Aryl morpholino triazenes inhibit cytochrome P450 1A1 and 1B1

Article abstract of DOI:10.1016/j.bmcl.2016.05.064

Many cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1) inhibitors, such as resveratrol, have planar, hydrophobic, aromatic rings in their structure and exhibit anti-cancer activity. Aryl morpholino triazenes have similar structural features and in addition

Full text of DOI:10.1016/j.bmcl.2016.05.064

Accepted Manuscript
Aryl Morpholino Triazenes Inhibit Cytochrome P450 1A1 and 1B1
Daniel Lee, Pedro Perez, William Jackson, Taylor Chin, Michael Galbreath,
Frank R. Fronczek, Ralph Isovitsch, Devin S. Iimoto
PII:
S0960-894X(16)30563-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.064
BMCL 23923
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 March 2016
20 May 2016
21 May 2016
Please cite this article as: Lee, D., Perez, P., Jackson, W., Chin, T., Galbreath, M., Fronczek, F.R., Isovitsch, R.,
Iimoto, D.S., Aryl Morpholino Triazenes Inhibit Cytochrome P450 1A1 and 1B1, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.064
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Aryl Morpholino Triazenes Inhibit Cytochrome P450 1A1 and 1B1
Daniel Leea, Pedro Pereza, William Jacksona, Taylor China╪, Michael Galbreatha, Frank R.
Fronczekb, Ralph Isovitscha, and Devin S. Iimotoa*
aDepartment of Chemistry, Whittier College, Whittier, CA, United States 90608
bDepartment of Chemistry, Louisiana State University, Baton Rouge, LA, United States 70803
╪in memorium
Key Words: triazenes; cytochrome P450 1A1; cytochrome P450 1B1; cancer; inhibition;
synthesis
*corresponding author
Devin S. Iimoto
Department of Chemistry
Whittier College
Whittier, CA 90608
USA
e-mail: diimoto@whittier.edu
phone: (562) 907-4200 x4421

ABSTRACT
Many cytochrome P4501A1 and 1B1 (CYP1A1 and CYP1B1) inhibitors, such as
resveratrol, have planar, hydrophobic, aromatic rings in their structure and exhibit anti-cancer
activity. Aryl morpholino triazenes have similar structural features and in addition contain a
triazene unit consisting of three consecutive, conjugated nitrogen atoms. Several aryl
morpholino triazenes, including 4-[(E)-2-(3,4,5-trimethoxyphenyl)diazenyl]-morpholine (2),
were prepared from a reaction involving morpholine and a diazonium ion produced from
different aniline derivatives, such as 3,4,5-trimethoxyaniline. The aryl morpholino triazenes
were then screened at 100 M for their ability to inhibit CYP1A1 and CYP1B1 using
ethoxyresorufin as the substrate. Triazenes that inhibited the enzymes to less than 80% of the
uninhibited enzyme activity were assayed to determine their IC50 value. Compound 2 was the
only triazene to inhibit both CYP1A1 and CYP1B1 to the same degree as resveratrol with IC50
values of 10 M and 18 respectively. Compounds 3 and 6 selectively inhibited CYP1B1
over CYP1A1 with IC values of 2 M and 7 M respectively. Thus, aryl morpholino triazenes
are a new class of compounds that can inhibit CYP1A1 and CYP1B1 and potentially prevent
cancer.

CYP1A1 and CYP1B1 are two extrahepatic enzymes that oxidize a wide range of
endogenous and foreign compounds, some of which become carcinogenic within the human
body. 1-3 For example, polyaromatic hydrocarbons (PAHs), found in tobacco smoke,
automobile exhaust, and charbroiled meat, can be oxidized by CYP1A1 and CYP1B1 into
carcinogenic epoxydiols through several reactions. 4-6 First, CYP1A1 and CYP1B1 form an
epoxide ring on the PAH, and then an epoxide hydrolase converts the epoxide to a dihydrodiol.
Finally, CYP1A1 and CYP1B1 catalyze the reaction of the dihydrodiol into different
expoxydiols. 7-9 These epoxydiols are good electrophiles that react with the primary amino
groups in adenosine and guanosine to form 2’-deoxyguanosine and 2’-deoxyadenosine in DNA.
10-12 These DNA adducts cause mis-replication, which lead to mutations in DNA that can cause
cancer. 10 Others have found that continuous exposure to PAHs cause sustained expression and
activity of CYP1A1 and CYP1B1 which produce other, as of yet unidentified, DNA adducts that
could also lead to cancer by a different mechanism. 13
Because CYP1A1 and CYP1B1 oxidation of PAHs has been linked to tumor formation,
various compounds have been studied for their ability to inhibit these enzymes and thus be tested
for their cancer preventative properties. 14,15 Many of these CYP1A1 and CYP1B1 inhibitors
share several structural motifs: they are planar and contain one or more hydrophobic aromatic
rings. 16,17 For example, resveratrol, a stilbene molecule that is planar with two aromatic rings,
inhibits CYP1A1 and CYP1B1 and it is hypothesized that this inhibition is linked to its cancer
preventative properties. 18-20 Other stilbene derivatives have been shown to prevent the growth
of various types of cancer cells such as breast, 21 colon, 22 and skin by potentially inhibiting
CYP1A1 and CYP1B1. 23-27 Other CYP1A1 and CYP1B1 inhibitors include napththoquinone

and anthraquinone derivatives, flavonoids, coumarin derivatives, alkaloids, and other
compounds. 28
Kinetic studies have shown that stilbene derivatives act as competitive inhibitors and/or
noncompetitive inhibitors indicating that this type of inhibitor binds to the active site and/or
another binding site. 19,25 Because the CYP1A1 and CYP1B1 inhibitors mentioned above are
planar, hydrophobic compounds, they likely bind well to the narrow, hydrophobic active sites of
CYP1A1 and CYP1B1 or another hydrophobic binding site. 1,2 Significantly, x-ray diffraction
analysis of CYP1A1 and CYP1B1 crystals with -napthoflavone, an inhibitor of both enzymes,
bound to the active site, indicate that one key interaction between -napthoflavone and the
enzyme active site is a - interaction between the aromatic ring in the inhibitor with Phe224 in
CYP1A1 and Phe231 in CYP1B1. 1,2
Aryl morpholino triazenes contain an aromatic ring plus three consecutively bonded,
conjugated nitrogens. 29-31 Because these triazenes are relatively planar and because the triazene
unit could form - interactions with phenylalanine residues or other groups with a system, it
is possible that these linear triazenes could bind to the active site or another hydrophobic binding
site in CYP1A1 and CYP1B1, and act to inhibit these enzymes. Several aryl morpholino
triazenes were synthesized and some of these linear triazenes, especially one with methoxy
substituents on the benzene ring, inhibited CYP1A1 and CYP1B1 with IC50 values at the
micromolar level similar to resveratrol. Thus, aryl morpholino triazenes are a new class of
CYP1A1 and CYP1B1 inhibitors that could have cancer preventative properties.
The aryl morpholino triazenes, compounds 1-6, were prepared according to the literature
procedures (Figure 1). 31-35 More specifically, compound 2, 4-[(E)-2-(3,4,5-
trimethoxyphenyl)diazenyl]-morpholine (Figure 1), was synthesized at 0° C by combining a

mixture of 3,4,5-trimethoxyaniline and 6 M HCl with an aqueous solution of NaNO2 to produce
a diazonium ion that underwent coupling with morpholine to give 2 in 78% yield. The synthesis
and yield of compound 2 are representative of the technique that was used to prepare the linear
triazenes in this work. 31-34,36
NH2
O
1
R1
R2
N
N
1. 6 M HCl, NaNO2 (aq)
N
2. morpholine
3. sat. aq. NaHCO3
R1
R3
R2
R3
1: R1,R2,R3=H
2: R1,R2,R3=OCH3
3: R1,R3=H, R2=NO2
4: R1,R3=H, R2=C(O)CH3
5: R1,R3=H, R2=COOH
6: R1,R3=H, R2=Br
Figure 1. Preparation and chemical structures of compounds 1-6.
Compound 2 exhibited the predicted spectroscopic characteristics. For example, the 13C-
NMR spectrum exhibited four resonances in the region 98-154 ppm that were assigned to the
aromatic carbons. The assignment of overlapping resonances for the methyl and morpholine
protons in the region 3.7-3.9 ppm of the 1H-NMR spectrum was aided by an HMQC-NMR
experiment. The identity of 2 was supported by an elemental analysis that gave experimental
values for the percent carbon, hydrogen, and nitrogen that were within ± 0.20 of the theoretical
values, and a ESI-HR-MS that had an [M+Na]+ peak at m/z = 304.1274, which was
approximately 2 ppm from the calculated value.
Compound 2 was recrystallized from cyclohexane to yield brown fragments that were
suitable for X-ray analysis. The ORTEP representation of 2 is shown in Figure 2, while its
37
crystallographic parameters (along with its CCDC deposition number) are in reference 37.

Figure 2. ORTEP view of compound 2. Thermal ellipsoids are represented at the 50%
probability level.
The N-N double bond, N(2)-N(3), adopted an (E)-configuration, and had bond angles that
deviated from ideal trigonal planar geometry by approximately 6° as evidenced by a N(3)-N(2)-
N(1) bond angle of 114.20 (8)° and a N(2)-N(3)-C(5) bond angle of 111.86 (8)°. The geometry
and bond angles of the N-N double bond in 2 compared favorably to related linear triazenes. 30,39
A degree of -delocalization across the linear triazene moiety of 2 was observed. For
example, the N-N double bond, N(2)-N(3), at 1.2679 (13) Å, and the N-N single bond, N(1)-
N(2), at 1.3501 (12) Å, where respectively longer and shorter than the literature bond length
values for a N-N double bond (1.222 Å) and a N(sp2)-N(sp3) single bond (1.420 Å). 40 The bond
angles of the morpholine nitrogen atom N(1) (120.45 (8), 112.20 (8), 115.78 (8) °), indicated its
partial sp2 character and its participation in -delocalization with N(2)-N(3). This type of -
delocalization has been observed in other linear triazenes. 41-43

The morpholine ring adopted a chair-like conformation with the N(2) and N(3) group in a
pseudo-equatorial position on N(1). Overall, the bond lengths and angles of compound 2
compared well with similar compounds found in the literature. 30-32,44
Various triazenes, their synthetic precursors, and resveratrol were screened for their
ability to inhibit CYP1A1 and CYP1B1 at a concentration of 100 M (Table 1). 45 Resveratrol
was tested because it has been touted as a natural anticancer agent through its ability to inhibit
CYP1A1 and CYP1B1, and can serve as a control for how effective different triazenes might be
in inhibiting these enzymes and thus at possibly preventing cancer. 19,20 The initial screening was
done at 100 M because resveratrol inhibits CYP1A1 and CYP1B1 with an IC50 in the
micromolar range and this will help determine which triazenes would have about the same or
even a greater ability to inhibit these enzymes compared to resveratrol. 19,20 The synthetic
precursors were tested to determine if the triazene unit plays a role in the inhibitory properties of
these compounds. Finally, the assay was done at pH = 7.4, so isomerization of the triazenes
from (E) to (Z) would not be expected. 47,48
At 100 M, compound 2 inhibited both CYP1A1 and CYP1B1 at a level close to or
greater than resveratrol with only 8% of the uninhibited activity present for cytochrome P450
1A1 and 14% present for cytochrome P450 1B1. In comparison, resveratrol inhibited the
enzymes such that 11% of the uninhibited cytochrome P450 1A1 activity was present while 56%
of the cytochrome P450 1B1 activity was present. Other triazenes showed more modest
inhibition of the enzymes (Table 1) with compounds 1 and 6 inhibiting cytochrome P450 1A1
more than compounds 3, 4, and 5. Compound 3 inhibited cytochrome P450 1B1 more than 1, 5,
and 6. Compound 4 did not inhibit cytochrome P450 1B1.

Table 1. Screening values and IC50 values for compounds 1-6, resveratrol, and the precursors
to compounds 1-6 with CYP1A1 and CYP1B1.
1A1 Screen
1A1
IC50
M
1B1 Screen
1B1
IC50
M
Percent of
Uninhibited
Enzyme
Activity
11%
Percent of
Uninhibited
Enzyme
Activity
56%
74%
14%
16%
100%
81%
59%
Resveratrol
34
120
10
115
82
55
163
18
Compound 1 (Triazene) 56%
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
8%
79%
70%
67%
53%
2
ND
400
7
167
63
3.4.5-trimethoxyaniline 95%
NDa
ND
ND
ND
ND
ND
100%
94%
75%
92%
100%
100%
ND
ND
>120
ND
ND
ND
4-bromoaniline
4-carboxyaniline
4-nitroaniline
Aniline
100%
100%
100%
100%
91%
Morpholine
aND means not determined. Any compound that did not significantly inhibit one of
the CYP enzymes, defined as a screening value of 80% or higher, was not tested for
its IC50 value.
After screening these compounds for their ability to inhibit CYP1A1 and CYP1B1 at 100
M, the compounds which were able to inhibit the enzymes to less than 80% of the uninhibited
enzyme activity were tested to determine their IC50 value. Compound 2, as expected from the
screening data, was the only compound that inhibited both CYP1A1 and CYP1B1 as well as or
more so than resveratrol. Its IC50 was 10 M with CYP1A1 and 18 M with CYP1B1
compared to resveratrol’s IC50 values which were determined to be 34 M with CYP1A1 and 55
M with CYP1B1. The literature IC50 values for resveratrol with CYP1A1 and CYP1B1 varied
from <2 M to 23 M with CYP1A1 and 1.4 M to >20 M with CYP1B1, indicating that there

is some variability in determining their IC50 values. 19,20,49 Other triazenes, as predicted by their
more limited ability to inhibit CYP1A1 at 100 M, had higher IC50 values ranging from 63 M
for compound 6 to 115 M for compound 3 (Table 1). In addition, most of the other triazenes
had higher IC50 values with CYP1B1 than did compound 2 with the exception of 3 and 6
(discussed later).
The aryl morpholino triazenes, particularly compound 2, are able to inhibit CYP1A1 and
CYP1B1 because it is likely that the planar aryl ring allows these triazenes to interact with the
large hydrophobic surface of a narrow active site similar to the binding of planar compounds like
resveratrol. In addition, the triazene unit of three nitrogens could form  -  interactions with
one of the phenylalanine residues in the active site, similar to the - interaction between -
napthoflavone and the respective phenylalanines in these enzymes. 1,2 This is supported by the
ability of compound 1 (unsubstituted triazene) to modestly inhibit CYP1A1 (Table 1).
To provide further evidence that the triazene unit is important for inhibiting CYP1A1 and
CYP1B1, the compounds that serve as the synthetic precursors for compounds 1-6, aniline,
3,4,5-trimethoxyaniline, 4- bromoaniline, 4-nitroaniline, 4-carboxyaniline, and morpholine, were
tested for their ability to inhibit these enzymes. The triazene unit is absent on all these
precursors, and notably, none of the precursors inhibited CYP1A1 to a significant extent (Table
1). In addition, all of these compounds did not inhibit CYP1B1 to any great extent, except for 4-
carboxyaniline, which inhibited the enzyme only modestly (Table 1).
In addition, the substituents on the aryl ring can modulate the binding of the triazene unit
to the enzyme because they can act as either electron donating groups or electron withdrawing
groups. The triazene compounds tested in these experiments have the substituted groups in the

para position on the aryl ring, which should modulate the binding of the triazene more than
groups in any other position because this provides the greatest degree of resonance interaction.
Electron donating groups, such as the methoxy group (compound 2), are capable of
adding electron density to the triazene unit through resonance and likely increase the -
interactions with the active site phenylalanine residue or other groups in another binding site
with  electrons. In addition, the methoxy group, via its methyl, make the triazene even more
hydrophobic, which may enhance its ability to interact with the hydrophobic surfaces of the
active sites in both enzymes. Chun et al. showed that when a tetrahydroxystilbene,
oxyresveratrol (IC50 = 34 M), is converted to 2,4,3’,5’-tetramethoxystilbene, the IC50 is
lowered 1000 fold to 6 nm with regards to the activity of CYP1B1. 26 Chun et al. postulated that
this is due to the increase in lipophilic properties of 2,4,3’,5’-tetramethoxystilbene as compared
to oxyresveratrol. 26 Both the electron donating and lipophilicity factors likely explain why
compound 2 inhibits CYP1A1 and CYP1B1 more than any of the other triazenes.
On the other hand, electron withdrawing groups can remove electron density from the
triazene unit through induction and/or resonance, and weaken the -interactions with the
enzyme phenylalanine residue. Compounds 3 (nitro), 4 (acetyl), and 5 (carboxy) contain the
respective electron withdrawing groups and can diminish the electron density at the triazene unit
and weaken its interactions with the enzyme active site. The screening data indicate that all
three compounds inhibit CYP1A1 to a lesser degree than compound 1 (triazene) and compounds
4 and 5 inhibit CYP1B1 to a lesser degree than compound 1 and provide some support for the
idea that electron withdrawing groups on the aryl ring weaken the interaction of the triazene with
the enzyme. One exception to this is that compound 3 inhibits CYP1B1 to a greater extent than
compound 1 and there may be other, as of yet unidentified, interactions possibly in the active site

or another binding site that could enhance the interaction of this compound with CYP1B1.
Finally, compound 6 (bromo) inhibits CYP1A1 and CYP1B1 to the same extent as compound 1
and given that bromo is both a weak electron donating and electron accepting group, it is not
surprising that its inhibitory properties are similar to compound 1.
Interestingly, several triazenes inhibited CYP 1B1 more strongly than resveratrol and
just as strongly if not more so than compound 2. Compound 3 had an IC50 value of 2 M while
compound 6 had an IC50 of 7 M with CYP1B1. The IC50 value for compound 6 was lower
than expected based upon the screening value obtained at 100 M relative to the IC50 and
screening values of other triazenes. One explanation for this observation is that this compound
may bind to more than one site; some CYP1A1 and CYP1B1 inhibitors have exhibited properties
of competitive and noncompetitive inhibitors. 19,25 Thus, at lower concentrations of the
compound, it may only bind to one of the sites and give different inhibitory properties compared
to other inhibitors that were tested.
More importantly, the IC50 values show that compounds 3 and 6 are more specific to
CYP1B1, which may be advantageous when specifically targeting this enzyme. While Walsh et
al. have described specific amino acid differences in the active sites between CYP1A1 and
CYP1B1, it is not possible to know how these specific differences may or may not contribute to
the selectivity of compounds 3 and 6 for binding to CYP1B1 over the CYP1A1 without further
investigation. 2 Furthermore, it is possible these inhibitors may inhibit CYP1B1 differently than
CYP1A1 (noncompetitive, competitive, or both) and may thus bind at a different site on
CYP1B1 than the other triazenes or a site much different than what is present on CYP1A1.
A final observation is that while compound 2 inhibits CYP1A1 and CYP1B1 at levels
similar to or more strongly than resveratrol, its inhibitory properties are much less than 2,4,3’,5’-

tetramethoxystilbene against CYP1B1 which is at the nanomolar level. 26 While the fourth
methoxy group on the stilbene would make the stilbene even more hydrophobic as compared to
three methoxy groups, it is unlikely that it would cause an increased affinity of the stilbene over
the triazene by 1000 fold. Most likely the difference between the stilbene and the triazene is that
while the stilbene is completely planar, the morpholine ring is conformationally flexible, making
the entire molecule less planar. It is likely that the bulky morpholine moiety reduces the binding
of the triazene to the narrow active site over the more planar stilbene compound. However, the
potential ability of the three nitrogens to form interactions with the enzyme active site
phenylalanine residue(s) likely compensates for the bulkiness of the morpholine conformation
that probably reduces its affinity for the active site or another hydrophobic binding site.
In conclusion, aryl morpholino triazenes are a new class of compounds that inhibit
CYP1A1 and CYP1B1 at the micromolar level. It is likely that these triazenes inhibit these
enzymes because they contain a planar aryl group and a triazene unit that is capable of forming
interactions with the enzyme active site amino acids or amino acids in another binding site.
Compound 2 was the only compound that inhibited both enzymes at a level close to resveratrol,
IC50 = 10 M with CYP1A1 and IC50 = 18 M with CYP1B1, which indicates that the
trimethoxy groups, which are electron donating and highly hydrophobic, provide a favorable
interaction with the hydrophobic enzyme active site or another binding site. Finally, compounds
3 and 6 selectively inhibit CYP1B1 (IC50 of 2 M and 7 M respectively) over CYP1A1 (IC50
of 115 M and 63 M respectively) and inhibit CYP1B1 to a degree that is similar to resveratrol.
This suggests that there are different binding properties and interactions between these triazenes
for similar binding sites, such as the active site, on the two different enzymes and/or the mode of
inhibition might be different for each enzyme, such as competitive, noncompetitive, or both. All

of this indicates that aryl morpholino triazenes could also have a similar therapeutic value to
resveratrol in preventing cancer. Future work will involve designing triazenes that will have
enhanced therapeutic value compared to the triazenes discussed above.
Acknowledgements
This work was funded by monies from the Roy W. Newsom Chair in Chemistry at Whittier
College and by a grant from the Whittier College Faculty Research and Development Funds.
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trimethoxyaniline (2.04 g, 11.1 mmol) to 3.7 mL of 6M HCl and heated to form a tan
suspension, to which 4.0 mL of H2O was added. Upon stirring, a light orange solution
resulted, which was cooled to 0°C to produce an orange solid. To this solid, a solution of
NaNO2 (0.81 g, 11.7 mmol in 2.0 mL of H2O) was added dropwise with vigorous stirring
over 10 minutes to yield a deep orange solution. 1.1 mL of morpholine (1.06 g, 12.2
mmol) was added dropwise to this solution with stirring over 10 minutes, yielding a red
solution. This solution was brought to room temperature and a saturated aqueous solution
of NaHCO3 was used to bring it to pH = 8. The orange solid that formed was collected by
vacuum filtration, washed well with water, and air-dried. The crude product was
recrystallized using cyclohexane to yield 2.44 g (78%) 2 as a light orange powder. mp
82-83°C; IR (KBr) 3002, 2970, 2946, 1595 (C=C), 1496 (N=N) cm-1; TLC Rf (silica,
ethyl acetate) 0.82; 1H-NMR (300 MHz, deuteriochloroform): d 3.76 (m, 4 H), 3.84 (m,

13 H), 6.74 (s, 2 H); 13C-NMR (deuteriochloroform): d 48.2, 56.1, 61.1, 66.5, 98.0,
136.8, 146.2, 153.5; ESI-HR-MS: m/z for [M+Na]+ = 304.1274, Calcd. m/z for [M+Na]+
= 304.1268. Anal. Calcd. for C13H19N3O4: C, 55.51; H, 6.81; N, 14.94. Found: C, 55.69;
H, 6.84; N, 14.96.
36.
37.
Barra, M.; Lim, H. Trends Org. Chem. 2006, 11, 17.
A brown fragment of 2 (0.35  0.30  0.19 mm) was used for data collection at T =
90 K on a Bruker APEX-II CCD diffractometer equipped with an Oxford Cryosystems
Cryostream chiller and graphite-monochromated Cu Kα radiation, λ = 1.54178 Å). A total
of 13,874 measured reflections with max = 69.2 yielded 2,539 unique data. The structure
was solved by direct methods, and structure refinement was carried out using SHELXL-97.
38 All hydrogen atoms were visible in difference maps, but were placed in calculated
positions in the refinement.
Single-crystal X-ray diffraction data for 2. C13H19N3O4, FW 281.31, monoclinic, P21/c, a
= 10.1801(5) Å, b = 14.9700(8) Å, c = 9.0890(5) Å, β = 97.629(2)°, V = 1372.87(13) Å3,
Z = 4, calc = 1.361 Mg/m3, μ = 0.85 mm−1, F(000) = 600, Rint = 0.028 for 2,444
reflections with I > 2σ(I), R1/wR2 = 0.030/0.076, Goodness-of-fit on F2 = 1.06, 185
parameters.
CCDC 837085 contains the supplementary crystallographic data for compound 2. These
data can be obtained free of charge from the Cambridge Crystallographic Data Centre via
http://www.ccdc.cam.ac.uk/products/csd/request/.
38.
39.
Sheldrick, G.M. Acta Cryst. 2008, A64, 112.
Hörner, M.; Brancher, A.L.; Silva, A.; Borhinhao, J.; Mössmer, C.M. Acta Cryst. 2004,
C60, o303.

40.
Allen, F.H.; Kennard, O.; Watson, .G.; Brammer, L.; Orpen, A.G.; Taylor, R. J. Chem.
Soc. Perkin Trans. 1987, 2, S1.
41.
42.
43.
Fu, J.; Lau, K.; Barra, M. J. Org. Chem. 2009, 74, 1770.
Little, V.R.; Jenkins, H.; Vaughan, K. J. Chem. Crystallogr. 2008, 38, 447.
Zhang, D.-C.; Fei, Z.-H.; Zhang, T.-Z.; Zhang, Y.-Q.; Yu, K.-B. Acta Cryst. 1999, C55,
102.
44.
45.
Mukai, C.; Zamora, E.; Blatti, J.L.; Fronczek, F.R.; Isovitsch, R. J. Chem. Crystallogr.
2013, 43, 412.
Cytochrome P450 1A1 and 1B1 assays were modified from those described by Chun et
al. 46 Assays were run in triplicates in the absence of inhibitor and in the presence of
inhibitor at each inhibitor concentration. Each reaction consisted of 0.10 M phosphate
buffer pH 7.4, 5.0 M ethoxyresorufin (ER), 6.25 mM MgCl2, 7.1 mM EDTA, 28.0 µM
NADPH, and either 10 nM CYP1A1 and 5 nM CYP1B1. NADPH was added to initiate
the reaction at 37˚C. The reaction was stopped at 2 minutes by adding 2.0 mL of chilled
methanol. The test tubes were then centrifuged for 25 minutes at 500 x g to remove the
precipitate from solution. The resorufin product was then excited at 574 nm and the
emission was read at 586 nm using a Fluoromax-4 spectrofluorometer. Decreasing
amounts of resorufin fluorescence corresponded with decreased enzyme activity and thus
increased enzyme inhibition.
46.
Chun, Y.J.; Ryu, S.Y.; Jeong, T.C.; Kim, M.Y. Am. Soc. Pharmacol. Exp. Toxicol. 2001,
29, 389.
47.
48.
Fu, J.; Lau, K.; Barra, M. J. Org. Chem. 2009, 74, 1770.
Zhang, H.; Barra, M. J. Phys. Org. Chem. 2005, 18, 498.

49.
Chen, Z-H; Hurh Y-J; Na, H-K; Kim J-H; Chun, Y-J; Kim, D-H; Kang, K-S; Cho, M-H;
Surh, Y-J. Carcinogenesis 2004, 25, 2005.
Figure 1. Preparation and chemical structures of compounds 1-6.
Figure 2. ORTEP view of compound 2. Thermal ellipsoids are represented at the 50%
probability level.

Graphical Abstract
NH2
O
N
R1
R2
N
1. 6 M HCl, NaNO2 (aq)
N
2. morpholine
3. sat. aq. NaHCO3
R1
R3
R2
R3
1: R1,R2,R3=H
2: R1,R2,R3=OCH3
3: R1,R3=H, R2=NO2
4: R1,R3=H, R2=C(O)CH3
5: R1,R3=H, R2=COOH
6: R1,R3=H, R2=Br
CYP 1A1 and 1B1
CYP 1B1
CYP 1B1