18 the 2-aroylthiophene substrates
used for measuring CYP 2C8 and CYP 2C9 dependent activ-
ities were 3-[2,3-dichloro-4-(2-thenoyl)phenoxy]ethanol and
tienilic acid, respectively. In the case of CYP 2C18 and CYP
2C19, 3-[2,3-dichloro-4-(2-thenoyl)phenoxy]propan-1-ol was
used as substrate.18 IC50 values of sulfaphenazole derivatives
were determined after addition of variable concentrations of
these derivatives to incubates containing 250, 6, 15, and 100
µM substrate of CYP 2C8, 2C9, 2C18, and 2C19, respectively.
These concentrations correspond to the KM values of the
hydroxylation of these substrates.18 The studied compound
dissolved in CH3OH or DMSO was added to the incubation
mixture (final solvent concentration 1%). The residual activity
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