Quinazoline derivatives as selective CYP1B1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.02.032

CYP1B1 is implicated to have a role in the development of breast, ovarian, renal, skin and lung carcinomas. It has been suggested that identification of potent and specific CYP1B1 inhibitors can lead to a novel treatment of cancer. Flavonoids have a compact rigid skeleton which fit precisely within the binding cavity of CYP1B1. Systematic isosteric replacement of flavonoid ‘O’ atom with ‘N’ atom led to the prediction that a ‘quinazoline’ scaffold could be the basis for designing potential CYP1B1 inhibitors. A total of 20 quinazoline analogs were synthesized and screened for CYP1B1 and CYP1A1 inhibition in Sacchrosomes. IC₅₀ determinations of six compounds with capability of inhibiting CYP1B1 identified quinazolines 5c and 5h as the best candidates for CYP1B1 inhibition, with IC₅₀ values in the nM range. Further selectivity studies with homologous CYPs, belonging to the CYP1, CYP2 and CYP3 family of enzymes, showed that the compounds are likely to be free from critical drug-drug interaction liability. Molecular modelling studies were performed to rationalize the observed enzymatic inhibitions. Further biological studies in live yeast and human cells, harboring CYP1A1 and CYP1B1 enzymes, have illustrated the most potent compounds' cellular permeability and capability of potently inhibiting CYP1B1 enzyme expressed within live cells.

Full text of DOI:10.1016/j.ejmech.2017.02.032

Accepted Manuscript
Quinazoline derivatives as selective CYP1B1 inhibitors
Mohd Usman Mohd Siddique, Glen McCann, Vinay Sonawane, Neill Horley, Linda
Gatchie, Prashant Joshi, Sandip B. Bharate, Venkatesan Jayaprakash, Barij N. Sinha,
Bhabatosh Chaudhuri
PII:
S0223-5234(17)30097-1
10.1016/j.ejmech.2017.02.032
EJMECH 9228
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 December 2016
Revised Date: 30 January 2017
Accepted Date: 11 February 2017
Please cite this article as: M.U. Mohd Siddique, G. McCann, V. Sonawane, N. Horley, L. Gatchie,
P. Joshi, S.B. Bharate, V. Jayaprakash, B.N. Sinha, B. Chaudhuri, Quinazoline derivatives as
selective CYP1B1 inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.02.032.
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GRAPHICAL ABSTRACT
O
O
A
NH
A
O
B
N
B
Br
Alpha-naphthoflavone (3)
Quinazoline 5c
Saccharosomes
CYP1A1: IC50 = 10 nM
CYP1B1: IC50 = 50 nM
CYP1A1: IC50 = 8200 nM
CYP1B1: IC50 = 3 nM
CYP1A2, 2D6, 3A4, 2C9,
2C19: IC50 = >10,000 nM
CYP1B1: IC50 = >10,000 nM
CYP1B1: IC50 = >10,000 nM
Live yeast cells
Live human cells
CYP1B1: IC50 = 60 nM
CYP1B1: IC50 = 25 nM
5C with CYP1B1
We report discovery of quinazoline class of cell permeable selective CYP1B1 inhibitors.

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Quinazoline derivatives as selective CYP1B1
inhibitors
Mohd Usman Mohd Siddique,a Glen McCann,b Vinay Sonawane,b Neill Horley,b Linda
Gatchie,bd Prashant Joshi,c Sandip B. Bharate,c* Venkatesan Jayaprakash,a Barij N. Sinha,a
Bhabatosh Chaudhuribd*
aDepartment of Pharmaceutical Sciences & Technology, Birla Institute of Technology, Mesra,
Ranchi-835215, India
bLeicester School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK
cMedicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180001, India.
d CYP Design Limited, Innovation Centre, 49 Oxford Street, Leicester, LE1 5XY, UK
*Corresponding author
Prof. Bhabatosh Chaudhuri
Leicester School of Pharmacy,
De Montfort University, Leicester, LE1 9BH, UK
Fax: Fax: +44(0) 116 257 7287; Tel: 44(0)116 250 7280
*E-mail: E-mail: bchaudhuri@dmu.ac.uk (BC), sbharate@iiim.ac.in (SBB)
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ABSTRACT:
CYP1B1 is implicated to have a role in the development of breast, ovarian, renal, skin and lung
carcinomas. It has been suggested that identification of potent and specific CYP1B1 inhibitors
can lead to a novel treatment of cancer. Flavonoids have a compact rigid skeleton which fit
precisely within the binding cavity of CYP1B1. Systematic isosteric replacement of flavonoid
‘O’ atom with ‘N’ atom led to the prediction that a ‘quinazoline’ scaffold could be the basis for
designing potential CYP1B1 inhibitors. A total of 20 quinazoline analogs were synthesized and
screened for CYP1B1 and CYP1A1 inhibition in SacchrosomesTM. IC50 determinations of six
compounds with capability of inhibiting CYP1B1 identified quinazolines 5c and 5h as the best
candidates for CYP1B1 inhibition, with IC50 values in the nM range. Further selectivity studies
with homologous CYPs, belonging to the CYP1, CYP2 and CYP3 family of enzymes, showed
that the compounds are likely to be free from critical drug-drug interaction liability. Molecular
modelling studies were performed to rationalize the observed enzymatic inhibitions. Further
biological studies in live yeast and human cells, harboring CYP1A1 and CYP1B1 enzymes, have
illustrated the most potent compounds’ cellular permeability and capability of potently inhibiting
CYP1B1 enzyme expressed within live cells.
Keywords. quinazoline, CYP1B1, cancer chemoprevention, Sacchrosomes, selective CYP1B1
inhibitor
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1. INTRODUCTION
Cytochrome P450 (CYP) enzymes are key metabolizing enzymes involved in the metabolism of
drugs and endogenous substrates [1]. CYPs are hemoproteins which depend on a reductase for
their function. Hydroxylation or mono-oxygenation is one of the many reactions that some CYPs
perform and, hence, CYPs are often referred to as oxidoreductases. The CYP1 family of
monooxygenases, 1A1, 1B1 and 1A2, are involved in the hydroxylation of a large number of
pro-carcinogens, such as polycyclic aromatic hydrocarbons [BaP], oxides, amines, and estrogens
to cytotoxic, mutagenic and carcinogenic chemicals. The CYP1 enzymes are highly expressed in
hormonal cancers related to the prostate, ovary, mammary, uterus and pituitary, and also in skin
and lung cancers, while they are expressed at very low levels or not at all in healthy tissues [2-7].
The CYP1B1 isoform is mainly responsible for the metabolism of estradiol. Metabolomics of
estradiol indicate that it is regio-specifically converted to 2-hydroxy estradiol by CYP1A1 and
CYP1A2 and to the carcinogenic form 4-hydroxy estradiol (4-OHE2) by CYP1B1. Formation of
4-OHE2 in mammary, ovarian and prostate tissues is thought to initiate the process of
carcinogenesis [8]. In CYP1B1 overexpressing cells, this catechol derivative of estradiol is
oxidized to a carcinogenic quinone intermediate, 3,4-estradiol quinone, which reacts with DNA
and tubulin leading to cancer-inducing toxicity [9-16]. Hence, overexpression of CYP1B1 is a
critical factor for hormonal metabolism and toxicity in mammary, endometrium and ovarian
tissues as well as it serves as a potential biomarker for hormonal carcinogenesis [3, 11, 17-19].
Interestingly, in the past few years cumulative evidence has emphasized that natural flavonoids
have a profound role in CYP1 inhibition. Flavonoids (Figure 1) such as quercetin [20] (1),
chrysin [21] (2), ANF [22-24] (3), 7,8-dehydrorutaecarpine (4) have been identified as CYP1B1
inhibitors. Cytochrome P450 (CYP) enzymes, generally share common structural features and
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mechanism for catalysis. They are involved in the metabolism of compounds with diverse
structures. CYP1B1 isoform, in particular, has a binding cavity of rounder shape than that of
CYP1A1 and CYP1A2 allowing accommodation of multiple conformations of estradiol [25].
However, X-ray crystallographic studies of CYP1B1 with ANF have revealed that that ANF
interacts with a narrow slot-like substrate binding cavity of CYP1B1 which is similar to that of
CYP1A2 [23]. Thus ANF is an equipotent inhibitor of CYP1B1 and CYP1A2 [25]. ANF
disrupts the interaction between CYP1 enzymes’ planar substrates and CYP1’s reactive heme
iron-oxo intermediate [23]. Exploration of the isosteric replacement of chromane nucleus of
ANF with N atom at 1 and 3 positions showed that the isosteres of ANF also mimic ANF-like
interactions with CYP1B1’s active site. As a part of an ongoing program on discovery of new
CYP1B1 inhibitors [25], the present manuscript provides design, synthesis and biological
evaluation of a quinazoline class of compounds for selective inhibition of CYP1B1 over
CYP1A1 and other drug metabolizing CYP enzymes.
2. RESULTS AND DISCUSSION
The analysis of common structural features of potent CYP1B1 inhibitors such as quercetin (1),
chrysin (2), alpha-napthoflavone (3) and 7,8-dehydrorutecarpine (4) indicate that the isosteric
replacement of ‘O’ atom with ‘N’ atom results in replacement of the chromane core with the
quinolone nucleus. Further, addition of nitrogen atom in C-3 position of chromane ring generates
quinazoline nucleus. This is a simple pharmacophore that comprises two aromatic rings suitably
linked with a polar linker of 2-3 bond lengths. It was thought that this could be the basis of a
‘new scaffold’ for CYP1B1 inhibition (Figure 1). Therefore, using CYP1B1 as a target protein,
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we designed the ‘quinazoline’ scaffold, with the view that possibly it would mimic the flavone
scaffolds 1-3.
Figure 1.
There are numerous reports on the synthesis of quinazolin-4- (3H)-ones [26-35]. Amongst these
reports, the most common approach has been the condensation of 2-aminobenzamide or O-
nitrobenzamides with aldehydes [32, 36], aryl acid chloride [37, 38], benzyl bromides [39],
benzylamine [40] and alcohols [32, 41-45]. Another approach comprises an Ullmann-type
coupling of 2-bromobenzamide with benzyl amine. Many of these Ullmann coupling protocols
require copper catalyst, ligand and high heating. Therefore, a simpler method was developed for
synthesis of quinazoline core, as shown in Scheme 1. It involved the condensation of 2-
aminobenzamide (6) with appropriate substituted aldehydes (7) using I2/sodium acetate in DMF
at high temperature. The rare substituted aldehydes, 9h-t, were synthesized separately and then
the quinazoline core, 5h-t, was constructed using I2/sodium acetate in DMF at high temperature,
as depicted in Scheme 1. The final compounds were purified by simply washing with ethyl
acetate and petroleum ether (40: 60) in 1:1 ratio. No column chromatography has been carried
out for purification of the final compounds. All compounds were characterized by NMR and MS
analysis.
Scheme 1.
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All 20 synthesized quinazoline derivatives, 5a-t, were tested for inhibition of CYP1B1 and
CYP1A1 using SacchrosomesTM at 10 µM final concentration of compound. Results are shown
in Table 1. In general, it was observed that all compounds displayed selective inhibition of
CYP1B1 over CYP1A1. The IC50 values of the six most potent CYP1B1 inhibitors 5c, 5f, 5g,
5h, 5i and 5j were then determined (Table 2). Furthermore, these six most potent CYP1B1
inhibitors were tested for inhibition of CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2C19
SacchrosomesTM at 10 µM concentration of compounds. The compounds 5g, 5i and 5j showed
more than 50% inhibition of CYP1A2. None of these compounds showed significant (>50%)
inhibition of CYP3A4 and CYP2D6 at 10 µM final concentration. Inhibition of CYP2C9 and
CYP2C19 at 10 µM was insignificant (Table 2). The results indicated that specific quinazoline
derivatives, 5c, 5h, and 5i, were potent CYP1B1 inhibitors. Their IC50 values in in vitro enzyme
assays, using SacchrosomesTM, ranged from 2 nM to 4 nM.
Table 1.
Table 2.
The C ring of quinazoline 5c interacts with reactive heme group via van der Waals interactions
(distance, <5 ºA) (Figure 2A). In quite similar fashion to benzo[a]chromane ring of ANF, the
heteroatom containing B ring of quinazoline allows the aromatic C rings to flank toward heme
moiety, which further allows compound 5c, quinazoline core to form hydrophobic π-π
interactions with Phe231. These interactions are common in both compound 5c and 5j as shown
in Figure 2B.
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Figure 2.
Overall, these interactions of quinazoline derivatives with CYP1B1 would explain the potent
inhibition of CYP1B1. The observed difference in efficacy to inhibit CYP1 family enzymes by
compounds 5c, 5h and 5i over other CYP1, CYP2 and CYP3 family members is likely to be due
to the differences in amino acid residues in the primary sequences of these isoforms and also the
structural architecture of their binding sites. The CYP1 family enzymes especially CYP1B1
isoform have narrow slot like structure where substrate/inhibitors get stuck, while in case of
CYP2 and CYP3 wide, open and large cavity which attract wide range of substrates and
inhibitors. This flexibility in structural architecture allows ligands to bind flexibly, which reduces
the interaction between ligand and heme molecule needed for the oxidoreductase function.
Overall, it can be predicted that the observed selectivity over the CYP2 and CYP3 isoforms is
likely to make 5c, 5h and 5i free from any putative drug-drug interactions.
All quinazolines were then tested in live human cells with the aim of finding out if (a) there was
a correlation between the inhibition of CYP1B1 enzyme observed in SacchrosomesTM, that is,
endoplasmic reticulum bound CYP-reductase complexes which were isolated from recombinant
baker’s yeast (Saccharomyces cerevisiae) cells, and the CYP1B1 enzyme expressed within ‘live’
human cells, and (b) the compounds were human cell permeable which would make them
amenable for further pre-clinical cellular studies. Adherent human embryonic kidney cells
(HEK293), adapted for growth in ‘suspension’, were transfected with plasmids
(pcDNA3.1/CYP1B1 and pcDNA3.1/CYP1A1) which encode the human CYP1B1 and CYP1A1
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genes. Growth of human cells in ‘suspension’ is essential for evaluating the compounds’
inhibitory potential in ‘live’ cells. Only in ‘live’ cells, are the enzyme activities of CYPs are
completely retained. Moreover, in ‘live’ cells, the natural interactions that exist between CYPs
and other proteins within eukaryotic human cells are fully maintained. One could assume that
cellular properties of such recombinant cells are akin to those of hepatocytes. However, there is
also a major difference. Whereas hepatocytes are derived from a conglomeration of degenerate
livers, often derived from drug addicts and alcoholics and therefore have a high batch-to-batch
variation in CYP activities, these recombinant cells express highly reproducible activity of a
particular CYP. In fact, these cells could be engineered to produce multiple human CYPs
(unpublished observations, CYP Design Ltd).
Transiently transfected recombinant HEK293 cells, gown in suspension, were incubated with the
compounds for 30 min. After incubation, the activities of CYP1B1 and CYP1A1 were measured
via the 7-ethoxy-resorufin-O-deethylase (EROD) assay, using 5 µM concentration of 7-ethoxy-
resorufin as substrate. The percentage inhibition of CYP1B1 and CYP1A1 in live human cells
(HEK293 cells) is shown in Table 3. As observed with yeast microsomes, most compounds
displayed selectivity towards CYP1B1 over CYP1A1 in live human cell assays, broadly
corroborating the results obtained using SacchrosomesTM isolated from baker’s yeast cells.
Nonetheless, comparison of Table 1 and 3 shows that there are major differences, too,
underlining the fact that the recombinant human cells would be a far better screening tool than
commercially available microsomal CYP enzymes (SupersomesTM, BaculosomesTM
,
BactsosomesTM, and SacchrosomesTM) which are isolated from recombinant insect, bacterial and
yeast cells.
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With the success in identifying a new structural class of potent CYP1B1 inhibitors using
SacchrosomesTM and live human cells, it was queried if live recombinant yeast cells, which can
be grown cheaply in bulk and with relative ease could be used, instead of expensive microsomal
enzyme, for rapid screening of CYP enzyme inhibitors. Hence, the quinazoline compound library
was also tested for CYP1A1 and CYP1B1 inhibition in live yeast cells (Table 3), wherein the
compound 5c was found to be the most potent inhibitor of CYP1B1 expressed within live yeast
cells (Table 4). The results indicate that live recombinant yeast cells have the potential to be used
as a substitute of the relatively expensive microsomal CYP1B1 enzyme for rapid screening of
CYP1B1 inhibitors as potential anticancer agents. The IC50 values of the best compound 5c,
together with other derivatives, with respect to CYP1B1 inhibition in live yeast cells and live
human cells are shown in Table 4. Compound 5c displayed the most potent inhibition of
CYP1B1 in live cells, IC50 being 310 and 25 nM in yeast and human cells, respectively.
The IC50 determinations (Table 3) indicated that compounds 5c, 5h and 5i showed potent
inhibition of CYP1B1 with IC50 values of 3, 2 and 4 nM, respectively. All three compounds were
highly selective for CYP1B1 when compared with inhibition of CYP1A1, CYP1A2, CYP3A4,
CYP2D6, CYP2C9 and CYP2C19. The compounds 5c and 5h were the most promising with
>2500-fold selectivity over other four CYPs.
Table 3.
3. CONCLUSION
In conclusion, isosteric replacement of the flavonoid core of the known potent CYP1B1
inhibitors, ANF, chrysin, quercetin and 7,8-dehydrorutecarpine has led to the identification of
quinazoline derivatives as a novel class of selective CYP1B1 inhibitors. Substitution on the
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phenyl ring at 2nd position of quinozoline significantly influences the potency and selectivity of
the compounds on in vitro % CYP inhibition. (i) Electron withdrawing groups (5b & 5c, 5k &
5p) at para position of the phenyl ring were found to improve the potency as well as selectivity
towards CYP1B1. (ii) Electron donating group (5d) at para position was found to improve
potency and selectivity towards CYP1A1. When the same substitution is at meta-position (5e),
the molecule tends to loose its selectivity between CYP1A1 & CYP1B1. In both the cases the
introduction of methoxy functional group ortho (5f and 5g) to hydroxy functional group, we
observed improved potency and decreased selectivity between the isoforms (1A1 and 1B1). (iii)
Further, replacement of hydroxyl hydrogen of 5d, 5f and 5g with bulkier groups provided mixed
responses. In all the cases 5d analogs (5i, 5j and 5h) displayed significant improvement in
potency. While 5i and 5h exhibited selectivity towards CYP1B1, 5j was found to be non-
selective towards 1A1, 1A2 & 1B1. A similar trend was observed for human live cell % CYP
inhibition with the exception that 5d and 5g analogues displaying the improved potency as well
as selectivity towards 1B1 isoform. The availability of newly identified ‘quinazoline derivatives’
as potent CYP1B1 inhibitors would allow further studies on CYP1B1 enzyme-mediated
modulation of xenobiotics. These studies are likely to reveal their possible usefulness in the
treatment of xenobiotics-associated disorders that lead to hormonal cancers.
4. EXPERIMENTAL SECTION
4.1. General. All chemicals were purchased from Spectro-Chem, Hi-media, Acros or Sigma
Aldrich and solvents from Rankem and used as it was or as specified below. All the reactions
were carried out in air and in dried flasks. Pre-coated silica gel aluminium plates (Merck) were
used for reaction monitoring and visualised under UV light. Column chromatography was
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performed using silica gel (60-100 mesh) obtained from Merck. Carbon (13C) and Proton (1H)
nuclear magnetic resonance (NMR) spectra were recorded on JEOL 400 MHz or Bruker 300
MHz instruments using dimethylsulfoxide (DMSO-D6) as solvent and tetramethylsilane as an
13
internal standard (1H NMR: DMSO δ 2.5 ppm, TMS: δ 0.00 ppm and C NMR DMSO: δ 39.5
ppm).
4.2. General procedure for the synthesis of 9h-9t. To the solution of 4-hydroxy
benzaldehydes, vanillin or iso-vanillin (1 equivalent) in acetone was added potassium carbonate
(2 equivalents). Then, corresponding substituted benzyl chlorides or acetamide was added
followed by addition of potassium iodide (1.5 equivalents). Reaction mixture was refluxed for 8-
10 h. After completion of reaction, the resultant mixture was concentrated and was diluted with
water. Product was extracted with ethyl acetate (3 x 20 ml). The organic layers were combined
and treated with brine and dried over sodium sulfate and concentrated. The crude mixture was
purified over gel silica gel column (60-120 mesh) using petroleum ether: ethyl acetate (9:1).
4.3. General procedure for the synthesis of 5a-t. To the stirring solution of 2-aminobenzamide
(1 equivalent) in DMF, sodium acetate (2 equivalents) was added. After stirring for- 10 min,
substituted benzaldehydes (1 equivalent, 7a-g and 9h-t) and iodine (2 equivalents) were added.
The reaction mixture was stirred at 70-80 °C for 20-24 h. Then the reaction mixture was poured
on to crushed ice. The resulting mixture was treated with sodium thiosulphate (10% w/v in
water) to reduce the remaining iodine. The precipitate was filtered off and washed with
petroleum ether (40:60): ethyl acetate in 1:1 ratio.
4.3.1. 2-(pyridin-3-yl) quinazolin-4(3H)-one (5a): Light pink powder; 0.724g (yield: 88.45%);
m.p. 280-282 °C; ESI-MS: m/z 224.10 [M+H]+ calcd 224.08; 1H NMR (400 MHz, DMSO-d6): δ
9.24-7.60 (m, 8H, Ar-H), (br. s., 1H, CO-NH); 13C NMR (101 MHz, DMSO-d6): δ 91.74 (Ar-C-
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), 94.16 (Ar-C-), 96.51 (Ar-C-), 97.58 (Ar-C-), 98.14 (Ar-C-), 99.36 (Ar-C-), 105.33 (Ar-C-),
106.01 (Ar-C-), 113.58 (Ar-C-), 119.36 (-C-N), 121.42 (-C=N), 122.44 (-C=N), 132.78 (-C=O)
4.3.2. 2-(4-nitrophenyl)quinazolin-4(3H)-one (5b): Yellowish orange powder, 0.713g (yield:
72.00%); m.p. >300 °C; ESI-MS: m/z 268.1 [M+H]+ calcd 268.07; 1H NMR (400 MHz, DMSO-
d6): δ 8.34-7.54 (m, 8H, Ar-H), (br. s., 1H, -CONH); 13C NMR (101 MHz, DMSO-d6): δ 121.43
(Ar-C-), 123.67 (Ar-C-), 126.70 (Ar-C-), 127.89 (Ar-C-), 128.27 (Ar-C-), 129.82 (Ar-C-),
135.34 (Ar-C-), 139.06 (-C-N), 149.49 (-C-NO2), 151.27 (-C=N), 162.63 (-C=O).
4.3.3. 2-(4-bromophenyl)quinazolin-4(3H)-one (5c): Yellowish powder; 0.983g (yield:
1
89.44%); m.p. 309-311°C; H NMR (400 MHz, DMSO-d6): δ 8.57-7.05 (m, 8H, Ar-H), 12.61
(br. s., 1H, CO-NH).
4.3.4. 2-(4-hydroxyphenyl) quinazolin-4(3H)-one (5d): Cream colored powder; 0.814g (yield:
1
93.13%); m.p. 300 °C; ESI-MS: m/z 239.0 [M+H]+ calcd 239.08; H NMR (400 MHz, DMSO-
d6): δ 8.03-6.93 (m, 8H, Ar-H), 10.09 (s, 1H, OH), 12.31 (s, 1H, CO-NH); 13C-NMR (101 MHz,
DMSO-d6): δ 115.88 (Ar-CH-), 121.23 (Ar-C-), 123.51 (Ar-C-), 126.04 (Ar-CH-), 127.82 (Ar-
CH-), 130.34 (Ar-CH-), 135.03 (Ar-CH-), 138.70 (Ar-CH-), 149.65 (-C-N), 152.64 (-C=N),
160.83 (-C-OH), 162.85 (-C=O).
4.3.5. 2-(3-hydroxyphenyl) quinazolin-4(3H)-one (5e): Cream powder; 0.734g (yield:
1
83.38%); m.p. 261-263 °C; ESI-MS: m/z 239.1 [M+H]+ calcd 239.08; H NMR (400 MHz,
DMSO-d6): δ 7.79-6.94 (m, 8H, Ar-H), 8.10 (s, 1H, OH), 9.79 (br, s., 1H, CO-NH).
4.3.6. (2-(3-hydroxy-4-methoxyphenyl)quinazolin-4(3H)-one (5f): White powder; 0.914g
1
(yield: 92.30%); m.p. 240-241 °C; ESI-MS: m/z 269.10 [M+H]+ calcd 269.09; H NMR (400
MHz, DMSO-d6): δ 3.83 (s, 3H, OCH3), 6.89 (s, 1H, Ar-OH), 8.11-7.48 (m, 7H, Ar-H), 9.77- (s,
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13
1H), 12.35 (br. s., 1H, CO-NH); C NMR (101 MHz, DMSO-d6): δ 56.20 (Ar- OCH3), 111.53
(Ar-CH-), 115.01 (Ar-CH-), 119.75 (Ar-CH-), 121.20 (Ar-C-), 125.52 (Ar-C-), 126.37 (Ar-CH-),
126.60 (Ar-CH-), 127.75 (-C-N), 135.36 (Ar-C-OCH3), 146.88 (-C=N), 151.14 (Ar-C-OH),
162.81 (-C=O).
4.3.7. (2-(4-hydroxy-3-methoxyphenyl)quinazolin-4(3H)-one) (5g): White powder; 0.901g
1
(yield: 91.47%); m.p. 209-211 °C; ESI-MS: m/z 269.10 [M+H]+ calcd 269.09; H NMR (400
MHz, DMSO-d6): δ 3.72 (s, 1H, Ar-OH), 3.83 (s, 3H, OCH3), 9.35-7.04 (m, 7H, Ar-H), 12.31 (s,
13
1H, CO-NH); C NMR (101 MHz, DMSO-d6): δ 56.27 (Ar- OCH3), 111.79 (Ar-CH-), 115.92
(Ar-CH-), 121.10 (Ar-CH-), 121.99 (Ar-C-), 123.88 (Ar-C-), 12.35 (Ar-CH-), 127.75 (Ar-CH-),
135.05 (-C-N), 147.99 (Ar-C-OCH3), 150.46 (-C=N), 152.55 (Ar-C-OH), 162.91 (-C=O).
4.3.8. 2-(4-(prop-2-ynyloxy)-phenyl)quinazolin-4(3H)-one (5h): White powder; 0.912g (yield:
1
94.70%); m.p. 214-217 °C; ESI-MS: m/z 277.00 [M+H]+ calcd 277.09; H NMR (400 MHz,
DMSO-d6): δ 3.63 (s, 1H, -CH), 4.90 (s, 2H, OCH2), 8.12-7.12 (m, 8H, Ar-H), 12.43 (s, 1H,
13
CO-NH); C NMR (101 MHz, DMSO-d6): δ 56.12 (OCH2), 70.96 (≡CH), 79.31 (≡C-), 115.11
(Ar-CH-), 121.19 (Ar-C-), 125.75 (Ar-C-), 127.78 (Ar-CH-), 129.86 (Ar-CH-), 134.60 (Ar-CH-),
135.03 (Ar-CH-), 136.72 (Ar-CH-), 149.35 (Ar-CH-), 152.24 (-C-N), 152.87 (-C=N), 160.35 (-
C-O), 162.74 (-C=O).
4.3.9. 2-(4-(2,4-dichlorophenoxy)phenyl)quinazolin-4(3H)-one (5i): White powder; 1.391g
1
(yield 96.95%); m.p. 107-109 °C; ESI-MS: m/z 397.00 [M+H]+ calcd 397.04; H NMR (300
MHz, DMSO-d6): δ 5.27 (s, 2H, OCH2), 7.90-7.23 (m, 11H, Ar-H), 9.88 (s, 1H, CO-NH); 13C
NMR (75 MHz, DMSO-d6): δ 66.81 (OCH2),115.30 (Ar-CH-), 127.71 (Ar-C-), 129.09 (Ar-CH-
), 130.15 (Ar-CH-), 131.70 (Ar-C-Cl), 131.90 (Ar-C-Cl), 132.88 (-C-N), 133.94 (-C=N),162.96
(-C=O), 191.49 (=C-O-).
13

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4.3.10. 2-(4-(2,4-difluorobenzyloxy)phenyl)quinazolin-4(3H)-one (5j): Light brown powder;
1
0.412g (yield: 77.1%); m.p. 201-212 °C; ESI-MS: m/z 365.00 [M+H]+ calcd 365.11; H NMR
(400 MHz, DMSO-d6): δ 5.20 (s, 2H, , OCH2), 7.16-8.174 (m, 11H, Ar-H) 12.41 (s, 1H, CO-
NH); 13C NMR (101 MHz, DMSO-d6): δ 63.86 (OCH2), 114.98 (Ar-CH-), 117.53 (Ar-C-),
121.23 (Ar-C-), 125.92 (Ar-C-), 126.35 (Ar-CH-), 127.84 (Ar-CH-), 130.05 (-C-N), 134.91 (-
C=N), 149.22 (Ar-C-F), 152.14 (Ar-C-F), 161.02 (-C=O), 162.82 (=C-O-);
4.3.11. (4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenyl benzene sulfonate) (5k): Light brown
crystalline powder; 1.214g (yield: 89.2%); m.p. 215-217 °C; ESI-MS: m/z 397.1 [M+H]+ calcd
1
397.07; H NMR (400 MHz, DMSO-d6): δ 8.11-7.21 (m, 13H, Ar-H), 12.58 (br. s., 1H, CO-
NH); 13C NMR (101 MHz, DMSO-d6): δ 121.23 (Ar-C-), 122.43 (Ar-C-), 122.71 (Ar-CH-),
126.32 (Ar-CH-), 127.40 (Ar-CH-), 127.83 (Ar-CH-), 128.53 (Ar-CH-), 128.68 (Ar-CH-),
130.22 (Ar-CH-), 130.42 (Ar-CH-), 132.32 (=C-S-), 134.41 (-C-N), 135.27 (=C-O-), 135.76 (-
C=N), 151.36 (-C=O).
4.3.12. 2-(4-(4-fluorobenzyloxy)-3-methoxyphenyl)quinazolin-4(3H)-one (5l): Light yellow
1
powder; 0.53g (yield: 76.7%); m.p. 281-283 °C; ESI-MS: m/z 377.12 [M+H]+ calcd 377.13; H
NMR (400 MHz, DMSO-d6): δ 3.385 (s, 3H, OCH3), 5.132 (s, 2H, OCH2), 7.183-7.814 (m, 11H,
Ar-H), 12.415 (s, 1H, CO-NH); 13C NMR (101 MHz, DMSO-d6): δ 56.23 (Ar- OCH3), 69.77
(OCH2), 111.39 (Ar-CH-), 113.37 (Ar-CH-), 115.95 (Ar-CH-), 121.19 (Ar-C), 126.36 (Ar-C-),
127.85 (Ar-CH-), 130.70 (-C-N), 134.94 (-C=N), 149.38 (Ar-C-OCH3), 150.86 (-C=O), 152.31
(Ar-C-F), 162.89 (=C-O-).
4.3.13. 2-(4-(2,4-dichlorobenzyloxy)-3-methoxyphenyl)quinazolin-4(3H)-one (5m): White
powder; 1.413g (yield: 90.20%); m.p. 105-107 °C; ESI-MS: m/z 427.00 [M+H]+ calcd 427.05;
1H NMR (300 MHz, DMSO-d6): δ 3.84 (s, 3H, OCH3), 5.25 (s, 2H, OCH2),7.72- 7.29 (m, 10H,
14

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Ar-H), 9.86 (s, 1H, CO-NH); 13C-NMR (75 MHz, DMSO-d6): δ 55.71 (Ar- OCH3), 67.10
(OCH2), 109.96 (Ar-CH-), 112.73 (Ar-CH-), 125.92 (Ar-C-), 127.73 (Ar-C-), 129.06 (Ar-CH-),
130.24 (Ar-CH-), 131.60 (Ar-C-Cl), 132.97 (Ar-C-Cl), 133.83 (-C-N), 133.91 (-C=N), 149.44
(Ar-C-OCH3), 152.79 (-C=O), 191.56 (=C-O-).
4.3.14. 2-(4-(benzyloxy)-3-methoxyphenyl)quinazolin-4(3H)-one (5n): White powder, 0.263g
1
(yield 81.30%); m.p. 206-209 °C; ESI-MS: m/z 359.2 [M+H]+ calcd 359.13; H NMR (400
MHz, DMSO-d6): δ 3.85 (s, 3H, OCH3), 5.14 (s, 2H, OCH2), 7.15-8.10 (m, 12H, Ar-H), 12.41 (s,
1H, CO-NH); 13C NMR (101 MHz, DMSO-d6): δ 56.50 (Ar- OCH3), 70.36 (OCH2), 111.11 (Ar-
CH-), 113.38 (Ar-CH-), 121.21 , 121.50 (Ar-C-), 125.73 (Ar-C-), 126.37 (Ar-CH-), 126.62 (Ar-
CH-), 127.73 (Ar-CH-), 128.47 (Ar-CH-), 129.01 (Ar-CH-), 134.78 (Ar-CH-), 137.25(-C-N),
149.40 (-C=N), 150.93 (Ar-C-OCH3), 152.59 (-C=O), 163.23 (=C-O-).
4.3.15. 2-(4-(2,4-difluorobenzyloxy)-3-methoxyphenyl)quinazolin-4(3H)-one
(5o):
White
powder, 0.208g (yield 71.10%); m.p. 243-245 °C; ESI-MS: m/z 395.10 [M+H]+ calcd 395.12; 1H
NMR (400 MHz, DMSO-d6): δ 3.90 (s, 3H, OCH3), 5.21 (s, 2H, OCH2), 7.25-8.15 (m, 10H, Ar-
H), 12.43 (s, 1H, CO-NH).
4.3.16. 2-methoxy-4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenyl
methanesulfonate
(5p):
yellowish powder; 0.217g (yield: 85.12%); m.p. 197-200 °C; ESI-MS: m/z 347.00 [M+H]+ calcd
1
347.07; H NMR (400 MHz, DMSO-d6): δ 3.39 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 7.44-8.14
(m, 7H, Ar-H), 12.63 (s, 1H, CO-NH); 13C NMR (101 MHz, DMSO-d6): δ 40.87 (CH3-SO3),
57.18 (Ar- OCH3), 113.08 (Ar-CH-), 121.16 (Ar-CH-), 121.44 (Ar-C-), 124.58 (Ar-C-), 126.41
(Ar-CH-), 127.40 (Ar-CH-), 128.13 (Ar-CH-), 129.20 (Ar-CH-), 132.11 (Ar-CH-), 133.02 (Ar-
C-OSO2), 135.19 (-C-N), 140.53 (-C=N), 149.13 (Ar-C-OCH3), 151.95 (-C=O), 162.85 (=C-O-).
15

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4.3.17. 2-(3-methoxy-4-(prop-2-ynyloxy)phenyl)quinazolin-4(3H)-one (5q): White powder;
1
0.141g (yield: 62.70%); m.p. 209-211 °C; ESI-MS: m/z 307.10 [M+H]+ calcd 307.12; H NMR
(400 MHz, DMSO-d6): δ 3.59 (s, 1H, -CH), 3.86 (s, 3H, OCH3), 4.87 (s, 2H, OCH2), 7.14 - 8.10
(m, 7H), 12.44 (s, 1H, CO-NH); 13C NMR (101 MHz, DMSO-d6): δ 56.23 (Ar- OCH3), 79.45
(OCH2), 111.36 (≡CH), 113.67 (≡C-), 121.24 (Ar-CH-), 126.20 (Ar-C-), 126.37 (Ar-C-), 126.78
(Ar-CH-), 127.89 (-C-N), 135.35 (-C=N), 149.36 (Ar-C-OCH3), 152.26 (-C=O), 162.86 (=C-O-).
4.3.18. 2-(3-(4-fluorobenzyloxy)-4-methoxyphenyl)quinazolin-4(3H)-one (5r): White powder;
1
0.234g (yield: 84.00%); m.p. 282-285 °C; ESI-MS: m/z 377.10 [M+H]+ calcd 377.13; H NMR
(400 MHz, DMSO-d6): δ 3.87 (s, 3H, OCH3), 5.11 (s, 2H, OCH2), 7.08- 8.09 (m, 11H, Ar-H),
12.38 (s, 1H, CO-NH); 13C NMR (101 MHz, DMSO-d6): δ 56.15 (Ar- OCH3), 69.93 (OCH2),
112.07 (Ar-CH-), 116.16 (Ar-CH-), 122.54 (Ar-C-), 126.37 (Ar-C-), 128.06 (Ar-CH-), 130.89 (-
C-N), 134.92 (-C=N), 147.61 (Ar-C-OCH3), 148.89 (-C=O), 152.17 (=C-O-), 163.11 (Ar-C-F).
4.3.19. 2-(3-(2,4-dichlorobenzyloxy)-4-methoxyphenyl)quinazolin-4(3H)-one (5s): Cream
powder; 0.508g (yield: 85.00%; m.p.: 223-225 °C; ESI-MS: m/z 427.10 [M+H]+ calcd 427.06;
1H NMR (400 MHz, DMSO-d6): δ 3.85 (s, 3H, OCH3), 5.25 (2H, s, OCH2), 7.14-8.13 (m, 11H,
Ar-H), 12.42 (s, 1H, CO-NH); 13C NMR (101 MHz, DMSO-d6): δ 56.32 (Ar- OCH3), 67.74
(OCH2), 112.30 (Ar-CH-), 112.80 (Ar-CH-), 121.20 (Ar-C-), 122.44 (Ar-C-), 125.20 (Ar-CH-),
126.37 (Ar-CH-), 126.74 (Ar-CH-), 127.85 (Ar-CH-), 128.17 (Ar-CH-), 129.46 (Ar-CH-),
131.99 (Ar-C-Cl), 134.13 (Ar-C-Cl), 135.13 (-C-N), 147.68 (-C=N), 149.39 (Ar-C-OCH3),
152.17 (-C=O), 163.13 (=C-O-).
4.3.20. 2-(3-(2,4-difluorobenzyloxy)-4-methoxyphenyl)quinazolin-4(3H)-one
(5t):
White
1
powder; 0.205g (yield 76.7%); m.p. >300 °C; ESI-MS: m/z 395.10 [M+H]+ calcd 395.12; H
NMR (400 MHz, DMSO-d6): δ 3.82 (s, 3H, OCH3), 5.20 (s, 2H, OCH2), 7.12-8.35 (m, 11H, Ar-
16

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H), 12.53 (s, 1H, CO-NH); 13C NMR (101 MHz, DMSO-d6): δ 56.34 (Ar- OCH3), 64.12
(OCH2), 111.94 (Ar-CH-), 112.92 (Ar-CH-), 117.42 (Ar-CH-), 122.50 (Ar-C-), 126.14 (Ar-C-),
127.77 (Ar-CH-), 129.94 (Ar-C-F), 134.52 (Ar-C-F), 135.61 (-C-N), 143.24 (-C=N), 147.68 (Ar-
C-OCH3), 152.53 (-C=O), 161.73 (=C-O-).
4.4. Typical procedure for In-vitro CYP450 enzyme inhibition. SacchrosomesTM (human CYP
enzymes bound to yeast microsomal membranes; CYP Design Ltd) were used to determine the
percentage inhibition of a CYP450 by a compound or to determine its IC50 values using 96-well
flat-bottomed microtitre plates [25]. Both percentage inhibition and IC50 values effectively
reflect the inhibitory potential of a compound and possible effectiveness of a compound in a
biological process. Percentage inhibition was determined at 10 µM concentration. IC50 values
were determined using six different concentrations. The DMSO concentrations never exceeded
0.5%. The fluorogenic substrates used were 7-ethoxyresorufin (7-ER) or 3-cyano-7-
ethoxycoumarin
(CEC),
7-ethoxy-methyloxy-3-cyanocoumarin
(EOMCC)
or
dibenzylfluorescein (DBF), depending on the CYP450 used in the assay. Formation of
fluorescent compounds from fluorogenic substrates, upon CYP metabolism, was monitored using
a fluorescent plate reader [25].
4.5. Transfection of mammalian expression plasmids that encode human CYP1A1 &
CYP1B1 genes isolated from a human liver cDNA library in HEK293 cells grown in
suspension cells. Actively dividing HEK293 cells, adapted for growth in ‘suspension’, growing
in log phase were seeded in appropriate volumes in Erlenmeyer flasks (Corning #431143) and
incubated at 37°C, 8% CO2 and shaken at 130 rpm in an orbital shaker (Panasonic). Before
transfection,
the
mammalian
expression
plasmids
(pcDNA3.1/CYP1B1
and
pcDNA3.1/CYP1A1; the basic plasmid pcDNA3.1 was obtained from Invitrogen), encoding
17

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human CYP1A1 and CYP1B1 genes (isolated from a human liver cDNA library), were prepared
as endotoxin-free DNA (Zymo PURE™ Plasmid Maxiprep Kit). To initiate transfection, the
respective plasmid DNA-cationic lipid complexes, prepared aseptically as per manufacturer’s
instructions (Invitrogen #16447-100) in OptiPRO SFM reduced serum medium (Invitrogen
#12309-09), were added slowly to the respective flasks containing HEK293 suspension cells.
Suspension cells were incubated at 37°C and checked for optimal expression of CYP enzymes at
regular intervals, 24, 48, 72, 96 h post transfection. Transfected cells, after 48-96h time period,
were spun down in sufficient volumes at 200 x g for 5 min. Cell precipitates were washed with
pre-warmed phosphate buffered saline and the process repeated. Cells were then gently re-
suspended in pre-warmed growth media for screening of CYP inhibitors [25].
4.6. Screening of potential CYP inhibitors using recombinant human live cells. For
screening of potential compounds, recombinant HEK293 cells (100 - 200 x 103 cells per well),
expressing CYP enzymes, were seeded in 50 µL volume in triplicates in black 96-well plates
with transparent bottom (Corning #3904). The test compounds either at single point
concentrations (10 µM) or at various concentrations (from 1 nM to 30 µM) for determination of
IC50 values were added in 25 µL volume to the wells followed by incubation at 37°C, 8% CO2
for 30 min. After incubation, a fluorogenic substrate was added at 5 µM concentration in 25 µL
to the wells and contents were mixed by shaking. The plate was read on a plate-reader (Biotek,
Synergy HT) for 60 min using suitable wavelengths for emission/excitation of the fluorescence
products formed. IC50 values were calculated using Graph-Pad Prism Software (Version 6.0)
[25].
4.7. Growth of yeast strains for expression of CYP1B1 enzyme. Yeast strains (CYP Design
Ltd), harboring human CYP gene expression cassettes, were streaked out from a glycerol stock,
18

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kept at -80°C, and grown on SD-minimal medium agar plates with required supplements, at 30ºC
for 3 days. Single colonies were picked and grown, at first, as pre-cultures in minimal medium
and then grown in full YPD medium for expression of CYP enzymes [25].
4.8. Determination of IC50 values using recombinant yeast live cells. For IC50 determinations,
4×108 cells (equivalent to approximately 25 OD600) were taken from the exponential growth
phase, approximately 16-20 h after induction of a CYP protein. These cells, enough for 100
assays, were aliquoted appropriately into Eppendorf tubes. Cells were centrifuged on a bench top
microfuge for 30 sec at 13,000 rpm (15.7 g). Supernatants were removed carefully so as to not
dislodge the pellet. Cell pellets were then re-suspended in 650 µl of TE buffer (50 mM Tris-HCl
pH. 7.4, 1 mM EDTA). Cells were diluted 1:10 before carrying out an IC50 determination using a
protocol similar to the determination of IC50s using SacchrosomesTM. IC50 values were calculated
as described earlier using Graph-Pad Prism Software (Version 6.0) [25].
4.9. Molecular modelling. The human CYP family of enzymes are oxidoreductases involved in
the metabolism of xenobiotics, mainly hydroxylation of unreactive carbon atoms in aromatic and
aliphatic rings or aliphatic chains. The crystal structures of CYP enzymes were retrieved from
the protein data bank: CYP1A1 (PDB ID: 4I8V),[24] CYP1B1 (PDB ID:3PM0),[23] CYP1A2
(PDB ID:2HI4),[22] CYP3A4 (PDB ID: 4NY4)[46] and CYP2D6 (PDB ID: 4WNT),[47] The
structures were subjected to protein preparation wizard facility under default conditions
implemented in Maestro v9.0 and Impact program v5.5 (Schrodinger, Inc., New York, NY,
2009). The prepared protein was further utilized to construct grid file by selecting co-crystallized
ligand as centroid of grid box. For standardization of molecular docking procedure co-
crystallized ligands such as ANF (CYP1A1, CYP1B1 and CYP1A2), ajmalicine (CYP2D6) and
bromocriptine (CYP3A4) were extracted from prepared enzyme-ligand complexes and re-docked
19

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The rest of the chemical structures were sketched, minimized and docked using GLIDE XP. The
ligand-protein complexes were re-minimized using MacroModel. In order to determine
selectivity, the corresponding binding sites of CYP enzymes 1A1, 1A2, 2D6 and 3A4 were
aligned and analysed with respect to CYP1B1 [25].
Supporting information available. Spectral data scans. This material is available free of charge
via the internet at http://sciencedirect.com.
ACKNOWLEDGEMENTS
BC would like to thank the UK Higher Education Innovation Fund (HEIF) and a British Council-
UKIERI grant for support. MUMS acknowledge UGC for an award of JRF fellowship, and BNS
and VJ acknowledge DST-FIST (SR/FST/CSI-242/2012) for NMR facility at the Birla Institute
of Technology, Mesra.
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TABLE CAPTIONS:
Table 1.
Table 2.
Percentage in-vitro inhibition of CYP1A1, CYP1B1, CYP1A2, CYP3A4, CYP2D6,
CYP2C9 and CYP2C19 SacchrosomesTM, at 10 µM.
The IC50 values of the six compounds, with the highest percentage inhibition of
CYP1B1 (Table 1), in CYP1A1, CYP1B1, CYP1A2, CYP3A4, CYP2D6, CYP2C9
and CYP2C19 fluorescence assays, using SacchrosomesTM.
Table 3.
Table 4.
Inhibition of CYP1A1 and CYP1B1, within live yeast and human HEK293 cells, by
quinazolines. a
IC50 valuesa for inhibition of CYP1A1 and CYP1B1 by quinazolines in live yeast
cells and human cells.
FIGURE CAPTIONS:
Figure 1.
Figure 2.
Chemical structures of potent CYP1B1 inhibitors 1-4 and design of ‘quinazoline’ 5
scaffold as a prospective CYP1B1 inhibitor.
Molecular modelling studies (A) Interactions of 5c with CYP1B1. (B) Interactions
of 5j with CYP1B1. ( )
Scheme 1. Synthesis of quinazolines 5a-t. Reagents and conditions: (a) I2, Sodium acetate,
DMF 60-70°C, 24 h (b) K2CO3, acetone, room temperature, 12-24 h, yield 73-90%
(c) I2, Sodium acetate, DMF 60-70°C, 24 h.
26

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TABLES
Table 1. Percentage in-vitro inhibition of CYP1A1, CYP1B1, CYP1A2, CYP3A4, CYP2D6, CYP2C9 and
CYP2C19 SacchrosomesTM, at 10 µM.
% CYP inhibition in SacchrosomesTM at 10 µMa
Entry
1A1
61.6
3.6
1B1
56.4
71.5
93.3
47.3
61.0
82.1
84.8
91.1
94.5
89.3
14.4
76.1
59.2
62.2
0.2
1A2
22.1
30.6
40.7
12.8
14.5
42.0
55.8
36.4
63.8
85.6
0.5
2D6
8.8
7.6
10.9
7.9
6.5
19.8
15.6
9.0
9.5
15.5
5.6
6.8
7.2
4.5
3.8
5.9
7.8
8.4
9.1
3.5
3A4
2.2
3.8
43.3
0.5
0.8
33.6
6.1
0.2
2.6
38.5
0.6
1.5
1.9
2.3
4.1
3.2
2.7
1.8
0.9
5.2
2C9
2
2C19
3
25a
5b
5c
5d
5e
5f
0
4
52.8
77.6
62.2
71.9
81.9
8.5
0
0
0
0
0
0
3
2
0
4
5g
5h
5i
0
5
17.9
87.4
0.6
5
3
8
9
5j
2
0
5k
5l
6.3
2.1
1.5
3
0
21.1
51.0
0.2
0.5
0
5m
5n
5o
5p
5q
5r
5s
15.2
0.3
0
1
0
3
32.6
54.4
15.9
9.4
77.9
81.6
2.1
9.6
0
4
23.2
0.8
0
0
0
1
29.1
10.1
0
3
44.6
19.3
12.2
0
4
5t
a the percentage inhibition values are an average of two independent observations.
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Table 2. The IC50 values of the six compounds, with the highest percentage inhibition of CYP1B1 (Table 1), in
CYP1A1, CYP1B1, CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2C19 fluorescence assays, using
SacchrosomesTM
.
CYP inhibition in SacchrosomesTM (IC50 in µM)a
Entry
1A1
1B1
1A2
2D6
>10
>10
>10
>10
>10
>10
>10
3A4
>10
>10
>10
>10
>10
>10
>10
2C9
>50
>50
>50
>50
>50
>50
>10
2C19
>50
>50
>50
>50
>50
>50
>10
8.2±0.05
0.003±0.0002
0.054±0.002
3.1 ±0.025
>10
5c
5f
4.4 ±0.03
7.4 ±0.06
405 ±1.3
>10
10
5g
0.002 ±0.0001
0.004 ±0.0002
0.268 ±0.02
0.05 ± 0.01
10
5h
5i
14.3 ±0.2
0.477 ±0.06
0.01± 0.002
10
5
5j
0.03 ± 0.01
ANF
aα-naphthoflavone (ANF) was used as a control in these studies; The IC50 values represent mean and standard
deviations from three independent experiments.
28