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Truncated Itraconazole Analogues Exhibiting Potent AntiHedgehog Activity and Improved Drug-Like Properties Jiachen Wen, Divya Chennamadhavuni, Shana R. Morel, and M. Kyle Hadden ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00188 • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 25, 2019
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Truncated Itraconazole Analogues Exhibiting Potent AntiHedgehog Activity and Improved Drug-Like Properties Jiachen Wen, Divya Chennamadhavuni, Shana R. Morel, M. Kyle Hadden* Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Rd, Unit 3092, Storrs, CT 060293092, United States KEYWORDS. Hedgehog pathway, Itraconazole, Structure–activity relationship, Metabolite, Druggability. ABSTRACT: We conducted a structure–activity relationship study to explore simplified analogues of the itraconazole (ITZ) scaffold for their ability to inhibit the hedgehog (Hh) signaling pathway. These analogues were based on exploring the effects of chemical modifications to the linker and triazolone/side chain region of ITZ. Analogue 11 was identified as the most potent compound in our first generation, with an IC50 value of 81 nM in a murine Hh-dependent basal cell carcinoma. Metabolic identification studies led us to identify the truncated piperazine (26) as the major metabolite in human liver microsomes (HLMs) and an improved Hh pathway inhibitor (IC50 = 22 nM). This work verifies that continued truncation of the ITZ scaffold is a practical method to maintain potent anti-Hh activity, while also reducing the molecular weight for the ITZ scaffold and achieving improved pharmacokinetic properties.
The Hedgehog (Hh) pathway is a signaling cascade critical for cell development and tissue patterning.1 Inappropriate modulation of Hh signaling has been implicated in developmental disorders and cancers including, basal cell carcinoma (BCC), medulloblastoma (MB), and leukemia.2−5 The approval of vismodegib and sonidegib as treatments for advanced BCC confirmed the clinical efficacy of small molecule pathway inhibitors for the treatment of Hh-dependent cancers.6-9 Of continued concern in the development of Hh pathway inhibitors is the identification of multiple mutations in Smoothened (Smo), the molecular target of vismodegib and sonidegib, which reduce binding affinity for the drugs and decrease overall efficacy.10−12
significantly more active than the comparable 4S analogues.14 We also Diphenylpiperazine Tether Triazolone/Side Chain
Dioxolane Region N N
N
O 2
O Cl
O 4
O
Cl
H3 C
Cl
N
N N
N
1, ITZ, IC50 = 140 nM, M.W. = 705.6
O
O
2S 4R
O
O
N
N
OH
N H
Cl
2, IC50 = 160 nM, M.W. = 634.6
H3 C
O O
Cl
O O
N
N
R
Current Work
Recent years have seen the identification and exploration of itraconazole (1, ITZ, Figure 1), an FDA approved antifungal agent, as an Hh pathway inhibitor.13−15 Strong evidence exists to suggest that ITZ binds and directly inhibits Smo, a key component of the Hh pathway;13 however, ITZ retains potent Hh inhibitory activity in vitro and in vivo in the presence of vismodegib-resistant Smo mutants.16,17 With this in mind, the ITZ scaffold represents a promising anti-cancer strategy that has the possibility to address the acquired resistance after treatment associated with the clinically approved Smo antagonists. As shown in Figure 1, the structure of ITZ can be separated into three general regions, the dioxolane region, the central diphenylpiperazine core, and the triazolone/side chain region. Our research group has been exploring structure-activity relationships (SARs) for the ITZ scaffold with respect to its anti-Hh activity.14,15 In these previous studies, we demonstrated that the stereochemistry around the dioxolane is critical for potent Hh inhibition. Analogues with the 4R orientation are
N
Previous Work
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Medicinal Chemistry Letters
X
Cl
3-20
Figure 1. Structure and regions of ITZ, lead analogue 2, and analogues made in current work.
demonstrated that the triazole ring is not required for ITZmediated Hh inhibition.14 Our most recent SAR suggests that modification in the triazolone/side chain area is well-tolerated. Potent compounds have been discovered through side chain group modification,14,18 (hetero)cycle replacement,15 and even direct truncation of the triazolone ring.14 In the current study, the simplified triazolone analogue 2, which retained the potent
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corresponding aniline 9 by palladium on charcoal. For analogues bearing an allyl group at the terminal phenol, free phenols were protected as allyloxy before coupling with 24. By treating with sodium borohydride and iodine, the allyl group was removed under a translocation mechanism, to give phenols 3, 4, 10−14, and 17.19 Annotated structures for the final analogues are listed in Table 1. For the analogues bearing a hydrazide, intermediate 21 was first coupled with phenyl chloroformate under alkaline conditions. The resulting amide 23 was coupled with dioxolane 22, followed by refluxing with hydrazine hydrate in 1,4-dioxane to afford hydrazide 24. The hydrazide terminal was later coupled with appropriate benzaldehydes to give final phenols 18 and 19. When 23 coupled with dioxolane in 2R,4R-trans configuration, followed by the same hydrazinolysis and condensation procedures, hydrazide 20 was synthesized (Structure in Table 1, complete syntheses are shown in the Supporting Information).
anti-Hh activity and a decreased molecular weight compared to ITZ, was used as our lead structure. We sought to begin probing SAR for the tether region adjacent to the triazolone by directly appending the aromatic functional group to the central piperazine through an amide bond. These modifications would also result in analogues with reduced molecular weight. Following our previously reported procedures, 1-(4hydroxyphenyl)piperazine 21 and the stereochemically defined dioxolane regions 22 (2S,4R-cis) and its enantiomer (2R,4Rtrans) were synthesized.14 As shown in Scheme 1, intermediate 21 was coupled with an appropriate carboxylic acid in the presence of the condensing agent 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDCI) and 4dimethylaminopyridine (DMAP) at room temperature. Selective addition to the piperazine secondary amine was observed for the majority of our analogues. Amide intermediates were then coupled with dioxolane 22 (2S,4R-cis) under alkaline conditions to afford analogues 5−8, 15 and 16. The analogue bearing a para-nitro (8) was reduced to the Scheme 1. General Synthesis of Amides and Hydrazidesa
O
O HO
N
e
NH
HO
N
OPh
N
25, b
f
O
O Cl
O
NH2
g O
O N
N H
24
a
HO
N
Cl
23
21
N
R n
N
O Cl
O O
N
N
Cl
Amide intermediates
N H
OH
N
18, 19
O O Cl
n = null, -CH2-, -CH2-CH2-, -CH=CH-, -CH2-CH2-CH2-
OTs b, c or d
Cl 22
NO2 R=
O O Cl
R= OCH3
O O
N
N
R n
R=
c
NH2 R=
R=
Allyloxy
R=
OH
d
Cl
3 - 17 aReagents
and conditions: (a) Appropriate carboxylic acid, EDCI, DMAP, TEA, DMF, RT, overnight, 31−89%; (b) Cs2CO3, DMSO, 60oC, 4 h, 51−93%; (c) Pd/C, hydrazine hydrate, EtOH, reflux, 4 h, 91%; (d) NaBH4, I2, THF, RT, 2 h, 31−62%; (e) Phenyl chloroformate, K2CO3, DCM, 0oC to RT, 4 h, 62%; (f) Hydrazine hydrate, 1,4-diox, reflux, 12 h, 89−91%; (g) Appropriate benzaldehyde, EtOH, reflux, 4 h, 62−92%.
As noted above, our previous SAR studies for the ITZ scaffold demonstrated that the triazolone/side chain region of ITZ is amenable to modification. In order to continue our SAR exploration of this region of the scaffold, we sought to synthesize and evaluate analogues that continue the scaffold truncation that we previously probed by removing the phenyl ring on the ‘right side’ of the scaffold and directly appending the 3-phenolic carboxylic acid of 2 to the piperazine amine to provide analogue 3. We evaluated this analogue for its ability to decrease Gli1 mRNA expression in the Hh-dependent murine BCC cell line ASZ001.20-22 Analogue 3 demonstrated comparable anti-Hh activity compared to 2 (IC50 values = 0.26 µM and 0.16 µM, respectively) verifying that this series of compounds retains potent Hh inhibition and encouraging us to synthesize additional analogues that incorporate the 4phenylpiperazinyl amide.
To further probe SAR for functionality on the aromatic moiety, aromatic carboxylic acids with a range of substitutions 4−9 were synthesized and evaluated. Analogue 4 (IC50 = 0.58 μM), with the phenol in the para-position, was less active than 3. Replacing the phenol with pyridines resulted in compounds 5−7 with reduced anti-Hh activity, with the 4-pyridine (7, IC50 = 0.57 μM) representing the best in this small collection. Replacing the phenol with a nitro group (8, IC50 = 0.16 μM) demonstrated improved activity compared to 3 and equivalent activity to the lead 2. Reduction of the nitro to the 4-aniline (9, IC50 = 0.28 μM) resulted in a modest decrease in activity. Next, we sought to increase the distance between the amide bond and the aromatic moiety to understand the optimal distance between these functional groups for potent Hh inhibition (10−20). The addition of a single methylene unit (10) resulted in a slight decrease in Hh inhibition compared to 2; however, the increase to two methylenes (11, IC50 = 0.081 µM)
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ACS Medicinal Chemistry Letters aIC
50 values represent the Mean ± SEM of at least two separate experiments performed in triplicate.
significantly enhanced the anti-Hh activity of the scaffold. Constraining the linker as a trans-alkene 12 resulted in an approximately 3-fold loss in Hh inhibitory activity, a trend that was also present when the phenol was located in the 4-position (13 and 14). Deletion of the phenol (15, IC50 = 0.23 μM) or masking it as the methoxy (16, IC50 = 0.46 μM) decreased activity, suggesting the free phenol is not essential for anti-Hh activity, but it is optimal in our scaffold. Due to the commercial availability of 3-hydroxybenzenebutanoic acid, analogue 17 with a propyl linker, but a para-phenol tail was synthesized and evaluated. A decrease in activity was observed with the extended linker in 17 (IC50 = 0.47 µM). Table 1. Anti-Hh Activity for ITZ and its Analoguesa H3C
O 2S
O Cl
Cl
H3C 4R
O
N
Cl
3 - 19
Cmpd ITZ 2 3
O 2R
O
N R
cis
Finally, we sought to vary the chemical composition of the linker by introducing heteroatoms; therefore, a small collection of hydrazide-containing analogues was synthesized and evaluated. Analogue 18 (IC50 = 0.12 μM) demonstrated superior Hh inhibitory activity to 2 and ITZ, once again suggesting this region of the scaffold is amenable to modification. Not surprisingly, analogue 19 (IC50 = 0.96 μM), which incorporates the para-phenol, was less active compared to the analogous meta-phenol 18. Our previous truncated ITZ analogue series identified the cis-dioxolane as the ideal orientation for these compounds; however, we also synthesized and evaluated the 2R,4R-trans-dioxolane hydrazide to determine whether this trend is replicated for the analogues described herein. This analogue, 20 (IC50 = 0.58 μM), was approximately 5-fold less active, highlighting the importance of the cis-orientation around the dioxolane ring for this series of compounds. Table 2. In Vitro Pharmacokinetic Dataa
Cl
4R
N
O
N R
trans
20
R -----
ASZ001, IC50 (μM) 0.14 ± 0.02 0.16 ± 0.03
Solubility, PBS (µM)
0.26 ± 0.08
Cmpd
4
0.58 ± 0.02
5
1.42 ± 0.30
6
1.30 ± 0.31
ITZb 2b 3 10 11 19
7
0.57 ± 0.11
8
0.16 ± 0.02
9
0.28 ± 0.11
10
0.38 ± 0.04
11
0.081 ± 0.022
12
0.22 ± 0.03
13
0.26 ± 0.03
14
0.36 ± 0.05
15
0.23 ± 0.16
16
0.46 ± 0.03
17
0.47 ± 0.11
18
0.12 ± 0.01
19
0.96 ± 0.15
20
0.58 ± 0.07
pH 4.0
pH 7.4
----0.31 0.25 0.13 0.05
0.80 0.02 0.17 0.13 0.03 0.02
Microsomal Stability Clint T1/2 (min) (µl/min/mg) 27.0 ± 6.2 25.2 ± 3.1 19.9 ± 0.6 34.7 ± 1.0 15.9 ± 0.2 43 ± 0.6 20.4 ± 0.4 33.9 ± 0.6 3.2 ± 0.1 218 ± 9.7 11.4 ± 0.2 60.0 ± 1.1
aMore
information about the assay protocols are shown in the Supporting Information. bValues for ITZ and 2 are taken from reference 15.
In addition to determining whether significant truncation to the triazolone/side chain region results in compounds that retain potent Hh inhibition, a key reason for the preparation of these analogues was to evaluate whether truncation also provides compounds with improved solubility and/or stability. Several representative compounds (3, 10, 11, and 15) were chosen to evaluate these two parameters for this series of analogues. Unfortunately, all tested analogues failed to improve the overall solubility as each analogue was less soluble than ITZ at both pH 4.0 and pH 7.4 (Table 2). Overall, our analogues were less stable than ITZ in human liver microsomes (HLMs) and demonstrated predicted intrinsic clearance levels significantly higher than the parent compound. Based on the reduced stability previously determined for amide-containing analogue 2, it was not too surprising that these compounds were also less stable. Disappointingly, our most active analogue 11, was also the least stable (T1/2 = 3.2 mins). The only structural difference between 11 and 10, which was approximately 6.5-fold more stable, is the addition of a single methylene between the amide and the phenol-substituted benzene ring. In order to more fully determine how the additional methylene influences stability, we performed follow-up metabolic identification (MetID) studies in HLMs for both 10 and 11. Incubation of 11 with HLMs resulted in the identification of two primary metabolites (Figure 2 and Figure S1−S4). Peak #1 indicated the metabolite (26, 30.09%) had undergone hydrolysis of the amide bond, an unexpected metabolite considering amides are generally deemed as a reliable tether and exist in many marketed drugs. The second major metabolite (Peak #2, 15.94%) resulted from single oxidation to the 3hydroxyphenethyl region. While the exact location of the hydroxyl/phenol in metabolite #2 could not be conclusively
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determined; CYP-mediated oxidation at the para-phenyl position is well-documented.23,24 Based on the measured m/z+ peaks, we presumed that 3,4-bis-phenol 28 is the most likely structure for metabolite #2. For analogue 10, our MetID studies suggested this analogue was involved in more complicated metabolic pathways, including hydrolysis, hydrogenation, de-oxidation, and then oxidation (Figure S5−S10). Interestingly, no direct oxidation to the phenol region was observed in the metabolites of 10. The difference in metabolic pathways for 10 and 11 may explain the difference in HLM stability. Of note, 26 was the primary metabolite for both 10 and 11, indicating the N-piperazine amide bond was a clear metabolic soft spot on our amide ITZ analogues.
Based on the results of the metabolism studies, our follow-up experiments were focused in two directions. First, we synthesized and evaluated the metabolites (26 and 28) of our most active analogue 11 to determine if they retain potent antiHh activity. In parallel, we sought to enhance the stability of 11 by eliminating the common metabolic soft spot, which led to the design of saturated analogue 31. The syntheses are described in Scheme 2. For the analogue truncated at the piperazine, selective protection of 21 as the secondary Boc amine afforded phenol 25. After coupling with dioxolane 22, Boc removal was performed by stirring with TMSOTf under alkaline conditions to give the free piperazine 26. Starting with 3,4-bis-allyloxybenzenepropanoic acid and using the same procedures as in Scheme 1, bis-phenol 28 was synthesized. For the des-amide analogue, intermediate 29 was synthesized after reduction and tosylation. Selective addition to the piperazine amine of 21 was observed by heating in the presence of 29 to give phenol 30. Dioxolane coupling and final deprotection were performed under the same conditions as described earlier to give amine 31. We were excited to determine that both metabolites retain potent inhibition of Hh signaling (Table 3). Analogue 28 (IC50 = 0.089 µM), which incorporates the bis-phenol, showed equivalent activity to the parent 11; whereas the truncated piperazine 26 (IC50 = 0.022 µM) was more potent than 11. Metabolite 23 was significantly less active than 11 or the other two metabolites (IC50 = 0.87 µM). In C3H10T1/2 cells, an Hhdependent mouse embryonic fibroblasts (MEFs), 11 and 26 were also more potent than ITZ when using endogenous oxysterols as the Hh pathway activator (Table S1). Based on the improved activity of the truncated analogue 26, which contains approximately half of the intact ITZ scaffold, we also evaluated this analogue in Sufu-/- mouse embryonic fibroblasts (MEFs) to determine whether Smo is still the most likely target of the metabolite. The anti-Hh activity of 26 was completely abolished in this cell line, suggesting 26 (and ITZ) is functioning upstream of Sufu, most likely at the level of Smo (Figure S11).
O O Cl
N
O
NH
Cl
Peak #1, 30.09% Hydrolysis O O Cl
O O
N
OH
N
Cl
11, 53.97% Oxidation
O O Cl
O O
N
N
OH O
Cl
Peak #2, 15.94%
Figure 2. Metabolite identification for 11 in the presence of HLMs. Percentages indicate the relative peak area under experimental conditions.
Scheme 2. Synthesis of Metabolites 26, 28 and Amine 31a
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ACS Medicinal Chemistry Letters O
O HO
N
b, e
Allyloxy
N
Cl
Allyloxy
27
O
O
O
N
OH
N
Cl
OH
28
d O
HO
N
NH
a
HO
N
21
b, c
N Boc
O Cl
25
O
N
NH
Cl
26
Allyloxy
TsO
f 29
HO
N
O
b, e
Allyloxy
N
O Cl
O
N
N
OH
Cl
30
31
aReagents and conditions: (a) Di-tert-butyl dicarbonate, TEA, DCM, RT, 2 h, 93%; (b) 22, Cs CO , DMSO, 60oC, 4 h, 39−98%; (c) 2 3 TMSOTf, 2,6-lutidine, DCM, 0oC to RT, overnight, 72%; (d) 3,4-Bis-allyloxybenzenepropanoic acid, EDCI, DMAP, TEA, DMF, RT, overnight, 81%; (e) NaBH4, I2, THF, RT, 2 h, 41−71%; (f) 29, K2CO3, DMF, 40oC, 4 h, 41%.
Table 3. Anti-Hh activitya, and Pharmacokinetic Datab for Metabolite-based ITZ Analogues O
H3 C
O Cl
O
N
N R
Cl
26, 28, and 31
Solubility, PBS (µM) pH 4.0
pH 7.4
0.022 ± 0.011
169.3
11.7
Microsomal Stability Clint T1/2 (min) (µl/min/mg) 50.7 ± 1.5 13.7 ± 0.4
28
0.089 ± 0.06
---
0.84
6.6 ± 0.1
104.7 ± 0.1
31
0.87 ± 0.6
0.13
0.02
14.0 ± 0.2
50.0 ± 0.8
Cmpd
R
ASZ001, IC50 (μM)
26
-H
aIC
50 values represent the Mean ± SEM of at bMore information about the assay protocols
least two separate experiments performed in triplicate. are shown in the Supporting Information.
The activity for both metabolites prompted us to study their PK profile (Table 3). Not surprisingly, analogue 26 demonstrated improved stability (T1/2 = 50.7 mins) compared to parent compound 11. The stabilities of compound 28 and 31 (T1/2 = 6.6 mins and 14.0 mins, respectively) were minimally improved compared to 11. In addition, significant improvements in solubility at pH 7.4 and pH 4.0 compared to ITZ were found for analogue 26. Finally, we evaluated the general toxicity of compounds 11 and 26 by exploring their anti-proliferative activity in HC-04 cells, a human hepatocytederived cell line that abundantly expresses drug metabolizing enzymes (Table S2).25 Both compounds demonstrated moderate toxicity (26, GI50 = 27.3 µM; 11, GI50 = 13.18 µM) in the HC04 cells, indicating their anti-Hh activity is not a result of broad toxicity and further highlighting their potential as selective anticancer therapeutics.
In conclusion, we have developed a series of ITZ analogues focused on continued truncation of the ‘right-side’ of the scaffold. Analogue 11 represents the most potent analogue in the first-round SAR study, with an IC50 value of 81 nM, approximately two-fold more potent than lead 2 and ITZ. Our initial SAR suggested a proper length linker with certain flexibility is critical for potent anti-Hh activity. In addition, a stereochemically defined dioxolane cap (2S,4R-cis) and a paraphenol tail are also important for the anti-Hh activity of 11. A metabolite of compound 11, analogue 26, demonstrated improved Hh pathway inhibition, increased stability and enhanced water solubility. Moreover, the molecular weight of 26 (MW = 423.3) is significantly decreased compared to ITZ (MW = 705.6) establishing analogue 26 as a promising candidate for further in vitro and in vivo analysis. Overall, these studies provide clear rationale for continued modification to the central core of the scaffold as a practical design strategy to
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retain potent anti-Hh activity while improving the overall druglike properties of the scaffold.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed MetID results, general experimental methods, detailed synthetic procedures, and compound characterization (1H, 13C NMR and MS). (PDF)
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected]. Phone: 1-860-486-8446. Fax: 1-860-486-6857.
ORCID M. Kyle Hadden: 0000-0001-9482-1712
Author Contributions J. W. synthesized and evaluated ITZ analogues and wrote the manuscript. D. C. synthesized ITZ analogues. S. R. M. evaluated ITZ analogues. All authors have given approval to the final version of the manuscript.
Funding Sources We gratefully acknowledge support of this work by the National Institutes of Health/National Cancer Institute (CA190617).
ACKNOWLEDGMENT The authors kindly thank Dr. Ervin Epstein (Children’s Hospital Oakland Research Institute) for providing the ASZ001 cells. HC04 cells were provided by Dr. José Manautou (University of Connecticut).
ABBREVIATIONS ITZ, Itraconazole; Hh, Hedgehog; BCC, basal cell carcinoma; MB, medulloblastoma; Gli, glioblastoma associated oncogene; Smo, Smoothened; SAR, structure–activity relationship; EDCI, 1-ethyl3-(3-dimethylaminopropyl)carbodiimide; DMAP, 4dimethylaminopyridine; Boc, tert-butyloxycarboryl; TMSOTf, trimethylsilyl trifluoromethanesulfonate; HLMs, human liver microsomes; MEFs, mouse embryonic fibroblasts; MetID, metabolic identification.
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ACS Medicinal Chemistry Letters (20) DeBerardinis, A. M.; Madden, D. J.; Banerjee, U.; Sail, V.; Raccuia, D. S.; De Carlo, D.; Lemieux, S. M.; Meares, A.; Hadden, M. K. Structure-activity relationships for vitamin D3-based aromatic Aring analogues as hedgehog pathway inhibitors. J. Med. Chem. 2014, 57, 3724−3736. (21) Wen, J.; Hadden, M. K. Structure–activity relationship studies of vitamin D3 analogues containing an ether or thioether linker as Hedgehog pathway inhibitors. ChemMedChem 2018, 13, 748−753. (22) Teske, K. A.; Dash, R. C.; Morel, S. R.; Chau, L. Q.; WechslerReya, R. J.; Hadden, M. K. Development of posaconazole-based analogues as hedgehog signaling pathway inhibitors. Euro. J. Med. Chem. 2019, 163, 320−332. (23) Bathelt, C. M.; Ridder, L.; Mulholland, A. J.; Harvey, J. N. Mechanism and structure–reactivity relationships for aromatic
hydroxylation by cytochrome P450. Org. Biomol. Chem. 2004, 20, 2998−3005. (24) Bathelt, C. M.; Ridder, L.; Mulholland, A. J.; Harvey, J. N. Aromatic hydroxylation by cytochrome P450: model calculations of mechanism and substituent effects. J. Am. Chem. Soc. 2003, 49, 15004−15005. (25) Lim, P. L.; Tan, W.; Latchoumycandane, C.; Mok, W. C.; Khoo, Y. M.; Lee, H. S.; Sattabongkot, J.; Beerheide, W.; Lim, S. G.; Tan, T. M.; Boelsterli, U. A. Molecular and functional characterization of drugmetabolizing enzymes and transporter expression in the novel spontaneously immortalized human hepatocyte line HC-04. Toxicol. In Vitro 2007, 21, 1390−1401.
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