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Structure-Activity Relationships for Itraconazole-Based Triazolone Analogues as Hedgehog Pathway Inhibitors Jennifer Pace, Kelly A. Teske, Lianne Chau, Radha Charan Dash, Angela M. Zaino, Robert Wechsler-Reya, and M. Kyle Hadden J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01283 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019
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Journal of Medicinal Chemistry
Structure-Activity Relationships for Itraconazole-Based Triazolone Analogues as Hedgehog Pathway Inhibitors Jennifer R. Pace1†, Kelly A. Teske1‡, Lianne Q. Chau2, Radha Charan Dash1, Angela M. Zaino1, Robert J. Wechsler-Reya2, and M. Kyle Hadden1
1 Department
of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, CT 06269, United States
2 Tumor
Initiation and Maintenance Program, NCI-Designated Cancer Center, Sanford Burnham
Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla CA 92037, United States
*To whom correspondence should be addressed.
[email protected] Phone: 1-860-4868446. Fax: 1-860-486-6857.
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ABSTRACT The FDA-approved antifungal agent, itraconazole (ITZ), has been increasingly studied for its novel biological properties. In particular, ITZ inhibits the hedgehog (Hh) signaling pathway and has the potential to serve as an anti-cancer chemotherapeutic against several Hh-dependent malignancies. We have extended our studies on ITZ analogues as Hh pathway inhibitors through the design, synthesis, and evaluate of novel des-triazole ITZ analogues that incorporate modifications to the triazolone/side chain region of the scaffold. Our overall results suggest that the triazolone/side chain region can be replaced with various functionality (hydrazine carboxamides and meta-substituted amides) resulting in improved potency when compared to ITZ. Our studies also indicate that the stereochemical orientation of the dioxolane ring is important for both potent Hh-pathway inhibition and compound stability. Finally, our studies suggest that the ITZ scaffold can be successfully modified in terms of functionality and stereochemistry to further improve its anti-Hh potency and physicochemical properties.
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INTRODUCTION Itraconazole (ITZ) is an FDA-approved antifungal agent that has several additional biological activities, including anti-cancer and anti-viral properties.1–3 With respect to its anticancer properties, ITZ inhibits the hedgehog (Hh) pathway, an embryonic cell-signaling cascade responsible for cell proliferation, differentiation, and tissue growth.1 Aberrant Hh pathway activation has been identified in a variety of cancers; most notably, basal cell carcinoma (BCC) and medulloblastoma (MB).4 Previous studies suggest that ITZ inhibits Hh signaling through direct binding interactions with Smoothened (Smo), a key regulatory protein within the Hh pathway. These studies also suggest that ITZ binds Smo in a distinct manner from other Hh pathway inhibitors that function through Smo antagonism.1,5,6,7 A common pitfall for Smo antagonists has been the emergence of resistance due to mutations in the binding site on Smo;8,9 however, ITZ maintains potent Hh pathway inhibition in the presence of both wild type and mutant forms of Smo, presumably through its distinct binding interactions. N N
N
O
2 4
O
Cl
Linker Region
O O
Cl
N
N
N
1, ITZ IC50 = 140 nM
Dioxolane Region
Triazolone/ Side Chain Region
O O Cl
Cl
N 2' N
O O
N
N
N
N N
2a, cis,des-triazole ITZ IC50 = 24 nM 2b, trans,des-triazole ITZ IC50 = 22 nM
Chart 1. Initial SAR for the ITZ scaffold. ITZ consists of a diverse scaffold that is amenable to modification. Previously, we performed structure activity relationship (SAR) studies for ITZ, which indicated the optimal functionality and stereochemistry for potent Hh pathway inhibition (Chart 1).10 These SAR studies
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resulted in a novel class of Hh pathway inhibitors: des-triazole ITZ analogues. In addition to removing the triazole moiety, we identified that the stereochemistry around the dioxolane (4R) is important for potent Hh pathway inhibition. Our studies also demonstrated that the absolute stereochemistry of the side chain is less important; however, the methyl of the sec-butyl region is crucial for maintaining potent Hh pathway inhibition. In addition, several analogues that truncated or completely removed the triazolone and side chain retained potent Hh pathway inhibition.9 Herein, we present our continued SAR for the des-triazole ITZ scaffold. These studies focus on the synthesis and evaluation of analogues containing a wide range of structural modifications to the triazolone/side chain region of lead ITZ analogues 2a and 2b. Our goals with these analogues were multi-fold. In addition to exploring SAR for this region of the scaffold, we sought to reduce the number of synthetic steps necessary to generate potent analogues and by extension reduced the molecular weight of the scaffold. We also sought to evaluate key early stage pharmacokinetic (PK) parameters for our analogues to ensure that future generations of des-triazole ITZ analogues were developed with both potency and drug-like properties in mind.
RESULTS Chemistry The preparation of des-triazole ITZ analogues containing a modified triazolone region starts with direct coupling of the desired dioxolane (3 or 4) with the unprotected linker region (5) to provide key intermediates 6 or 7.11-13 The dioxolane-containing nitro was reduced to the aniline in the presence of 10% Pd/C and hydrazine monohydrate (Scheme 1).12,13 These key aniline intermediates (8 and 9) can be utilized to provide the most efficient synthetic route towards a diverse set of ITZ analogues that contain modifications to the triazolone/side chain region.
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The majority of des-triazole analogues synthesized and evaluated in this study simplified the ITZ scaffold by replacing the triazolone and side chain with a substituted aromatic moiety directly appended to the aniline of 8 or 9. Synthesis towards these amide analogues (10-19) utilized standard
amidation
conditions:
dimethylaminopropyl)carbodiimide
aniline
(8
or
hydrochloride
9),
activating
(EDCI·HCl),
agent
1-ethyl-3-(3-
4-dimethylaminopyridine
(DMAP), and various carboxylic acids stirred in DCM at room temperature for 12 hours (Scheme 2).14 While the yields obtained with this procedure were less than ideal (< 50%) further optimization was not undertaken.
Scheme 1. Synthesis of Aniline Intermediatesa O O Cl
Cl
OTs
+ HO
N
3, trans 4, cis
N
NO2
a
5
O
O
O Cl
N
O
N
NO2
Cl
b
O Cl
N
O
N
NH2
Cl
6, trans 7, cis
8, trans 9, cis
aReagents
and conditions: (a) Cs2CO3 (10 eq), DMSO, 90 °C, 12 h, 34-85%; (b) 10% Pd/C, hydrazine monohydrate, EtOH, reflux, 3.5 h, 42%-94%.
Scheme 2. Initial Synthesis of des-Triazole Amide Analoguesa O
8 or 9
O
+ HO
aReagents
a R
O Cl
Cl
O O
N
N
N H
R
10-19 see Table 1 for structrues
and conditions: (a) EDCI, DMAP, DCM, RT, 12 h, < 50%.
The synthetic route described in Scheme 2 worked well to generate all the amidecontaining analogues with the exception of those incorporating a phenol-substituted phenyl ring.
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Under these standard conditions, the amide couplings with the phenol carboxylic acids did not proceed, presumably because the free phenol becomes activated allowing for multiple side reactions to occur. Synthesis of these analogues was achieved by protecting the free phenols of the corresponding aromatic esters with a benzyl ether moiety (Scheme 3a). Ideally, the benzyl (Bn) group could be removed under mild conditions (Pd/C and H2) that would not affect the intact dioxolane.14 This synthetic approach provided the desired phenol-substituted analogues; however, these compounds, except for compound 30, were obtained in very low yields after the final hydrogenation reaction (< 20%). Scheme 3. Couplings Procedures for Phenols and Second Generation ITZ Amidesa (A)
O
O
8 or 9
+
a
OH
BnO
O
O Cl
O
N
N
N H
Cl
OBn
20-25 O b
O
O Cl
O
N
N
Cl
N H
OH
26-31 See Table 1 for Structures
(B)
O
8 or 9
O
+ HO
c R
O Cl
Cl
O O
N
N
N H
R
26-36 see Table 1 for structures
aReagents
and conditions: (a) EDCI, DMAP, DCM, RT, 12 h, < 50%; (b) 10% Pd/C, THF:EtOH, H2, < 20%; (c) HATU, NMM, DMF, RT, 12 h, 8-31%.
In an attempt to obtain higher yields for the analogues incorporating a phenol, we utilized N-methylmorpholine
(NMM)
and
1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5,6]pyridinium3-oxidhexafluorophosphate (HATU) in dimethyl formamide (Scheme 3b).15,16 While obtaining higher yields was unsuccessful, these conditions did allow for an efficient direct coupling of the carboxylic acid of the free phenol to the aniline intermediates (8 or 9) to
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yield our final analogues (26-31). With the increased efficiency of these conditions (NMM/HATU), they were also utilized for several subsequent second generation des-triazole ITZ amide analogues (34-36) in combination with our original amide coupling conditions (EDCI/DMAP). Table 1. First Generation des-Triazole ITZ Amide Analogues. N
R1 O
Cmpd
N
R2
R1
R2
Cmpd
R1
O 2
6
O Cl
7
2
16
O
Cl
O Cl
17
Cl
O
O
Cl
18
O
O Cl
O O Cl
4
2
O
O
Cl
4
Cl 2
11
O Cl
4
O
Cl
12
O Cl
Cl
4
O
Cl 2
O Cl
O
Cl
14
O Cl
4
O
Cl 2 4
15
O
Cl
Cl
O
N H
OH
N H O
4
O
Cl
O
N H OH
N
O
Cl O O
Cl
O O
Cl O O
Cl
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Cl
OH
N H O
2 4
31
OH
N H
O
2 4
30 O
O
O
2 4
29 Cl
N H
O
O
N
N OH
N H
O
Cl O
O
O 2
28 Cl
N H
4
Cl O
2
O 4
O
O
N
N H
N H
O
O
O
N O
4
2 4
27
O
13
Cl
N H
O 2
O
O
O
N H
O
Cl O 2
26
N H
O
O Cl
O 4
Cl O 2
19
O
Cl O
10
Cl 2
N
N H
O
O 4
Cl 2
9
O
O 2 4
O
N H
O
Cl
O
O
Cl
4
2 4
2
8
O
O 4
O
Cl
R2
O
N H OH
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Des-triazole ITZ analogues containing carbamate, hydrazine carboxamide, or an unsubstituted triazolone were synthesized in a similar manner as previously described for the synthesis of ITZ and other analogues (Scheme 4).11,12 The carbamates 37 and 38 were generated through the addition of phenyl chloroformate to anilines 8 and 9, respectively. Addition of hydrazine monohydrate to the carbamates provided the hydrazine carboxamides 39 and 40. Finally, the intact triazolones were generated following the addition of formamidine acetate to yield 41 and 42.11,12
Table 2. Second Generation des-ITZ Amide Analogues. N
R1 O
N
Compound
R2
R1
R2
Compound
R1
O 2
32
4
2
35
O
Cl
Cl
2
4
Cl
2
Cl
2
4
O
Cl O
34
Cl O
36
O
4
O
Cl O
33
R2
O
Cl
4
O
Cl
Cl
Scheme 4. Synthesis of des-Triazole Triazolone Precursorsa O
8 or 9
a Cl
Cl
Cl
O
Cl
O O
O
O
N 39, trans 40, cis
N
N H
N
N H
b
O
37, trans 38, cis O
O O
N
NHNH2
c
O Cl
aReagents
Cl
O O
N
N
N
NH N
41, trans 42, cis
and conditions: (a) Pyr (17 eq), ClCOOPh (1.1 eq), 3 h, 78-81%; (b) NH2NH2-H2O (5.5 eq), reflux, 3 h, 88-89%; (c) formamidine acetate (4.5 eq), acetic acid, reflux, 3 h, 20-46%.
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Biological Evaluation Hh inhibition studies. All des-triazole ITZ analogues were initially evaluated for Hh pathway inhibition by monitoring endogenous Gli1 mRNA levels in the murine BCC cell line ASZ-001. Our previous SAR studies indicated that the ASZ cells represent a more appropriate cellular model for measuring des-triazole ITZ analogue-mediated Hh pathway inhibition compared to the C3H10T1/2 and NIH-3T3 mouse embryonic fibroblasts (MEFs) commonly used for evaluating inhibition of Hh signaling. More specifically, the ability of lead analogues 2a and 2b to downregulate Gli1 mRNA expression in the ASZ cell line correlated more closely with our results in the primary Hh-dependent murine MB cells isolated from conditional patched knockout (Math1Cre;Ptcfl/fl, hereafter termed Ptch-CKO) mice.17-19 As shown above for lead analogue 2, our initial SAR studies suggested that the 4R-trans orientation around the dioxolane was optimal for potent Hh pathway inhibition.9 In addition, our compounds suggested that truncation of the triazolone region of the scaffold was tolerated. With this in mind, our initial plan for this generation of ITZ analogues was to synthesize and evaluate a series of compounds in which the triazolone moiety was replaced with substituted phenyl groups directly appended to the linker region through an amide bond. Substituted amides were chosen based on their general stability (chemical and biological), enhanced solubility, and ease of synthesis. Our first series of des-triazole ITZ analogues focused on analogues that contained the 2R,4R-trans-dioxolane for the reasons noted above (6, 8, 10-14, 26-28, Table 1).10 Both truncated analogues (6 and 8) exhibited moderate Hh inhibition (IC50 = 0.31 µM and 0.9 µM, respectively); however, the majority of compounds containing an amide in place of the triazolone were inactive at the concentrations tested (Table 3). Despite reduced potency within this trans series of des-
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triazole ITZ amides, the SAR data did indicate that substitutions in the meta-position of the phenyl ring were most favorable as 13 (meta-pyridyl) was moderately active (IC50 = 4.3 µM) and 27 (meta-phenol) demonstrated potent Hh inhibition (IC50 = 0.19 µM) compared to the ortho- and para-substituted analogues. These results suggest that functional groups capable of hydrogen bonding at the meta-position may be advantageous to binding with Smo, the hypothesized target of ITZ and des-triazole ITZ analogues.
Table 3. In Vitro Activity of First Generation des-ITZ Amide Analogues Compound IC50 (µM)a, b Ptch-CKO Compound IC50 (µM)a, b GI50 (µM)a,b 6 0.31 ± 0.04 --16 >10 7 0.26 ± 0.04 --17 3.9 ± 0.3 8 0.92 ± 0.04 --18 0.19 ± 0.09 9 0.39 ± 0.09 --19 >10 10 >10 --26 >10 11 >10 --27 0.19 ± 0.01 12 >10 --28 >10 13 4.3 ± 0.2 >10 29 2.2 ± 0.3 14 >10 --30 0.16 ± 0.03 15 >10 --31 3.0 ± 0.1
Ptch-CKO GI50 (µM)a,b ----1.1 ± 0.6 ----2.8 ± 1.5 ----0.35 ± 0.06 ---
aIC
values represent the Mean ± SEM of at least two separate experiments performed in triplicate. bAll analogues evaluated following 48 h incubation. 50
As noted, we initially focused our synthesis and evaluation of ITZ amides on the transdioxolane because of the activity of this stereochemical orientation in our first generation series;10 however, several cis-dioxolane ITZ analogues also demonstrated potent Hh inhibition. With this in mind, we prepared cis-dioxolane amides with the same substituents to determine what effect this modification would have on Hh inhibitory activity of the scaffold (7, 9, 15-19, 29-31, Table 1). Interestingly, each of the cis-dioxolane analogues was more potent than its corresponding transdioxolane suggesting that this orientation was preferred when the triazolone was substituted with
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the simplified aromatic moiety. In addition, analogues containing the meta-pyridyl (18, IC50 = 0.19 µM) or meta-phenol (30, IC50 = 0.16 µM) were the most potent for the cis-dioxolane. Based on the SAR generated from our first series of amide-containing ITZ analogues, we synthesized and evaluated several more compounds that incorporated the cis-dioxolane and a meta-substituted aromatic moiety (Table 3). These analogues were designed to further probe the sterics, electronics, and hydrogen bonding of the substituents in the meta-position. Analogues containing a furan (35) or thiophene (36) were inactive, suggesting 5-membered heterocycles are potentially less active (Table 4). Masking the phenol of 30 as the methoxy (33) significantly decreased potency, supporting a role for the free phenol in the meta-position (IC50 values = 0.16 µM and 5.7 µM, respectively). The most potent analogue in this second series, and the most potent
amide analogue we prepared, was 34 (IC50 = 0.089 µM), which contains the meta-acetyl phenyl ring. Based on our results, potent inhibition of the Hh pathway can be correlated to the ability to hydrogen bond and/or increased polarity at the meta-position of the phenyl ring for cis-dioxolane analogues that incorporate an amide moiety in place of the triazolone/side chain.
Table 4. In Vitro Evaluation of Second Generation des-ITZ Amide Analogues Ptch-CKO Compound IC50 (µM)a, b GI50 (µM)a,b 32 >10 --33 5.7 ± 1.9 --34 0.089 ± 0.01 1.0 ± 0.3 35 >10 --36 >10 --aIC
values represent the Mean ± SEM of at least two separate experiments performed in triplicate. bAll analogues evaluated following 48 h incubation. 50
In parallel with our studies of ITZ analogues that contain amides as isosteres of the triazolone ring, we synthesized and evaluated the stereochemically defined unsubstituted triazolones (41 and 42). Our earlier SAR demonstrated that the side chain was not required for Hh
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inhibitory activity,10 but the defined cis- and trans-dioxolane des-triazole analogues were not evaluated (40 and 41). We also explored the Hh inhibitory activity of several synthetic precursors to the triazolone (37-40). In addition to a shorter synthetic route, the functional groups on these precursors may aid in improving the overall drug-like properties of this scaffold. Carbamates have been widely studied within the field of medicinal chemistry and are key structural motifs in many FDA-approved compounds and prodrugs because they offer chemical stability and enhanced cell permeability.10 Hydrazine carboxamides are derivatives of urea and have thus been studied extensively within the field of drug development and may aid in hydrogen-bonding and solubility.20 Not surprisingly, both of the intact triazolones (41 and 42) were potent inhibitors of Hh signaling (IC50 values = 0.14 and 0.21 µM, respectively), similar to our previous results for the undefined triazolone analogue (IC50 = 0.12 µM9) (Table 5). The most potent analogues for the triazolones and precursors were the two hydrazine carboxamides (39 and 40) and similar to the ITZ amides, the cis-hydrazine was more potent than the corresponding trans-analogue. In fact, the most potent analogue generated through these studies was the cis-carboxamide (40, IC50 = 0.009 µM). Based on our results from the ITZ amide analogues, the enhanced inhibitory activity for the carboxamides was not surprising given their increased polarity and hydrogen bond donating/accepting properties. In addition, the carboxamide is an acyclic functionality, providing strong evidence that anti-Hh activity can still be achieved without the cyclic functionality of the triazolone and aromatic isosteres. The carbamate analogues were inactive, which may be attributed to the presence of the bulky non-substituted hydrophobic ring. Based on their activity in ASZ cells, several compounds were evaluated for their antiproliferative activity in Hh-dependent MB Ptch-CKO cells. Overall, the ability of our ITZ analogues to reduce the growth of the Ptch-CKO cells was greatly reduced compared to their anti-
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Hh activity in the ASZ cell line. The one compound that did not follow this trend was 30, which contains the meta-phenol (GI50 = 0.35 µM). Similar to the ASZ cells, the cis-amide analogues 18 and 30 were more potent than the corresponding trans-amides, 13 and 27. Interestingly, this activity trend did not hold for the carboxamides 39 and 40; for these analogues, the trans-dioxolane 40 was more potent in the Ptch-CKO cells. This difference in anti-Hh activity could be attributed to differing permeability across the various cellular models or because down-regulation of Gli1 does not directly result in anti-proliferative activity in either of these cell models.
Table 5. In Vitro Evaluation of des-ITZ Intermediate Analogues Compound IC50 (µM)a, b Ptch-CKO GI50 (µM)a,b 37 >10 --38 >10 --39 0.11 ± 0.01 0.5 ± 0.4 40 0.009 ± 0.0002 2.8 ± 0.9 41 0.14 ± 0.03 --42 0.21 ± 0.04 --aIC
values represent the Mean ± SEM of at least two separate experiments performed in triplicate. bAll analogues evaluated following 48 h incubation. 50
Preliminary In Vitro Pharmacokinetic Assays. Several representative compounds were chosen for evaluation in a series of preliminary pharmacokinetic (PK) assays (Table 6). The amide substituents did not enhance solubility as all these analogues were significantly less soluble than ITZ and our initial analogues (2a and 2b). Interestingly, the trans isomers (13, 27, and 39) demonstrated enhanced metabolic stability compared to the cis analogues (18, 30, 34, and 40), suggesting that the stereochemistry around the dioxolane moiety plays a role in compound metabolism and clearance. We previously verified that removal of the triazole abolishes inhibition of CYP3A4 for the ITZ scaffold and this also translates to the des-triazole ITZ analogues prepared in our current series. Neither ITZ nor any of the analogues evaluated demonstrated the ability to
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passively diffuse through a hexadecane membrane in a parallel artificial membrane permeability assay (PAMPA). Finally, as these compounds are being developed to target MB, we evaluated whether ITZ and several analogues are substrates of the two primary efflux pumps in the blood brain barrier, Breast Cancer Resistance Protein (BCRP) and P-glycoprotein (P-gp). ITZ was not a substrate for either efflux pump; however, none of the analogues evaluated were soluble enough under the assay conditions to determine efflux parameters. Interestingly, our results demonstrating that ITZ is not a P-gp substrate are different than those previously reported, in which P-gp efflux was identified as a mechanism through which ITZ is actively removed from mouse brain.20
Table 6. Pharmacokinetic Evaluation of des-Triazole ITZ Analogues.a T1/2 hERG Efflux Pump Clearance CYP3A4 PAMPA (HLM, Inhibition Substratesa,b a a a (mL/min/mg) (µM) -log Pe min)a (%)a BCRP P-gp ITZ 0.8 27.0 ± 6.2 --0.04 20 ND 7.7 ± 0.2 NDb ND 2b 1.56 ----1.02 ND 9.0 ± 0.1 ND ND 13 0.0042 59.9 ± 2.0 11.6 ± 0.4 >30 ND 7.9 ± 0.03 ND ND 18 0.0069 8.9 ± 0.03 78.3 ± 0.2 >20 231 30 1.2 ------30 0.0016 19.9 ± 0.6 34.7 ± 1.0 11.4 30 30 2.2 ------40 0.0014 11.5 ± 0.1 60.4 ± 0.7 >30 ND ------aAdditional experimental information for all assays provided in the supplementary information. bND = Not determined based on poor compound solubility under the assay conditions. Solubility (µM)a
Computational Modeling We recently developed a homology model of Smo and used it to perform docking and molecular dynamics studies for the structurally related triazole anti-fungal posaconazole (PSZ) and several PSZ analogues.7 These studies provided strong evidence that the triazolone/side chain region of the scaffold penetrates deeply into the Smo binding site on the seven-transmembrane domain (7TM) while the dioxolane/triazole region extends out of the binding pocket and is solvent exposed. This binding conformation also supports the in vitro SAR that determined for both of these
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scaffolds with respect to Hh inhibition. To explore whether our amide-based ITZ analogues adopt the same binding conformation in the Smo 7-TM we performed docking studies for 30 and 34 with our homology model (Figure 1).
Figure 1. Predicted binding mode of 30 and 34 in complex with Smo. Compared to 30, compound 34 penetrates more deeply inside the binding pocket (A). Key intermolecular interactions between Smo:34 (B) and Smo:30 (C) include a network of π –π (orange mesh), cation–π (pink mesh) and hydrogen bonds (yellow dotted line). The pdb file of our homology model of Smo is included in the Supplementary Information.
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Compounds 30 and 34 adopt similar orientations and form comparable binding interactions with Smo inside the previously determined binding pocket. The dichlorophenyl ring is oriented towards the extracellular surface with the linker/amide region penetrating deeply into the Smo binding pocket. Interestingly, the dioxolane region of 30 is significantly more exposed to the solvent accessible surface of the binding pocket when compared to compound 34, which penetrates more deeply into the binding grove. Our docking studies predict that the shallower penetration of compound 30 is due to a hydrogen bond between the meta-phenol and the side chain of E518; an intermolecular interaction not present in the Smo:34 structure. The dioxolane and dicholorophenyl rings of both compounds are primarily stabilized by a network of cation–π interactions with the positively charged basic side chain residues (K395 and R296) present at the opening of the binding site. The biaryl piperazine groups of 30 and 34 exhibit favorable non-polar interactions with several hydrophobic side chains in the binding pocket (I215, M301, F222, V386 and L515). The deeper penetration of analogue 34 allows for a cation–π interaction between the protonated nitrogen of the piperazine and the side chain of F484, an additional intermolecular interaction that may underlie its enhanced activity. The amide nitrogen of both compounds forms a hydrogen bond with D384, a linker and phenyl moiety of both the compounds demonstrate a network of π-π stacking and hydrogen bonds with Y394 and D384, respectively.
DISCUSSION AND CONCLUSIONS We have extended our studies on des-triazole ITZ analogues as Hh pathway inhibitors by designing, synthesizing, and evaluating a new series of compounds that incorporate extensive modifications to the triazolone/side chain region of the scaffold. Our primary goal for these
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analogues was three-fold. First, we sought to further probe the sterics, electronics, and hydrogen bonding requirements in this region of the ITZ scaffold for optimal anti-Hh potency. Second, we wanted to identify a simplified triazolone mimic that could be prepared more rapidly than the triazolone/side chain region of the parent scaffold. Finally, we evaluated preliminary PK properties of our most potent analogues to determine how our modifications affect drug-like properties of the scaffold and to determine what potential PK liabilities should be addressed in subsequent compounds. Interestingly, des-triazole analogues that simplified the triazolone/side chain to an aromatic amide demonstrated a preference for the 2S,4R-cis-orientation around the dioxolane ring. The most potent des-triazole ITZ amide analogues contained the 4R-cis-dioxolane region and meta-substitutions on the phenyl ring. In addition, the anti-Hh activity of the scaffold increases with increasing polarity at the meta-position. The most potent amide analogues incorporate either a meta-phenol (30) or meta-acetyl (34) functional group. In addition to the amide-containing compounds, several analogues that truncate the triazolone of ITZ were potent inhibitors of the Hh pathway, verifying that this region of the scaffold is amenable to a wide range of structural modifications. The most potent analogue in the ASZ cell line was the cis-carboxamide 40. Finally, these compounds were significantly more potent in the immortalized murine ASZ BCC cell line compared to the primary murine Ptch-CKO cells. We previously noted a similar difference in cellular activity for the ITZ scaffold across several Hh-dependent cell lines, highlighting the importance of comparing activity across several model systems.9 As many of our analogues exhibit potent inhibition of Hh activity, a key factor for delineating between their potential as therapeutic agents relies on which compounds exhibit improved drug-like properties. This is especially important for this scaffold, considering ITZ is a ‘large’ small molecule (MW = 705 g/mol) and is poorly soluble. Unfortunately, the majority of
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our compounds were less soluble than ITZ and the most potent (30, 34, 40) demonstrated comparable or reduced metabolic stability compared to ITZ. Not surprisingly, each analogue was also poorly permeable and solubility issues prevented us from determining whether the analogues were substrates of the two primary efflux pumps in the blood brain barrier. Taken together, these results highlight the potential of the ITZ scaffold to treat Hh-dependent cancers while also demonstrating the need for continued improvements in the drug-like properties and formulation of the ITZ scaffold.
EXPERIMENTAL SECTION Chemical Synthesis. General Information. All chemicals were purchased from either Sigma-Aldrich or Fisher Scientific. ACS grade methanol, ethyl acetate, toluene, anhydrous DMF, DCM, and DMSO were purchased from Fisher Scientific or Sigma-Aldrich. All reactions were performed under an argon atmosphere. NMR data were collected on a Bruker AVANCE 500 MHz spectrometer, and analysis was carried out using MestReNova. HRMS data was obtained at the Mass Spectrometry Facility at the University of Connecticut. FT-IR analysis was carried out on a Bruker Alpha Platinum ATR instrument using OPUS software (v7.2). The preparation of previously characterized
ITZ
intermediates
(3-5)
followed
known
procedures
with
minor
modifications.11,12,21,22 All ITZ analogues evaluated in the biological assays were greater than 95% pure based on the HPLC methods described below.
Purity Analysis of Final Analogues. Purity analysis of all final des-triazole ITZ analogues was determined via one of the methods described below.
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Method A. ITZ analogues were dissolved in HPLC-grade MeCN and injected (25 μL of a 1 mM solution) into an Agilent Manual FL-Injection Valve (600 bar) on an Agilent 1100/1200 Series HPLC equipped with an Agilent Eclipse Plus C18 (4.6 x 100 mm) column and Agilent 1100 Series Photodiode Array Detector. The mobile phase consisted of 70% MeCN/30% H2O. All analogues were run at a flow rate of 1.0 mL/min for 20 min and purity was assessed at 254 nm. Method B. ITZ analogues were dissolved in HPLC-grade MeCN and injected (25 μL of a 1mM solution) into an Agilent HPLC system coupled to an Agilent ESI single quadrupole mass spectrometer equipped with a Kinetix C18 (150 x 4.6 mm) column and an Agilent G1315 diode array detector. The mobile phase was a gradient of 25% - 100% MeCN (0.1 % formic acid) in H2O over 75 min. All analogues were run at a flow rate of 0.7 mL/min for 30 min and purity was assessed at 254 nm.
1-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)-4-(4nitrophenyl)piperazine (6). To a solution of 5 (0.77 g, 2.61 mmol) in DMSO (30 mL) was added Cs2CO3 (8.5 g, 26.14 mmol) and 3 (1.2 g, 2.87 mmol). The solution was warmed to 90 °C and stirred for 12 h. The mixture was cooled to room temperature and water was added slowly with vigorous stirring (~20 mL). A yellow precipitate formed, which was filtered and recrystallized in EtOH to yield 6 (1.2 g, 85%). 1H NMR (500 MHz, Chloroform-d) δ 8.20 (d, J = 9.4 Hz, 2H), 7.66 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 2.1 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 7.03 – 6.87 (m, 6H), 4.37 (h, J = 5.4 Hz, 1H), 4.16 (dd, J = 9.5, 5.1 Hz, 1H), 4.11 – 3.95 (m, 2H), 3.95 – 3.84 (m, 1H), 3.69 – 3.53 (m, 4H), 3.35 – 3.18 (m, 4H), 1.86 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 154.79, 153.29, 146.68, 138.10, 134.59, 132.83, 131.21, 128.89, 126.79, 125.98 (2), 118.58 (2), 115.56 (2), 112.90 (2), 109.99, 109.12, 73.94, 69.27, 66.99, 50.39 (2), 47.31 (2), 25.73. DART-HRMS: m/z calcd. for
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C27H27Cl2N3O5 [MH]+, 544.1406; Found: 544.1388. IR (solid) vmax: 3060, 2926, 2880, 2828, 1556, 1507, 1485, 1446, 1376, 1222, 1190, 1142, 1047, 995, 942, 821, 737. Purity, Method A: 95.2%.
1-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)-4-(4nitrophenyl)piperazine (7). To a solution of 5 (260 mg, 0.87 mmol) in DMSO (20 mL) was added Cs2CO3 (3.1 g, 9.58 mmol) and 4 (400 mg, 0.0958 mmol). The solution was warmed to 90 °C and stirred for 12 h. The mixture was cooled to room temperature and water was added slowly with vigorous stirring (~10 mL). A yellow precipitate formed, which was filtered and recrystallized in EtOH to yield 8 (175 mg, 34%). 1H NMR (500 MHz, Chloroform-d) δ 8.20 (d, J = 9.4 Hz, 2H), 7.68 (d, J = 8.5 Hz, 1H), 7.42 (d, J = 2.1 Hz, 1H), 7.24 (d, J = 8.5 Hz, 1H), 6.93 (dd, J = 9.1, 5.4 Hz, 4H), 6.78 (d, J = 9.0 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 3.99 (dd, J = 9.7, 5.4 Hz, 1H), 3.81 – 3.76 (m, 2H), 3.64 – 3.59 (m, 4H), 3.29 – 3.23 (m, 4H), 1.83 (s, 3H).
13C
NMR (126 MHz, CDCl3) δ 154.78, 153.09, 145.50, 139.34, 138.76, 134.35, 132.82,
130.97, 128.55, 126.70, 125.98 (2), 118.53 (2), 115.31 (2), 112.89 (2), 109.18, 75.05, 68.41, 67.33, 50.38 (2), 47.30 (2), 25.90. DART-HRMS: m/z calcd. for C27H27Cl2N3O5 [MH]+, 544.1406; Found: 544.1409. IR (solid) vmax: 2922, 2850, 1596, 1555, 1509, 1450, 1375, 1321, 1228, 1195, 1151, 1036, 944, 827, 753. Purity, Method B: 95.1%
4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)aniline (8). 10% palladium on carbon (24.4 mg, 5% mole ratio) was added to a dry round bottom flask. Ethanol (100 mL) was added followed by slow addition of 6 (250 mg, 0.459 mmol). Hydrazine monohydrate (0.14 mL, 4.59 mmol) was added
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dropwise and the mixture was stirred at reflux for 2 h. Upon cooling to RT, the mixture was filtered through celite. The celite was washed with ethanol (500 mL) to ensure complete elution of the aniline. The filtrate was concentrated to afford a yellow solid, which was recrystallized in EtOH to afford 8 (100 mg, 42%). 1H NMR (500 MHz, Chloroform-d) δ 8.16 (d, J = 9.2 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 1.9 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 6.94 (dd, J = 9.3, 3.0 Hz, 2H), 6.91 – 6.83 (m, 4H), 6.68 (d, J = 8.6 Hz, 1H), 4.33 (d, J = 6.9 Hz, 1H), 4.12 (dd, J = 9.5, 5.1 Hz, 1H), 4.08 – 3.89 (m, 2H), 3.89 – 3.78 (m, 1H), 3.58 (t, J = 5.2 Hz, 2H), 3.45 (s, 1H), 3.31 – 3.11 (m, 6H), 1.82 (s, 3H).
13C
NMR (126 MHz, CDCl3) δ 152.77, 146.21, 144.46, 132.83,
131.20, 128.91, 126.79, 125.99, 118.85 (2), 118.59, 118.25 (2), 116.23 (2), 115.56, 115.46 (2), 112.90, 73.97, 69.29, 67.04, 51.25 (2), 50.86 (2), 25.74. DART-HRMS: m/z calcd. for C27H29Cl2N3O3 [MH]+, 514.1664; Found: 514.1648. IR (solid) vmax: 2919, 2850, 1595, 1560, 1510, 1464, 1450, 1375, 1228, 1196, 1150, 1037, 877, 825, 754. Purity, Method A: 98.0%.
4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)aniline (9). 10% palladium on carbon (10 mg, 5% mole ratio) was added to a dry round bottom flask. Ethanol (50 mL) was added followed by slow addition of 8 (100 mg, 0.184 mmol). Hydrazine monohydrate (0.06 mL, 1.84 mmol) was added dropwise and the mixture was stirred at reflux for 2 h. Upon cooling to RT, the mixture was filtered through celite. The celite was washed with ethanol (300 mL) to ensure complete elution of the aniline. The filtrate was concentrated to afford a yellow solid, which was recrystallized in EtOH to afford 9 (89 mg, 94%). 1H NMR (500 MHz, Chloroform-d) δ 8.20 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.43 (s, 1H), 7.24 (d, J = 8.6 Hz, 1H), 6.92 (q, J = 8.5 Hz, 4H), 6.81 – 6.68 (m, 3H), 4.70 – 4.59 (m, 1H), 4.35 (t, J = 7.4 Hz, 1H), 3.99 (s, 1H), 3.78 (d, J = 6.8 Hz, 2H), 3.61 (t, J = 5.2 Hz,
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2H), 3.49 (s, 1H), 3.24 (d, J = 8.2 Hz, 6H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 140.38, 139.34, 136.37, 134.35, 130.97, 128.59, 128.35, 126.70, 125.99, 118.85 (2), 118.54, 118.21 (2), 116.24 (2), 115.21(2), 112.90, 75.07, 68.43, 67.40, 51.24 (2), 50.85 (2), 25.90. DART-HRMS: m/z calcd. for C27H29Cl2N3O3 [MH]+, 514.1664; Found: 514.1666. IR (solid) vmax: 2921, 2823, 1590, 1510, 1450, 1375, 1321, 1227, 1193, 1149, 1037, 944, 824, 753. Purity, Method B: 95.2%
General Amide Coupling Procedures. Method A. Aniline 8 or 9 (20 mg, 0.039 mmol), carboxylic acid (0.117 mmol), EDCI (0.117 mmol), and DMAP (0.117 mmol) were dissolved in anhydrous DCM (10 mL) and stirred under argon for 12 h at room temperature. The crude reaction was immediately purified via column chromatography (SiO2, 0-80% acetone in hexanes) to afford the ITZ amide analogue. Method B. Acid (0.038 mmol) and HATU (0.083 mmol) were added to a round bottom flask. Anhydrous DMF was added (5 mL) followed by NMM (0.15 mmol); and this mixture was stirred at room temperature for 30 mins. Aniline 8 or 9 (20 mg, 0.038 mmol) in anhydrous DMF (4 mL) was added to the mixture and stirred for 12 h at room temperature. The solution was added to ice cold water and washed with EtOAc (3 x 20 mL). The organic layers were combined and washed with H2O (2 x 20 mL) and brine (1 x 20 mL). The organic layers were dried over Na2SO4 and evaporated to dryness. Crude products was purified via preparative thin layer chromatography (Analtech Uniplate 20x20 cm 2000 micron, 3% acetone in hexanes) or column chromatography (SiO2, 0-80% acetone in hexanes) to afford the ITZ amide analogue.
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)benzamide (10). Amide coupling method A; Yield =
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13.0 mg, 54%. 1H NMR (500 MHz, Chloroform-d) δ 7.92 (d, J = 7.3 Hz, 2H), 7.73 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.63 – 7.57 (m, 3H), 7.54 (t, J = 7.5 Hz, 2H), 7.46 (d, J = 2.1 Hz, 1H), 7.28 (dd, J = 8.4, 2.0 Hz, 1H), 7.05 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 4.37 (q, J = 5.5 Hz, 1H), 4.17 (dd, J = 9.5, 5.0 Hz, 1H), 4.09 – 3.98 (m, 2H), 3.89 (t, J = 7.7 Hz, 1H), 3.41 – 3.33 (m, 4H), 3.33 – 3.22 (m, 4H), 1.86 (s, 3H).13C NMR (126 MHz, CDCl3) δ 165.48, 152.92, 148.50, 146.09, 138.15, 135.17, 134.56, 132.83, 131.69, 131.19, 130.61, 128.91, 128.78 (2), 126.95, 126.79 (2), 121.64 (2), 118.40 (2), 116.95 (2), 115.48 (2), 109.09, 73.96, 69.29, 67.03, 50.74 (2), 49.88 (2), 25.74.DART-HRMS: m/z calcd. for C34H33Cl2N3O4 [MH]+, 618.1926; Found: 618.1919. IR (solid) vmax: 3367, 3333, 2958, 2924, 2853, 1693, 1638, 1547, 1511, 1450, 1375, 1248, 1230, 1194, 1157, 1037, 941, 822, 703. Purity, Method A: 96.2%
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-naphthamide (11). Amide coupling method A; Yield = 6.0 mg, 23%. 1H NMR (500 MHz, Chloroform-d) δ 8.39 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 6.9 Hz, 1H), 7.75 (d, J = 7.0 Hz, 1H), 7.66 – 7.48 (m, 7H), 7.41 (d, J = 2.1 Hz, 1H), 7.24 (dd, J = 8.4, 2.1 Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.1 Hz, 2H), 4.32 (dq, J = 11.4, 6.5, 5.6 Hz, 1H), 4.12 (dd, J = 9.5, 5.1 Hz, 1H), 4.05 – 3.93 (m, 2H), 3.89 – 3.81 (m, 1H), 3.38 – 3.30 (m, 4H), 3.29 – 3.20 (m, 4H), 1.82 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 167.31, 152.94, 148.59, 146.09, 138.15, 134.75, 134.56, 133.81, 132.84, 131.20, 130.94, 130.75, 130.17, 128.91, 128.43, 127.33, 126.79, 126.59, 125.38, 125.02, 124.79, 121.41 (2), 118.41 (2), 117.01 (2), 115.49 (2), 109.09, 73.96, 69.29, 67.03, 50.74 (2), 49.92 (2), 25.74. DART-HRMS: m/z calcd. for C38H35Cl2N3O4 [MH]+, 668.2083; Found: 668.2075. IR
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(solid) vmax: 3294, 3247, 3042, 2985, 2873, 1639, 1586, 1509, 1450, 1373, 1226, 1192, 1146, 1035, 942, 821, 733. Purity, Method A: 97.0%.
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)picolinamide (12). Amide coupling method A; Yield = 9.0 mg, 37. 1H NMR (500 MHz, Chloroform-d) δ 9.91 (s, 1H), 8.61 (d, J = 4.7 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H), 7.90 (t, J = 7.7 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.4 Hz, 1H), 7.50 – 7.43 (m, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.23 (dd, J = 8.4, 2.1 Hz, 1H), 7.01 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 4.33 (p, J = 5.7 Hz, 1H), 4.12 (dd, J = 9.5, 5.1 Hz, 1H), 4.05 – 3.92 (m, 2H), 3.85 (t, J = 7.7 Hz, 1H), 3.38 – 3.29 (m, 4H), 3.29 – 3.21 (m, 4H), 1.82 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 161.67, 152.90, 150.10, 148.20, 147.94, 146.11, 138.16, 137.64, 134.56, 132.84, 131.20, 130.66, 128.91, 126.79, 126.26, 122.32, 120.91 (2), 118.38 (2), 116.97 (2), 115.48 (2), 109.09, 73.97, 69.30, 67.04, 50.75 (2), 49.91 (2), 25.74.DARTHRMS: m/z calcd. for C33H32Cl2N4O4 [MH]+, 619.1879; Found: 619.1872. IR (solid) vmax: 3367, 2923, 2851, 1672, 1585, 1552, 1509, 1454, 1385, 1268, 1227, 1192, 1148, 1020, 940, 912, 814, 747, 620. Purity, Method A: 96.9%.
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)nicotinamide (13). Amide coupling method A; Yield = 6.2 mg, 25%.
1H
NMR (500 MHz, Chloroform-d) δ 9.14 (s, 1H), 8.83 (s, 1H), 8.26 (d, J = 8.0
Hz, 1H), 7.72 (s, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 7.2 Hz, 1H), 7.46 (d, J = 2.2 Hz, 1H), 7.28 (dd, J = 8.4, 2.1 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 9.0
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Hz, 2H), 6.96 – 6.92 (m, 2H), 4.37 (p, J = 6.0 Hz, 1H), 4.17 (dd, J = 9.5, 5.0 Hz, 1H), 4.08 – 4.00 (m, 2H), 3.89 (dd, J = 8.4, 6.9 Hz, 1H), 3.41 – 3.36 (m, 4H), 3.32 – 3.27 (m, 4H), 1.86 (s, 3H).13C NMR (126 MHz, CDCl3) δ 184.50, 170.35, 152.52, 147.78, 146.05, 138.15, 136.05, 135.22, 134.57, 132.84, 131.20, 129.90, 128.91, 126.80, 123.69, 121.87 (2), 118.43 (2), 116.86 (2), 115.49 (2), 109.10, 73.96, 69.29, 67.03, 50.73 (2), 49.72 (2), 25.74. DART-HRMS: m/z calcd. for C33H32Cl2N4O4 [MH]+, 619.1879; Found: 619.1876. IR (solid) vmax: 3639, 3570, 2920, 2824, 1644, 1509, 1447, 1374, 1227, 1194, 1150, 1037, 941, 828, 733, 534. Purity, Method A: 96.9%.
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)isonicotinamide (14). Amide coupling method A; Yield = 12.0 mg, 49%. 1H NMR (500 MHz, Chloroform-d) δ 8.85 (d, J = 5.2 Hz, 2H), 7.75 (d, J = 6.0 Hz, 3H), 7.66 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 2.2 Hz, 1H), 7.27 (d, J = 2.2 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 4.38 (d, J = 5.8 Hz, 1H), 4.17 (dd, J = 9.7, 5.1 Hz, 1H), 4.08 – 3.99 (m, 2H), 3.92 – 3.87 (m, 1H), 3.42 – 3.36 (m, 4H), 3.32 – 3.27 (m, 4H), 1.86 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 163.41, 152.98, 150.79 (2), 148.93, 146.03, 142.18, 138.14, 134.57, 132.83, 131.19, 129.64, 128.90, 126.79, 121.81 (2), 120.79 (2), 118.43 (2), 116.79 (2), 115.50 (2), 109.09, 73.96, 69.30, 67.02, 50.72 (2), 49.66 (2), 25.73. DART-HRMS: m/z calcd. for C33H32Cl2N4O4 [MH]+, 619.1879; Found: 619.1871. IR (solid) vmax: 3302, 3290, 2950, 2936, 1662, 1587, 1512, 1458, 1377, 1249, 1225, 1194, 1151, 1035 943, 823, 690. Purity, Method A: 94.1%
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)benzamide (15). Amide coupling method A; Yield =
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5.0 mg, 20.6%. 1H NMR (500 MHz, Chloroform-d) δ 7.91 (d, J = 7.4 Hz, 2H), 7.73 (s, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.59 (dd, J = 8.0, 5.2 Hz, 3H), 7.54 (t, J = 7.5 Hz, 2H), 7.43 (d, J = 2.2 Hz, 1H), 7.25 (dd, J = 8.5, 2.2 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 6.78 (d, J = 8.9 Hz, 2H), 4.65 (p, J = 6.2 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.82 – 3.74 (m, 2H), 3.36 (d, J = 5.4 Hz, 4H), 3.28 (d, J = 5.2 Hz, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 165.58, 152.78, 151.10, 148.49, 139.40, 135.17, 134.36, 132.81, 131.68, 130.97, 130.61, 128.78, 128.58 (2), 126.95, 126.70 (2), 121.64 (2), 118.35 (2), 116.95 (2), 115.24 (2), 109.16, 75.07, 68.44, 67.39, 50.73 (2), 49.88 (2), 25.90. DART-HRMS: m/z calcd. for C34H33Cl2N3O4 [MH]+, 618.1926; Found: 618.1916. IR (solid) vmax: 3305, 2964, 2824, 1637, 1592, 1511, 1448, 1374, 1226, 1192, 1152, 1034, 943, 818, 728, 703. Purity, Method B: 95.6%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-naphthamide (16). Amide coupling method A; Yield = 3.0 mg, 11.5%. 1H NMR (500 MHz, Chloroform-d) δ 8.43 (s, 1H), 8.08 – 7.92 (m, 4H), 7.89 (s, 1H), 7.73 – 7.58 (m, 5H), 7.43 (d, J = 2.1 Hz, 1H), 7.25 (dd, J = 8.5, 2.1 Hz, 1H), 7.06 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.1 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.86 – 3.71 (m, 2H), 3.45 – 3.33 (m, 4H), 3.33 – 3.21 (m, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 165.53, 152.75, 148.50, 146.03, 139.34, 134.83, 134.36, 132.80, 132.69, 132.36, 130.97, 130.68, 128.96, 128.73, 128.59, 127.83, 127.41, 126.94, 126.71, 123.57, 121.69 (2), 118.36 (2), 116.96 (2), 115.24 (2), 109.16, 75.07, 68.44, 67.40, 50.74 (2), 49.87 (2), 25.91. DART-HRMS: m/z calcd. for C38H35Cl2N3O4 [MH]+, 668.2083; Found: 668.2116. IR (solid) vmax: 3326, 2920, 2851, 1663, 1596, 1558, 1513, 1450, 1373, 1229, 1195, 1151, 1037, 948, 822, 760. Purity, Method A: 96.3%.
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Journal of Medicinal Chemistry
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)picolinamide (17). Amide coupling method A; Yield = 9.0 mg, 37%. 1H NMR (500 MHz, Chloroform-d) δ 9.96 (s, 1H), 8.66 (dt, J = 3.8, 1.1 Hz, 1H), 8.34 (dt, J = 7.9, 1.1 Hz, 1H), 7.95 (td, J = 7.7, 1.7 Hz, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.57 – 7.48 (m, 1H), 7.43 (d, J = 2.1 Hz, 1H), 7.25 (dd, J = 8.5, 2.1 Hz, 1H), 7.06 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.40 – 4.30 (m, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.78 (dd, J = 8.6, 7.2 Hz, 2H), 3.39 (d, J = 12.8 Hz, 4H), 3.28 (s, 4H), 1.83 (s, 3H). 13C
NMR (126 MHz, CDCl3) δ 178.75, 147.93, 146.04, 139.33, 137.64, 134.73, 134.36, 132.79,
130.97, 130.88, 128.58, 126.71, 126.25, 122.98, 122.31, 120.91 (2), 118.33 (2), 118.16, 116.96 (2), 115.25 (2), 109.16, 75.06, 68.44, 67.39, 50.74 (2), 49.90 (2), 25.91. DART-HRMS: m/z calcd. for C33H32Cl2N4O4 [MH]+, 619.1879; Found: 619.1862. IR (solid) vmax: 3365, 2955, 2849, 1675, 1586, 1568, 1510, 1453, 1373, 1268, 1229, 1193, 1151, 1097, 1037, 942, 823, 747. Purity, Method B: 92.7%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)nicotinamide (18). Amide coupling method A; Yield = 6.0 mg, 24.7%. 1H NMR (500 MHz, Chloroform-d) δ 9.15 (s, 1H), 8.83 (s, 1H), 8.27 (s, 1H), 7.86 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 16.7 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H), 7.28 – 7.20 (m, 1H), 7.01 (dd, J = 29.6, 8.5 Hz, 4H), 6.83 – 6.73 (m, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.83 – 3.74 (m, 2H), 3.43 – 3.36 (m, 4H), 3.29 (t, J = 5.0 Hz, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 171.38,
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160.06, 148.66, 142.60, 139.33, 136.75, 135.23, 134.36, 132.80, 130.97, 130.06, 128.58, 127.85, 126.71, 121.92 (2), 118.62, 118.54 (2), 116.92 (2), 115.27 (2), 109.17, 75.05, 68.43, 67.36, 50.88 (2), 49.67 (2), 25.90. DART-HRMS: m/z calcd. for C33H32Cl2N4O4 [MH]+, 619.1879; Found: 619.1866. IR (solid) vmax: 3364, 3315, 2960, 2924, 2853, 1673, 1511, 1456, 1372, 1259, 1227, 1193, 1097, 1035, 826, 807. Purity, Method A: 96.5%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)isonicotinamide (19). Amide coupling method A; Yield = 8.0 mg, 33%. 1H NMR (500 MHz, Chloroform-d) δ 8.85 (s, 2H), 7.77 (d, J = 13.1 Hz, 3H), 7.68 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 2.1 Hz, 1H), 7.24 (dd, J = 8.5, 2.2 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.98 – 6.92 (m, 2H), 6.78 (d, J = 9.0 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.82 – 3.75 (m, 2H), 3.38 (q, J = 4.2, 3.0 Hz, 4H), 3.32 – 3.24 (m, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 163.39, 152.80, 150.80 (2), 148.93, 145.98, 142.18, 139.36, 134.36, 132.81, 130.97, 129.65, 128.58, 126.70, 121.81 (2), 120.80 (2), 118.38 (2), 116.79 (2), 115.27 (2), 109.16, 75.07, 68.46, 67.39, 50.71 (2), 49.65 (2), 25.90. DART-HRMS: m/z calcd. for C33H32Cl2N4O4 [MH]+, 619.1879; Found: 619.1866. IR (solid) vmax: 3256, 2963, 2854, 1660, 1587, 1510, 1449, 1376, 1261, 1226, 1193, 1150, 1034, 940, 826, 740. Purity, Method A: 95.1%.
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-hydroxybenzamide
(26).
Amide
coupling
method B; Yield = 10.0 mg, 16%. 1H NMR (500 MHz, Chloroform-d) δ 12.14 (s, 1H), 7.87 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.58 – 7.44 (m, 5H), 7.28 (dd, J = 8.4, 2.2 Hz, 1H), 7.07 (dd, J =
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Journal of Medicinal Chemistry
13.2, 8.5 Hz, 3H), 6.99 (t, J = 7.9 Hz, 2H), 6.95 (t, J = 8.0 Hz, 3H), 4.41 – 4.33 (m, 1H), 4.17 (dd, J = 9.4, 5.1 Hz, 1H), 4.09 – 3.98 (m, 2H), 3.93 – 3.85 (m, 1H), 3.44 – 3.35 (m, 4H), 3.29 (t, J = 5.0 Hz, 4H), 1.86 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 206.89, 168.26, 161.91, 152.97, 149.12, 146.04, 138.14, 134.52, 132.83, 131.20, 128.91, 128.83, 126.79, 125.25, 122.85 (2), 118.97, 118.85, 118.43 (2), 116.79 (2), 115.49 (2), 114.62, 109.09, 73.96, 69.29, 67.03, 50.72 (2), 49.65 (2), 29.72. DART-HRMS: m/z calcd. for C34H33Cl2N3O5 [MH]+, 634.1865; Found: 634.1875. IR (solid) vmax: 3310, 2956, 2916, 2848, 1646, 1596, 1511, 1453, 1373, 1250, 1228, 1196, 1152, 1036, 942, 821, 755. Purity, Method B: 98.1%.
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-3-hydroxybenzamide
(27).
Amide
coupling
method B; Yield = 6.0 mg, 25%. 1H NMR (500 MHz, Chloroform-d) δ 7.74 (d, J = 23.9 Hz, 1H), 7.63 – 7.54 (m, 3H), 7.47 – 7.39 (m, 4H), 7.10 (s, 1H), 7.04 (d, J = 8.6 Hz, 3H), 7.00 (d, J = 8.5 Hz, 2H), 6.96 – 6.92 (m, 2H), 5.21 (s, 1H), 4.37 (s, 1H), 4.16 (dd, J = 9.3, 5.3 Hz, 1H), 4.12 – 3.95 (m, 2H), 3.88 (d, J = 8.0 Hz, 1H), 3.37 (d, J = 5.2 Hz, 4H), 3.29 (t, J = 4.9 Hz, 4H), 1.88 (dd, J = 17.2, 1.6 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.41, 156.15, 151.12, 147.60, 145.64, 141.66, 138.16, 131.21, 130.09, 128.92, 128.26, 127.93, 127.18, 126.80, 125.31, 121.69 (2), 118.75, 118.42 (2), 116.95 (2), 115.50 (2), 114.43, 110.65, 73.97, 69.30, 67.04, 50.75 (2), 49.85 (2), 25.74. DART-HRMS: m/z calcd. for C34H33Cl2N3O5 [MH]+, 634.1876; Found: 634.1847. IR (solid) vmax: 3311, 2967, 2926, 2877, 1736, 1644, 1586, 1513, 1449, 1375, 1230, 1198, 1038, 945, 825, 746. Purity, Method A: 97.8%.
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N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-4-hydroxybenzamide
(28).
Amide
coupling
method B; Yield = 5.0 mg, 8%. 1H NMR (500 MHz, Chloroform-d) δ 7.84 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.28 (dd, J = 8.3, 2.1 Hz, 1H), 7.04 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.98 – 6.91 (m, 3H), 5.41 (s, 1H), 4.42 – 4.31 (m, 1H), 4.16 (dd, J = 9.4, 5.1 Hz, 1H), 4.10 – 3.96 (m, 2H), 3.89 (t, J = 7.7 Hz, 1H), 3.44 – 3.32 (m, 4H), 3.32 – 3.21 (m, 4H), 2.05 (s, 3H).
13C
NMR (126 MHz, CDCl3) δ 160.25, 152.99, 148.16, 145.95, 145.69,
138.03, 134.54, 133.92, 132.75, 131.14, 131.01 (2), 129.02 (2), 126.78, 126.06, 121.75 (2), 118.50 (2), 117.00 (2), 115.46 (2), 115.35, 109.08, 73.93, 69.24, 66.91, 50.79 (2), 49.94 (2), 25.62. DARTHRMS: m/z calcd. for C34H33Cl2N3O5 [MH]+, 634.1865; Found: 634.1779. IR (solid) vmax: 3288, 2918, 2828, 1627, 1515, 1375, 1230, 1038, 944, 824, 762, 527. Purity, Method A: 99.9%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-hydroxybenzamide
(29).
Amide
coupling
method B; Yield = 7.4 mg, 30%. 1H NMR (500 MHz, Chloroform-d) δ 12.14 (s, 1H), 7.87 (s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.51 (td, J = 17.0, 16.5, 7.9 Hz, 4H), 7.43 (s, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 16.4, 8.5 Hz, 3H), 6.95 (d, J = 8.3 Hz, 3H), 6.78 (d, J = 8.7 Hz, 2H), 4.65 (p, J = 6.2 Hz, 1H), 4.43 – 4.27 (m, 1H), 4.00 (dd, J = 9.5, 5.2 Hz, 1H), 3.84 – 3.74 (m, 2H), 3.53 (q, J = 7.1 Hz, 1H), 3.37 (d, J = 4.7 Hz, 4H), 3.28 (d, J = 4.5 Hz, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 168.27, 161.92, 152.80, 149.12, 146.00, 139.37, 134.52, 134.37, 132.82, 130.98, 128.84, 128.59, 126.71, 125.26, 122.86 (2), 118.98, 118.86, 118.39 (2), 116.79 (2), 115.26 (2), 114.64, 109.17, 75.07, 68.45, 67.39, 50.70 (2), 49.64 (2), 25.90. DART-HRMS: m/z calcd. for
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C34H33Cl2N3O5 [MH]+, 634.1876; Found: 634.1843. IR (solid) vmax: 2924, 2852, 1598, 1511, 1452, 1228, 1191, 1036, 824, 756. Purity, Method A: 99.3%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-3-hydroxybenzamide
(30).
Amide
coupling,
method B; Yield = 8 mg, 45.5%. 1H NMR (500 MHz, Chloroform-d) δ 7.70 (s, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.52 (d, J = 7.5 Hz, 2H), 7.46 (s, 1H), 7.38 (s, 1H), 7.34 (s, 2H), 7.24 – 7.17 (m, 1H), 7.04 – 6.96 (m, 3H), 6.90 (d, J = 7.9 Hz, 2H), 6.73 (d, J = 7.8 Hz, 2H), 5.72 (s, 1H), 4.65 – 4.56 (m, 1H), 4.31 (t, J = 7.8 Hz, 1H), 3.95 (dd, J = 9.1, 5.2 Hz, 1H), 3.74 (t, J = 7.9 Hz, 2H), 3.31 (d, J = 5.2 Hz, 4H), 3.23 (d, J = 5.2 Hz, 4H), 1.78 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 165.26, 156.37, 152.76, 148.58, 146.01, 139.34, 136.59, 134.36, 132.80, 130.97, 130.33, 130.05, 128.58, 127.50, 121.75 (2), 118.96, 118.47, 118.36 (2), 116.92 (2), 115.25 (2), 114.62, 109.16, 75.07, 68.44, 67.39, 50.71 (2), 49.81 (2), 25.90. DART-HRMS: m/z calcd. for C34H33Cl2N3O5 [MH]+, 634.1876; Found: 634.1884. IR (solid) vmax: 3279, 2925, 2852, 2824, 1639, 1590, 1446, 1409, 1373, 1228, 1190, 115, 1036, 944, 826, 747. Purity, Method A: 96.2%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-4-hydroxybenzamide
(31).
Amide
coupling
method B; Yield = 7.7 mg, 31%. 1H NMR (500 MHz, Chloroform-d) δ 7.82 (d, J = 7.6 Hz, 2H), 7.68 (d, J = 5.9 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 7.43 (s, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 6.8 Hz, 4H), 6.78 (d, J = 7.9 Hz, 2H), 4.69 – 4.62 (m, 1H), 4.35 (t, J = 7.5 Hz, 1H), 4.02 – 3.96 (m, 1H), 3.78 (t, J = 7.6 Hz, 2H), 3.36 (s, 4H), 3.27 (s, 4H), 1.83 (s, 3H).
13C
NMR (126 MHz, CDCl3) δ 165.16, 158.80, 152.84, 148.46, 146.02, 139.35, 134.37,
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132.81, 130.98 (2), 130.73, 129.08 (2), 128.59, 126.71, 122.23, 121.74, 118.38 (2), 116.98 (2), 115.55 (2), 115.26 (2), 109.16, 75.07, 68.44, 67.39, 50.74 (2), 49.89 (2), 25.90. DART-HRMS: m/z calcd. for C34H33Cl2N3O5 [MH]+, 634.1876; Found: 634.1843. IR (solid) vmax: 3068, 2927, 2853, 1729, 1607, 1511, 1230, 1099, 824. Purity, Method A: 95.7%.
3-chloro-N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)benzamide (32). Amide coupling method A; Yield = 6.0 mg, 24%. 1H NMR (500 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.60 – 7.54 (m, 3H), 7.47 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H), 7.24 (dd, J = 8.4, 2.1 Hz, 1H), 7.04 (d, J = 8.9 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.40 – 4.30 (m, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.82 – 3.73 (m, 2H), 3.41 – 3.32 (m, 4H), 3.30 – 3.23 (m, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 164.11, 152.76, 146.00, 139.34, 139.25, 134.35, 131.72, 130.97, 130.13, 130.10, 129.93, 128.58, 127.34, 126.71, 125.04, 124.94, 121.74 (2), 118.36 (2), 116.88 (2), 115.24 (2), 109.16, 75.06, 68.44, 67.39, 50.72 (2), 49.77 (2), 25.90. DART-HRMS: m/z calcd. for C34H32Cl3N3O4 [MH]+, 648.2032; Found: 648.1998. IR (solid) vmax: 3311, 3283, 3254, 2961, 2854, 1640, 1588, 1512, 1451, 1375, 1228, 1192, 1152, 1037, 943, 820, 755. Purity, Method A: 99.8%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-3-methoxybenzamide
(33).
Amide
coupling
method A; Yield = 4.0 mg, 16. 1H NMR (500 MHz, Chloroform-d) δ 7.74 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.51 – 7.46 (m, 1H), 7.45 – 7.41 (m, 3H), 7.24 (dd, J = 8.4, 2.1 Hz, 1H), 7.14 – 7.08 (m, 1H), 7.04 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 9.0
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Hz, 2H), 4.65 (p, J = 6.2 Hz, 1H), 4.39 – 4.31 (m, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.92 (s, 3H), 3.81 – 3.74 (m, 2H), 3.37 (s, 4H), 3.27 (s, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 165.31, 162.01, 160.04, 152.82, 146.19, 139.41, 136.67, 134.37, 132.81, 131.02, 129.76, 128.64, 126.72, 123.36, 121.59 (2), 118.59, 118.36 (2), 117.88, 116.94 (2), 115.24 (2), 112.48, 109.16, 75.07, 68.44, 67.39, 55.52, 50.72 (2), 49.86 (2), 25.93. DART-HRMS: m/z calcd. for C35H35Cl2N3O5 [MH]+, 652.1537; Found: 652.1532. IR (solid) vmax: 3289, 2922, 2823, 138, 1580, 1510, 1450, 1374, 1288, 1190, 1150, 1033, 943, 817, 746, 686, 524. Purity, Method A: 95.0%.
3-acetyl-N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)benzamide (34). Amide coupling method A; Yield = 3.4 mg, 26%. 1H NMR (500 MHz, Chloroform-d) δ 8.47 (s, 1H), 8.16 (dd, J = 7.7, 1.7 Hz, 2H), 7.88 (s, 1H), 7.71 – 7.58 (m, 4H), 7.43 (d, J = 2.1 Hz, 1H), 7.24 (dd, J = 8.5, 2.2 Hz, 1H), 7.04 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.2 Hz, 1H), 3.84 – 3.72 (m, 2H), 3.37 (dd, J = 6.4, 3.6 Hz, 4H), 3.27 (dd, J = 6.4, 3.6 Hz, 4H), 2.72 (s, 3H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 197.40, 164.46, 152.76, 148.66, 146.01, 139.35, 137.38, 135.62, 134.35, 132.80, 131.75, 131.37, 130.97, 130.21, 129.26, 128.59, 126.71, 126.30, 121.80 (2), 118.36 (2), 116.86 (2), 115.24 (2), 109.16, 75.07, 68.44, 67.39, 50.72 (2), 49.76 (2), 26.78, 25.90. DART-HRMS: m/z calcd. for C36H35Cl2N3O5 [MH]+, 660.2021; Found: 660.1904; IR (solid) vmax: 3295, 2928, 2827, 1685, 1511, 1229, 1037, 823. Purity, Method B: 97.2%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)furan-3-carboxamide (35). Amide coupling method
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A; Yield = 2.0 mg, 8%. 1H NMR (500 MHz, Chloroform-d) δ 8.06 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 7.8 Hz, 3H), 7.43 (d, J = 2.1 Hz, 1H), 7.36 (s, 1H), 7.24 (dd, J = 8.5, 2.1 Hz, 1H), 7.02 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.82 – 6.71 (m, 3H), 4.65 (p, J = 6.0 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.4 Hz, 1H), 3.82 – 3.73 (m, 2H), 3.40 – 3.31 (m, 4H), 3.31 – 3.21 (m, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 171.43, 152.75, 148.53, 148.31, 146.01, 144.95, 143.99, 139.34, 137.89, 134.35, 132.80, 130.97, 128.58, 126.79, 126.71, 121.68 (2), 118.35 (2), 116.89 (2), 115.24 (2), 109.16, 75.07, 68.44, 67.39, 50.72 (2), 49.81 (2), 25.90. DART-HRMS m/z calcd. for C32H31Cl2N3O5 [MH]+, 608.1719; Found: 608.1701. IR (solid) vmax: 3313, 2918, 2849, 1636, 1512, 1375, 1233, 1155, 1037, 873, 821, 752, 601. Purity, Method B: 95.6%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)thiophene-3-carboxamide (36). Amide coupling method A; Yield = 3.0 mg, 13%. 1H NMR (500 MHz, Chloroform-d) δ 7.99 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.54 (dd, J = 21.5, 6.1 Hz, 4H), 7.44 (dd, J = 8.9, 2.7 Hz, 2H), 7.24 (dd, J = 8.4, 2.1 Hz, 1H), 7.03 (d, J = 8.9 Hz, 2H), 6.95 (d, J = 8.9 Hz, 2H), 6.78 (d, J = 8.9 Hz, 2H), 4.65 (p, J = 6.2 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.6, 5.3 Hz, 1H), 3.78 (dd, J = 9.2, 6.5 Hz, 2H), 3.41 – 3.32 (m, 4H), 3.30 – 3.21 (m, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 164.63, 152.74, 148.46, 146.02, 142.97, 139.34, 134.35, 132.80, 130.97, 130.36, 128.58, 128.35, 126.83, 126.70, 126.03, 121.66 (2), 118.35 (2), 116.92 (2), 115.23 (2), 109.15, 75.07, 68.44, 67.39, 50.72 (2), 49.84 (2), 25.90. DART-HRMS: m/z calcd. for C32H31Cl2N3O4S [MH]+, 624.1491; Found: 624.1473. IR (solid) vmax: 2922, 2853, 1635, 1512, 1259, 1036, 818. Purity, Method A: 96.8%.
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phenyl
(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)carbamate (37). A solution of aniline 8 (500 mg) and pyridine (1.3 mL) in CHCl3 (30 mL) was cooled to 0 °C. Phenyl chloroformate (0.134 mL) was added dropwise and the mixture stirred at RT for 3 h. Water and petroleum ether (1:1) were added at which time an off-white precipitate formed. The solid was washed with H2O and isopropyl alcohol to directly provide the trans-dioxolane carbamate 37. Yield = 500 mg, 81%.1H NMR (500 MHz, Chloroform-d) δ 7.66 (d, J = 8.4 Hz, 1H), 7.49 – 7.37 (m, 5H), 7.26 (dd, J = 21.6, 7.6 Hz, 4H), 7.04 – 6.97 (m, 4H), 6.93 (d, J = 9.1 Hz, 2H), 6.86 (s, 1H), 4.37 (p, J = 6.3 Hz, 1H), 4.16 (dd, J = 9.4, 5.1 Hz, 1H), 4.09 – 3.97 (m, 2H), 3.92 – 3.86 (m, 1H), 3.34 (d, J = 5.0 Hz, 4H), 3.31 – 3.26 (m, 4H), 1.86 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 152.93, 150.75, 146.09, 138.17, 134.57, 132.85, 131.21, 129.39 (2), 128.92, 127.88, 126.80, 125.60, 121.66 (2), 120.35, 118.55, 118.39 (2), 117.17 (2), 115.50 (2), 114.55 (2), 109.10, 73.97, 69.31, 67.03, 50.74 (2), 49.98 (2), 25.74. IR (solid) vmax: 3332, 3292, 2926, 2825, 1733, 1706, 1510, 1490, 1450, 1414, 1227, 1179, 1161, 1034, 943, 823, 747, 687. Xevo ESI-HRMS: m/z calcd. for C34H34Cl2N3O5 [MH]+, 633.1797; Found: 634.1865. Purity, Method A: 99.1%.
phenyl
(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)carbamate (38). A solution of aniline 9 (500 mg) and pyridine (1.3 mL) in CHCl3 (30 mL) was cooled to 0 °C. Phenyl chloroformate (0.134 mL) was added dropwise and the mixture stirred at RT for 3 h. Water and petroleum ether (1:1) were added at which time an off-white precipitate formed. The solid was washed with H2O and isopropyl alcohol to directly provide the cis-dioxolane carbamate 38. Yield = 480 mg, 78%. 1H NMR (500
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MHz, Chloroform-d) δ 7.68 (d, J = 8.5 Hz, 1H), 7.43 (t, J = 7.7 Hz, 5H), 7.26 (dd, J = 22.2, 7.7 Hz, 4H), 7.01 (d, J = 8.4 Hz, 3H), 6.96 (s, 2H), 6.87 (s, 1H), 6.78 (d, J = 8.8 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.38 – 4.33 (m, 1H), 4.00 (dd, J = 9.6, 5.3 Hz, 1H), 3.82 – 3.75 (m, 2H), 3.35 (s, 4H), 3.28 (s, 4H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 150.75, 139.36, 134.37, 132.81, 131.34, 130.98, 129.39 (2), 128.59, 128.59, 127.51, 126.71, 126.52, 125.60, 125.16, 121.66 (2), 120.34 (2), 118.43 (2), 117.23 (2), 115.27 (2), 109.17, 75.06, 68.44, 67.38, 50.78 (2), 49.94 (2), 25.90. Xevo ESI-HRMS: m/z calcd. for C34H34Cl2N3O5 [MH]+, 633.1797; Found: 634.1866. IR (solid) vmax: 3332, 2958, 2926, 2853, 1757, 1736, 1553, 1510, 1491, 1417, 1229, 1195, 1181, 1162, 1024, 911, 828, 751, 688. Purity, Method A: 99.9%.
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)hydrazinecarboxamide
(39).
A
solution
of
hydrazine hydrate (0.132 mL) and 37 (500 mg) in 1,4-dioxane (30 mL) was stirred at reflux for 3 h. After cooling to room temperature, the mixture was poured into H2O (15 mL) and the resulting precipitate was filtered and washed with H2O and isopropyl alcohol to directly provide the transdioxolane carboxamide 39. Yield = 400 mg, 89%. 1H NMR (500 MHz, Chloroform-d) δ 8.07 (s, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.52 (s, 1H), 7.49 – 7.39 (m, 3H), 7.29 (s, 1H), 7.02 – 6.97 (m, 4H), 6.93 (d, J = 8.3 Hz, 2H), 4.37 (s, 1H), 4.16 (s, 1H), 4.05 (s, 1H), 3.96 – 3.78 (m, 2H), 3.33 (s, 4H), 3.28 (s, 4H), 1.86 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 153.65, 152.87, 147.69, 147.08, 146.15, 138.17, 134.56, 132.84, 131.20, 128.92, 126.80, 121.21 (2), 118.35 (2), 117.25 (2), 115.48 (2), 109.09, 73.97, 69.30, 67.04, 50.77 (2), 50.21 (2), 25.74. DART-HRMS: m/z calcd. for C28H32Cl2N5O4 [MH]+, 572.1753; Found: 572.1958. IR (solid) vmax: 2916, 2848, 1546, 1510, 1462, 1450, 1375, 1225, 1197, 1037, 945, 824. Purity, Method A: 95.0%.
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N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)hydrazinecarboxamide
(40).
A
solution
of
hydrazine hydrate (0.127 mL) and 38 (480 mg) in 1,4-dioxane (30 mL) was stirred at reflux for 3 h. After cooling to room temperature, the mixture was poured into H2O and the resulting precipitate was filtered and washed with H2O and isopropyl alcohol to directly provide the cis-dioxolane carboxamide 40. Yield = 380 mg, 88%. 1H NMR (500 MHz, Chloroform-d) δ 8.06 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.46 (d, J = 8.9 Hz, 2H), 7.43 (t, J = 4.0 Hz, 2H), 7.37 (s, 1H), 7.28 (d, J = 7.4 Hz, 1H), 7.24 (d, J = 8.2 Hz, 2H), 7.00 (dd, J = 8.9, 4.6 Hz, 2H), 6.94 (d, J = 13.2 Hz, 2H), 6.78 (d, J = 9.1 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.35 (dd, J = 8.5, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.82 – 3.73 (m, 2H), 3.37 – 3.30 (m, 4H), 3.30 – 3.21 (m, 5H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 153.56, 146.98, 139.35, 134.37, 132.81, 130.98, 129.40, 128.60, 126.72, 125.60, 121.66, 121.21 (2), 118.34 (2), 117.25 (2), 115.24 (2), 109.16, 75.07, 68.44, 67.40, 50.75 (2), 50.20 (2), 25.90. Xevo ESI-HRMS: m/z calcd. for C28H32Cl2N5O4 [MH]+, 572.1753; Found: 572.1711. IR (solid) vmax: 2920, 2876, 2851, 1554, 1511, 1464, 1450, 1377, 1225, 1184, 1151, 1035, 944, 826. Purity, Method A: 100%.
4-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one (41). A solution of formamidine acetate (364 mg) and 39 (400 mg) in anhydrous DMF (30 mL) was stirred at 130 °C for 3 h. The mixture was cooled to room temperature and diluted with H2O at which point a light brown precipitate formed. The solid was filtered and washed with CHCl3 to provide transdioxolane triazolone 41. Yield = 187 mg, 46%. 1H NMR (500 MHz, Chloroform-d) δ 7.66 (d, J =
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8.3 Hz, 1H), 7.52 – 7.42 (m, 3H), 7.28 (d, J = 8.4 Hz, 1H), 7.04 (dd, J = 46.8, 8.1 Hz, 5H), 6.94 (s, 2H), 4.37 (s, 1H), 4.20 – 4.12 (m, 1H), 4.09 – 3.98 (m, 2H), 3.89 (t, J = 7.5 Hz, 1H), 3.39 (d, J = 34.6 Hz, 4H), 3.28 (s, 4H), 1.86 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 162.76, 158.68, 145.98, 138.29, 134.57, 132.84, 131.20, 128.91, 126.80, 123.99, 121.47, 118.41 (2), 117.33 (2), 116.86 (2), 116.69, 115.51 (2), 109.29, 73.96, 69.30, 67.02, 50.71 (2), 49.79 (2), 25.73. Xevo ESI-HRMS: m/z calcd. for C29H29Cl2N5O4 [MH]+, 582.1597; Found: 582.1666 IR (solid) vmax: 2958, 1687, 1509, 1227, 1191, 1036, 941, 819, 455. Purity, Method A: 95.7%
4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one (42). A solution of formamidine acetate (342 mg) and 40 (380 mg) in anhydrous DMF (30 mL) was stirred at 130 °C for 3 h. The mixture was cooled to room temperature and diluted with H2O at which point a light brown precipitate formed. The solid was filtered and washed with CHCl3 to provide cis-dioxolane triazolone 42. Yield = 78 mg, 20%. 1H NMR (500 MHz, Chloroform-d) δ 7.68 (d, J = 8.5 Hz, 1H), 7.52 (dd, J = 30.0, 9.9 Hz, 1H), 7.43 (s, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 7.02 – 6.96 (m, 2H), 6.94 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 8.9 Hz, 2H), 4.70 – 4.61 (m, 1H), 4.38 – 4.30 (m, 1H), 3.99 (dd, J = 9.7, 5.3 Hz, 1H), 3.83 – 3.75 (m, 2H), 3.30 (d, J = 42.8 Hz, 8H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 162.70, 158.66, 152.78, 149.48, 148.66, 146.00, 139.36, 134.36, 132.81, 128.58, 126.71, 121.48, 118.36 (2), 117.32 (2), 116.86 (2), 115.27 (2), 109.17, 75.07, 68.45, 67.38, 50.70 (2), 49.78 (2), 25.90. DART-HRMS: m/z calcd. for C29H30Cl2N5O4 [MH]+, 582.1597; Found: 582.1728 . IR (solid) vmax: 2961, 1510, 1258, 1022, 1016, 794. Purity, Method A: 97.1%.
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BIOLOGICAL ASSAY PROTOCOLS. General Information for Cell Culture Studies Protocols for general cell culture, qPCR, and antiproliferation in the ASZ cells are as previously described.9,23,24 All Gli1 mRNA studies are normalized to actin as an internal control and concentrations of compound that are cytotoxic to the cells (based on visual assessment and/or a significant decrease in actin expression) are excluded from the analysis. Protocols for the initiation and growth of Math1-Cre-ER;Ptcfl/fl medulloblastoma tumors, isolation, and in vitro culture of MERP MB cells, and the antiproliferation and qPCR studies carried out in these cells were previously described.16–18 Data was analyzed using GraphPad Prism 5 and reported values represent mean SEM for at least two separated experiments carried out in triplicate.
DOCKING PROTOCOLS. The docking and analysis of ITZ analogues in our homology model of Smo was performed as previously described.7
ASSOCIATED CONTENT Supporting Information. Synthesis protocols and characterization of previously disclosed ITZ intermediates; general protocols for pharmacokinetic assays; 1H and 13C NMR spectra for all new intermediates and final des-ITZ analogues; anti-proliferation data within ASZ cells for compounds 27, 30, 34, and 39. Compound structures and associated biological data for compounds 1-42 are available within the molecular formula strings.
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AUTHOR INFORMATION Corresponding Author *Tel: 1-860-486-8446. Fax: 1-860-486-6857. Email:
[email protected] Author Contributions J.R.P. and K.A.T. synthesized ITZ analogues and performed biological evaluations. L.Q.C. performed compound evaluation in the Ptch-CKO cells. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Present Addresses †J.R.P.
Current Address: Tufts University Department of Chemistry, 62 Talbot Avenue, Medford
MA, 02155 ‡K.A.T.
Current Address: Western Michigan University Department of Chemistry, Kalamazoo,
MI, 49008
ACKNOWLEDGMENTS We gratefully acknowledge support of this work by the National Institutes of Health/National Cancer Institute (CA190617). J. R. P. gratefully acknowledges financial support from the Division of Medicinal Chemistry of the American Chemical Society (MEDI Pre-doctoral Fellowship). ASZ001 cells were provided by Dr. Ervin Epstein (Children’s Hospital Oakland Research Institute).
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ABBREVIATIONS ITZ; itraconazole; Hh: Hedgehog; Gli: glioblastoma associated oncogene; Ptch: patched; MEF: mouse embryonic fibroblast. Ptch-CKO; primary murine Hh-dependent medulloblastoma cells.
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Yauch, R. L.; Dijkgraaf, G. J. P.; Alicke, B.; Januario, T.; Ahn, C. P.; Holcomb, T.; Pujara, K.; Stinson, J.; Callahan, C. A.; Tang, T.; Bazan, J. F.; Kan, Z.; Seshagiri, S.; Hann, C. L.; Gould, S. E.; Low, J. A.; Rudin, C. M.; de Sauvage, F. J. Smoothened mutation confers resistance to a hedgehog pathway inhibitor in medulloblastoma. Science 2009, 326, 572– 574.
(10) Pace, J. R.; DeBerardinis, A. M.; Sail, V.; Tacheva-Grigorova, S. K.; Chan, K. A.; Tran, R.; Raccuia, D. S.; Wechsler-Reya, R. J.; Hadden, M. K. Repurposing the clinically efficacious antifungal agent itraconazole as an anticancer chemotherapeutic. J. Med. Chem. 2016, 59, 3635–3649. (11) Baji, H.; Kimny, T.; Gasquez, F.; Flammang, M.; Compagnon, P. L.; Delcourt, A.; Mathieu, G.; Viossat, B.; Morgant, G.; Nguyen-Huy, D. Synthesis, antifungal activity and structure-
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activity relationships of 2-(alkyl or aryl)-2-(alkyl or polyazol-1-ylmethyl)-4-(polyazol-1ylmethyl)-1, 3-dioxolanes. Euro. J. Med. Chem. 1997, 32, 637–650. (12) Shi, W.; Nacev, B. A.; Bhat, S.; Liu, J. O. Impact of absolute stereochemistry on the antiangiogenic and antifungal activities of itraconazole. ACS Med. Chem. Lett. 2010, 1, 155–159. (13) Shi, W.; Nacev, B. A.; Aftab, B. T.; Head, S.; Rudin, C. M.; Liu, J. O. Itraconazole side chain analogues: Structure–activity relationship studies for inhibition of endothelial cell proliferation, vascular endothelial growth factor receptor 2 (VEGFR2) glycosylation, and hedgehog signaling. J Med Chem 2011, 54, 7363–7374. (14) Montalbetti, C. A. G. N.; Falque, V. Amide bond formation and peptide coupling. Tetrahedron 2005, 61, 10827–10852. (15) Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu, Y.-T.; Lovey, R.; Hendrata, S.; Huang, Y.; Pan, W.; Parekh, T.; Pinto, P.; Popov, V.; Pike, R.; Ruan, S.; Santhanam, B.; Vibulbham, B.; Wu, W.; Yang, W.; Kong, J.; Liang, X.; Wong, J.; Liu, R.; Butkiewicz, R.C.; Hart, A.; Agrawal, S.; Ingravallo, P.; Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.; Prongay, A.; Madison, V.; Broske, L.; Cui, X.; Cheng, K.-C.; Hsieh, Y.; Brisson, J.-M.; Prelusky, D.; Korfmacher, W.; White, R.; BogdanowichKnipp, S.; Pavlovsky, A.; Bradley, P.; Saksena, A.K.; Ganguly, A.; Piwinski, J.; Girijavallabhan,
V.
and
Njoroge,
F.G.
Discovery
of
(1R,5S)-N-[3-Amino-1-
(Cyclobutylmethyl)-2,3-Dioxopropyl]Dimethylethyl)Amino]Carbonyl]Amino]-3,3-Dimethyl-1-Oxobutyl]-
3-[2(S)-[[[(1,16,6-Dimethyl-3-
Azabicyclo[3.1.0]Hexan-2(S)-Carboxamide (SCH 503034), a selective, potent, orally
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bioavailable hepatitis C virus NS3 protease inhibitor: A potential therapeutic agent for the treatment of hepatitis C infection. J. Med. Chem. 2006, 49, 6074–6086. (16) Venkatraman, S.; Velazquez, F.; Wu, W.; Blackman, M.; Madison, V.; Njoroge, F. G. Potent ketoamide inhibitors of HCV NS3 protease derived from quaternized P1 groups. Bioorg. Med. Chem. Lett. 2010, 20, 2151–2155. (17) Yang, Z.-J.; Ellis, T.; Markant, S. L.; Read, T.-A.; Kessler, J. D.; Bourboulas, M.; Schüller, U.; Machold, R.; Fishell, G.; Rowitch, D. H.; Wainwright, B. J.; Wechsler-Reya, R. J. Medulloblastoma can be initiated by deletion of patched in lineage-restricted progenitors or stem cells. Cancer Cell 2008, 14, 135–145. (18) Markant, S. L.; Esparza, L. A.; Sun, J.; Barton, K. L.; McCoig, L. M.; Grant, G. A.; Crawford, J. R.; Levy, M. L.; Northcott, P. A.; Shih, D.; Remke, M.; Taylor, M. D.; Wechsler-Reya, R. J. Targeting sonic hedgehog-associated medulloblastoma through inhibition of aurora and polo-like kinases. Cancer Res. 2013, 73, 6310–6322. (19) Brun, S. N.; Markant, S. L.; Esparza, L. A.; Garcia, G.; Terry, D.; Huang, J.-M.; Pavlyukov, M. S.; Li, X.-N.; Grant, G. A.; Crawford, J. R.; Levy, M. L.; Conway, E. M.; Smith, L. H.; Nakano, I.; Berezov, A.; Greene, M. I.; Wang, Q.; Wechsler-Reya, R. J. Survivin as a therapeutic target in sonic hedgehog-driven medulloblastoma. Oncogene 2015, 34, 3770– 3779. (20) Miyama, T.; Takanaga, H.; MAtsuo, H.; Yamano, K.; Yamamoto, K.; Iga, T.; Naito, M.; Tsuruo, T.; Ishizuka, H.; Kawahara, Y.; Sawada, Y. P-glycoprotein-meidated transport of itraconazole across the blood-brain barrier. Antimicrob. Agents Chemother. 1998, 42, 1738-1744.
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(21) Mishra, A.; Mishra, S.; Ninama, S.; Pritam, A. EXAFS study of copper thiosemicarbazide complexes. J. Phys.: Conf. Ser. 2014, 534, 012029. (22) Heeres, J.; Backx, L. J. J.; Van Cutsem, J. Antimycotic azoles. 7. Synthesis and antifungal properties of a series of novel triazol-3-ones. J. Med. Chem. 1984, 27, 894–900. (23) Tanoury, G. J.; Hett, R.; Wilkinson, H. S.; Wald, S. A.; Senanayake, C. H. Total synthesis of (2R,4S,2′S,3′R)-hydroxyitraconazole: Implementations of a recycle protocol and a mild and safe phase-transfer reagent for preparation of the key chiral units. Tetrahedron: Asymmetry 2003, 14, 3487–3493. (24) Banerjee, U.; Ghosh, M.; Kyle Hadden, M. Evaluation of vitamin D3 A-ring analogues as hedgehog pathway inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 1330–1334.
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Table of Contents Graphic: O O Cl
Cl
O O
N
N
2a, ASZ IC50 = 24 nM Ptch-CKO GI50 = 1.0 11 synthetic steps
O O Cl
N N
N
O O
N
N
N H
Cl
OH
30, ASZ IC50 = 160 nM Ptch-CKO GI50 = 0.35 7 synthetic steps
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