Structure-Activity Relationships of New Natural Product-Based Diaryloxazoles with Selective Activity against Androgen Receptor-Positive Breast Cancer Cells

Article abstract of DOI:10.1021/acs.jmedchem.7b01228

Targeted therapies for ER+/PR+ and HER2-amplified breast cancers have improved patient survival, but there are no therapies for triple negative breast cancers (TNBC) that lack expression of estrogen and progesterone receptors (ER/PR), or amplification or

Full text of DOI:10.1021/acs.jmedchem.7b01228

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Article
Structure-activity relationships of new natural product-based diaryloxazoles
with selective activity against androgen receptor-positive breast cancer cells
Andrew J. Robles, Shelby McCowen, Shengxin Cai, Michaels Glassman,
Francisco Ruiz, Robert H. Cichewicz, Stanton F McHardy, and Susan L. Mooberry
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01228 • Publication Date (Web): 20 Oct 2017
Downloaded from http://pubs.acs.org on October 23, 2017
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Structureꢀactivity relationships of new natural
productꢀbased diaryloxazoles with selective activity
against androgen receptorꢀpositive breast cancer
cells
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†
§,
⊥
ǁ,
∇
§
Andrew J. Robles, Shelby McCowen, Shengxin Cai, Michaels Glassman, Francisco Ruiz
§,
ǁ,
∇
*,†, §
*,†,‡
¥
II, Robert H. Cichewicz, Stanton F. McHardy,
and Susan L. Mooberry
†
‡
Department of Pharmacology, and UT Health San Antonio Cancer Center, UT Health San
Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, U.S.A.
§
Center for Innovative Drug Discovery, Department of Chemistry, The University of Texas at
San Antonio, 1 UTSA Circle, San Antonio, TX, 78249. U.S.A.
ǁ
Natural Product Discovery Group, Institute for Natural Products Applications and Research
∇
Technologies, and Department of Chemistry & Biochemistry, University of Oklahoma, 101
Stephenson Parkway, Norman, OK, 73019, U.SA.
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Key Words: tripleꢀnegative breast cancer, natural products, structureꢀactivity relationship,
Amyris texana, androgen receptor
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ABSTRACT
Targeted therapies for ER+/PR+ and HER2ꢀamplified breast cancers have improved patient
survival, but there are no therapies for triple negative breast cancers (TNBC) that lack expression
of estrogen and progesterone receptors (ER/PR), or amplification or overexpression of HER2.
Gene expression profiling of TNBC has identified molecular subtypes and representative cell
lines. An extract of the Texas native plant Amyris texana was found to have selective activity
against MDAꢀMBꢀ453 cells, a model of the luminal androgen receptor (LAR) subtype of TNBC.
Bioassayꢀguided fractionation identified two oxazole natural products with selective activity
against this cell line. Analog synthesis and structureꢀactivity relationship studies conducted
provided analogs with more potent and selective activity against two LAR subtype cell line
models, culminating in the discovery of compound 30 (CIDDꢀ0067106). Lead compounds
discovered have potent and selective antiproliferative activities, and mechanisms of action
studies show they inhibit the activity of the mTORC1 pathway.
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INTRODUCTION
Tripleꢀnegative breast cancers (TNBC) represent approximately 15% of breast cancers and are
defined by the lack of expression of estrogen/progesterone receptors (ER/PR) and human
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epidermal growth factor receptor 2 (HER2). However, the classification of these cancers as
tripleꢀnegative only provides information as to what these cancers are not, rather than what they
are. Patients with TNBC typically have poorer prognoses than patients with ER/PR+ and HER2ꢀ
amplified tumors; the median survival is a mere 13 months for patients with metastatic
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,3,4
TNBC.
There are no targeted therapies or validated pharmacological targets for TNBC.
Therefore, patients receive chemotherapy with cytotoxic and genotoxic agents, which include
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,3
taxanes, eribulin, anthracyclines, carboplatin, gemcitabine and/or cyclophosphamide.
However, drugꢀresistance invariably develops leaving few effective treatment options. Patients
with ER+/PR+ and/or HER2ꢀamplified breast cancers are now surviving longer as a direct result
of the development of targeted therapies that inhibit the molecular drivers of these cancers.
However, the highly heterogeneous nature of TNBC, which encompasses diverse cancers with
different molecular and phenotypic characteristics, has thus far precluded targeted therapies for
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these patients.
In 2011, Lehmann and Bauer et al conducted a large metaꢀanalysis of gene expression
profiles for TNBC patient tumor samples and identified 6 distinct molecular subtypes of TNBC,
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each with defined molecular alterations and phenotypic characteristics. More recently, Lehmann
et al refined these subtypes based on new analyses, identifying four molecular subtypes of
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TNBC. These include two basalꢀlike (BL1 and BL2), a mesenchymalꢀlike (M) and a luminal
androgen receptor (LAR) subtype. They also identified cell lines representing these subtypes,
thus allowing us to initiate a screening program to identify natural product extracts with selective
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activities against cell lines representing the different TNBC subtypes. In this study, a crude
extract of the Texas native plant Amyris texana was found to have selective activity against
MDAꢀMBꢀ453 cells, a model of the LAR subtype of TNBC. The LAR subtype accounts for
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approximately 16% of all TNBCs, or roughly 3% of all breast cancers. These tumors express
high levels of androgen receptors (AR), making it the TNBC subtype with the clearest
biomarker. Bioassayꢀguided fractionation identified one new and one known oxazole with
selective activity in this cell line. Based on these data, 32 analogs of the natural products were
synthesized and structureꢀactivity relationship studies were performed to identify a lead
candidate for in vivo efficacy experiments and investigations into the mechanisms of action for
this class of compounds against LAR cells.
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RESULTS AND DISCUSSION
Isolation and identification of novel cytotoxic oxazoles from Amyris texana
We initiated a screening program to identify crude extracts of plants and fungal cultures
with selective activity against cell lines modeling five molecular subtypes of TNBC. It was
hypothesized that based on the different molecular characteristics and drivers of each subtype,
extracts with selective activity against particular cell lines could be identified from novel natural
product libraries. The plant and fungal extract libraries screened for this project were generated
by the Mooberry and Cichewicz laboratories and previously yielded both new and known
compounds with selective in vitro activities against different TNBC molecular subtypes in
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vitro.
The crude supercritical CO extract of the leaves and branches of Amyris texana,
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commonly known as the Texas torchwood, displayed modest selective antiproliferative activity
against MDAꢀMBꢀ453 cells, a model of the LAR subtype, relative to cells representing other
molecular subtypes of TNBC. A second sample of the plant material was collected, extracted,
and also found to have selective antiproliferative activity against this cell line (Figure 1A). The
crude plant extract was subjected to bioassayꢀguided fractionation on silica gel VLC and HP20ss
columns, followed by C preparative HPLC and C semiꢀpreparative HPLC. Fractions F2 and
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F3 showed selective antiproliferative activity against MDAꢀMBꢀ453 cells relative to HCC70
cells which model the BL2 subtype (Figure 1B, 1C), and were selected for further purifications
to identify the active constituents. From these fractions, one new diaryloxazole (1), which we
named texazole A, and one known diaryloxazole (2) were purified (Figure 2). Isolation of 2 from
Amyris plumieri was first reported by Philip, Burke and Jacobs in 1984, but its cytotoxic activity
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against cancer cell lines was not investigated. The chemical structures of both 1 and 2 were
established unequivocally through analysis of the spectroscopic data of both the naturally
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sourced material (see Supplementary Figures S1, S2 and S3ꢀS8), as well as the spectroscopic
data for both 1 and 2 from materials produced through two different and independent synthesis
routes.
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The structure of compounds 1 and 2 were of interest to us not only because of their
selectivity for cells representing the LAR subtype, but also the opportunity they represent as
structural leads for further medicinal chemistry investigation based on multiple structural
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moieties for SAR studies and favorable drugꢀlike physicochemical properties. Compounds 1
and 2 are both comprised of an oxazole ring core substituted at the 2ꢀ and 5ꢀpositions. The
phenyl substituent at the 5ꢀposition either bears a 4ꢀsubstituted Oꢀprenyl group (compound 1) or
a 4ꢀsubstituted Oꢀgeranyl group (compound 2). The aryl substituent at the 2ꢀposition is either
phenyl (compound 1) or 3ꢀpyridyl (compound 2). Compounds 1 and 2 are also similar to the
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diaryloxazoles texalin (Figure 2), previously isolated from Amyris elemifera, and balsoxin
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(Figure 2), isolated from Amyris balsamifera.
Additionally, Cheplogoi and colleagues
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reported the isolation of uguenenazole (Figure 2) from Vepris uguenensis, suggesting similar
compounds may be present in multiple plant genera. Although other diaryloxazoles with
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algicidal and fungicidal activities have been isolated from A. texana, to our knowledge there
are no reports of these compounds having cytotoxic activity against cancer cells.
Evaluation of in vitro antiproliferative and cytotoxic activities of isolated natural products
The in vitro antiproliferative and cytotoxic activities of the natural products 1 and 2 were
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evaluated using a sulforhodamine B (SRB) assay.
Both compounds displayed modest
selective antiproliferative and cytotoxic activity against MDAꢀMBꢀ453 cells, a model of the
LAR subtype, compared to other TNBC cell lines (Figure 1D, 1E). Since our goal is to identify
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targeted therapies for the subtypes of TNBC, we sought to identify compounds that have potent
activities in a cell line representing one TNBC subtype, as compared to cells representing the
other subtypes. The discovery of such natural products is predicated on the hypothesis that these
compounds are likely to specifically target a molecular driver of the sensitive cells. The results
with 1 and 2 showed that both of these compounds had relatively low cytotoxic potency. The
concentration of 1 that inhibited growth by 50% (GI ) was approximately 40 ꢀM in MDAꢀMBꢀ
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453 cells, but greater than 200 ꢀM in HCC70 and MDAꢀMBꢀ231 cells (Table 1). The GI of 2
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was approximately 17 ꢀM in MDAꢀMBꢀ453 cells, but ranged from 77 – 100 ꢀM in HCC70,
HCC1937, and MDAꢀMBꢀ231 cells. These results demonstrate that 1 and 2 have at least 5ꢀ and
4.5ꢀfold selectivity for MDAꢀMBꢀ453 cells. However, we expected that more potent analogs
would be optimal for in vivo efficacy studies. Additionally, both compounds, but particularly 1,
have low aqueous solubility and analogs with improved physicochemical properties will be
needed for consideration of this class of compounds for more advanced in vivo profiling.
Synthesis of natural products 1 and 2 and structural analogs
Having isolated, characterized and screened natural products 1 and 2, we aimed to
prepare the two compounds via independent synthesis to confirm structure, purity and the
antiproliferative and cytotoxic activities with synthesized samples. To this end, we utilized two
different synthesis strategies to prepare 1 and 2, such that the regiochemistry of the diaryloxazole
substitution could be established, and two different synthesis routes would be available to
prepare analogs for SAR studies. Within our synthesis strategy, we focused on developing a
synthesis route that would allow us to easily incorporate structural changes at the 2ꢀposition of
the oxazole core as well as the 4ꢀphenolic ether side chain moieties to support preliminary SAR
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studies. In the first route to obtain 1 and 2 (Scheme 1), paraꢀanisaldehyde was subjected to Van
Leusen oxazole forming conditions to provide the desired oxazole intermediate 5 in excellent
yield. With this common intermediate in hand, two different synthesis methods were employed
to access the desired natural products 1 and 2. First, functionalization of the 2ꢀposition of the
oxazole was carried out using a palladium mediated cross coupling reaction with an aryl bromide
in the presence of CuI and K CO under microwave conditions to provide the desired coupled
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product 6 in low to moderate yields, depending on the aryl bromide substrate employed.
Multiple coupling conditions were screened for this transformation and we ultimately settled on
a palladiumꢀfree, copperꢀmediated cross coupling reaction reported by the Miura group, which
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proved to be more synthetically useful and reproducible. Under these conditions (CuI, PPh ,
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Na CO , DMF), compound 6 could be produced in moderate yield with a wide range of aryl and
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heteroaryl bromide coupling partners. Thus, the forward synthesis involved treatment of
compound 5 with excess BBr which cleanly produced the corresponding phenol (not depicted),
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which was alkylated under standard conditions to produce the geranyl or prenyl derivatives
represented by compound 7. Compound 7 was subjected to the CuI/PPh coupling conditions
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with either bromobenzene or 3ꢀbromopyridine to provide compounds 1 and 2. Alternatively,
compound 6 could undergo the BBr demethylation/alkylation procedure to provide the final
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compounds 1 and 2 directly. In addition to the two natural products 1 and 2 prepared using these
routes, the corresponding structural congeners 3 and 4 were also prepared. Analytical
characterization and spectral data of the syntheticallyꢀderived samples of compounds 1 and 2
supported the desired structures and were spectroscopically identical to those samples isolated
from the natural source (See Supplementary Figures S1ꢀS14). To further confirm the
regiochemistry of the oxazole substitution in 6 and develop an alternative route to analogs, we
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employed a similar route to that described by Copp and coꢀworkers during their reported
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synthesis of the oxazole texalin (Figure 2). Thus, pꢀmethoxyacetophenone was carried through
a 3ꢀstep sequence involving bromination, azide formation and reduction to form the desired
amine 8. Coupling of 8 and benzoic acid under HATU/DIPEA conditions afforded the desired
benzamide 9, which was subjected to Ac O/H SO cyclization conditions to afford oxazole 6.
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The H and C NMR, as well as the HPLC/MS data for both samples of 6 from the two different
synthetic routes were found to be identical, thus confirming the regiochemistry of oxazole 6 (See
Supplementary Figures S15 and S16). Utilizing the synthesis methods described in Scheme 1,
compounds 1ꢀ4 and additional structural analogs 10ꢀ20, highlighted in Chart 1, were also
prepared for SAR studies.
In vitro evaluation and SAR
The antiproliferative and cytotoxic activities of compounds 1 ꢀ 4 were evaluated in a
panel of TNBC cells lines with the SRB assay. All four compounds inhibited growth and were
cytotoxic to MDAꢀMBꢀ453 cells. The GI values of syntheticallyꢀderived 1 and 2 were 60 and
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11 ꢀM, respectively (Table 1). These potencies were comparable to the natural products, further
confirming the determined structures. Compounds 1 and 4, which have a phenyl group at the 2ꢀ
position of the oxazole, were less potent than compounds 2 and 3, which have a 3ꢀpyridine at the
2ꢀposition of the oxazole. (Table 1). This suggested that a 3ꢀpyridine group at the 2ꢀposition of
the oxazole is advantageous for potency in this cell line and also offers physicochemical property
advantages over the corresponding phenyl derivatives by providing lower lipophilicity and
higher topological polar surface area. Interestingly, while 2 retained the selectivity observed with
the natural product, compound 3 did not have the same selectivity for MDAꢀMBꢀ453 cells as
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compared to cells representing other TNBC molecular subtypes. This suggested that the longer
geranyl side chain, together with the 3ꢀpyridine moiety, are important for LAR subtype
specificity, however further SAR studies were required.
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Based on the preliminary data obtained from evaluation of the first four oxazoles, SAR
studies focused on changing both the heteroaryl group at the 2ꢀposition of the oxazole, as well as
the structure and electronic nature of the phenol side chain, as represented by analogs 10ꢀ20
(
Chart 1). The antiproliferative activities of each compound were evaluated and nine of these
compounds showed antiproliferative and/or cytotoxic activities at concentrations below 100 ꢀM
Table 1). Compounds 15 and 20 did not have any activity at 100 ꢀM so their biological effects
were not evaluated further. The GI values for the other nine compounds ranged from 2 – 40
(
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ꢀM in MDAꢀMBꢀ453 cells (Table 1). First, modification of the phenolic side chain to nꢀpropyl
(compound 10) produced an analog with similar potency to the natural product 2. Replacement
of the 3ꢀpyridine moiety with other 6ꢀmember heteraromatics such as pyrimidine provided potent
activity as well, assuming the prenyl phenolic side chain was present (compound 13 and 18).
Fiveꢀmembered heterocyclic analogs such as 1Hꢀpyrrazole showed a marked decrease in potency
(compound 16). A phenolic ester functionality such as acetate also decreased potency as
represented by compound 14. Installation of a 4ꢀpyrrolidine moiety (compound 11) produced
potency in the MDAꢀMBꢀ453 cell line virtually equivalent to natural product 2; even though
compound 11 possesses the methyl group at the R position. This result suggests that some
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structural modifications at both positions are tolerated and can produce analogs with good
potency. Further investigation into pyridine substitution patterns and the substituent effects lead
to the discovery of compound 17, which was the most potent analog identified during initial SAR
studies. Compound 17 showed selectivity for MDAꢀMBꢀ453 cells, consistent with the activities
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of the natural products (Figure 3). The activity of this compound also indicated that the relative
position of the pyridine nitrogen could be changed while still maintaining the potency and
selectivity for MDAꢀMBꢀ453 cells observed with the natural products. The GI for 17 was
5
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found to be 2 ꢀM in MDAꢀMBꢀ453 cells, but ranged from 6 – 9 ꢀM in the other TNBC cells,
indicating 3ꢀ to 4.5ꢀfold selectivity for MDAꢀMBꢀ453 cells compared to other TNBC cell lines
(Figure 3, Table 1). The structural nature and physicochemical properties of compound 17 were
also greatly improved relative to the natural products by lowering LogD, increasing tPSA and
providing a tertiary amine handle for salt formation, all of which likely contributed to the
improved aqueous solubility observed for 17 in comparison to earlier compounds. These results
indicated that analogs with improved pharmacokinetic properties could be synthesized without
compromising potency for MDAꢀMBꢀ453 cells, prompting further efforts into the synthesis of
LAR subtypeꢀselective oxazoles with improved pharmaceutical properties. Based on the
outcomes of the brief SAR study described in Table 1 and the identification of lead compound
17, we focused our medicinal chemistry efforts to study the impact other structural modifications
to the (pyridinꢀ3ꢀyl)methanamine moiety would have on potency, selectivity and
physicochemical properties. To this end, we designed a synthesis and SAR study holding the
prenylꢀphenolic side chain constant, while modifying the pyridylꢀmethanamine moiety. These
analogs were prepared as shown in Scheme 2 starting from 4ꢀhydroxybenzaldehyde. Treatment
of 4ꢀhydroxybenzaldehyde with NaH/prenylbromide in CH CN provided prenyl ether 21, which
3
was reacted under the standard Van Leusen conditions to form oxazole 22 in near quantitative
yield. Coupling of 22 with 2ꢀ or 3ꢀbromonicotinaldehyde under the CuI/PPh conditions
3
22
described previously produced the desired intermediate represented by 23. Reductive amination
with an amine (HNR R ) and NaHB(OAc) , followed by HCl salt formation afforded the desired
1
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3
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analogs 24-34. The analogs 24-34 prepared in this study highlighted in Scheme 2 are a small
group of acyclic and cyclic aliphatic amine derivatives that offer changes in tPSA, LogD,
hydrogen bond donors (HBD) and acceptors (HBA), while maintaining MW<500. As before,
these compounds were evaluated with the SRB assay against the panel of TNBC cell lines
representing different molecular subtypes, and all eleven displayed antiproliferative activity and
were selective for MDAꢀMBꢀ453 cells (Table 2). These analogs were substantially more potent
overall than our previous compounds, with GI values ranging from approximately 0.2 ꢀM to 2
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ꢀM in MDAꢀMBꢀ453 cells. A direct comparison of the 3ꢀpyridine analog 24 and the 2ꢀpyridine
analog 26 bearing the piperidine amine suggested that the position of the pyridineꢀnitrogen atom
was not a major factor for potency or selectivity, thus the 2ꢀpyridine template was used for the
remaining analogs. Both secondary and tertiary amine derivatives showed potent activity as
represented by compounds 25, 27, 31 and 32. Amine groups bearing an additional heteroatom or
polar functional groups such as 28, 29, 30, 33 and 34 offered compounds with further
improvements in potency and selectivity. While 1,2ꢀdiamine type analogs such as 28 and 33
show good potency, compounds bearing an additional carbonyl group such as compound 30 offer
improved potency and selectively for MDAꢀMBꢀ453 cells as compared to the other TNBC cells.
Thus, compound 30 showed the greatest combination of potency and selectivity for MDAꢀMBꢀ
435 cells (Figure 4, Table 2). The GI of 30 was 0.8 ꢀM in MDAꢀMBꢀ453 cells and 30 – 50 ꢀM
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in the TNBC cell lines representing four different molecular subtypes. This indicated that 30 has
approximately 37.5ꢀ to 62.5ꢀfold antiproliferative selectivity for MDAꢀMBꢀ453 cells (Figure 4,
Table 2). As outlined in our medicinal chemistry strategy, SAR studies leading to the
identification of compound 30 provided simultaneous improvements in both potency against
MDAꢀMBꢀ453 cells and physicochemical properties. Interestingly, Compounds 26, 27, 28 and
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0 had biphasic concentrationꢀresponse curves in MDAꢀMBꢀ453 cells but not in other TNBC
cell lines (Figure 4), suggesting these compounds may have multiple mechanisms of action.
These differences might explain the improved selectivity for MDAꢀMBꢀ453 cells.
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To further evaluate the selectivity of this second series of compounds for LAR models,
the six compounds with the greatest selectivity for MDAꢀMBꢀ453 cells were evaluated against a
second LAR cell line, SUMꢀ185PE. Compounds 24, 26, 27, 28, 30 and 31 potently inhibited
growth of this cell line as well, with compound 30 again demonstrating the best combination of
potency and selective activity in this cell line compared to cells modeling other TNBC molecular
subtypes (Figure 4, Table 2). The GI of 30 was 1.6 ꢀM in SUMꢀ185PE cells, indicating this
5
0
compound has 18.8 to 31.3ꢀfold selectivity against these cells as compared to cells representing
other TNBC subtypes (Table 2). The other five compounds had GI values ranging from 1 to 3
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ꢀ
M in SUMꢀ185PE cells. However, these compounds did not show the same degree of
selectivity for both LAR cell lines as compound 30. Compounds 24, 26, 27, 28, 30 and 31 were
also tested against the Aꢀ10 rat smooth muscle cell line to measure their selectivity compared to
nonꢀcancerous cells. The GI values in Aꢀ10 cells were comparable to those calculated for nonꢀ
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LAR cells, further demonstrating their selectivity.
Table 3 captures the key physicochemical property data on lead compounds 17 and 30 in
comparison to the original natural products 1 and 2. Decreases in lipophilicity (LogD) and
increases to tPSA were realized by the introduction of more polar functional groups, structural
modifications which simultaneously improved MDAꢀMBꢀ435 cell potency >100ꢀfold compared
to our starting natural products, and generated increased selectivity for LAR cells over other
TNBC cells. These results, taken together, suggested that 30 is a suitable candidate for future in
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vivo efficacy studies against LAR xenograft models and investigations into this class of
compounds’ mechanisms of action.
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Mechanism of Action Studies
Prior to evaluation of our lead molecule against an in vivo LAR model, we sought to gain
insight into the potential mechanisms of action of these compounds and why they have
selectivity for LAR cells and potentially identify a molecular targets, which might have
therapeutic value for the LAR subtype of TNBC. Cell cycle analyses were performed to
determine if these compounds produced changes in cell cycle distribution characteristic of other
anticancer agents and to give an initial indication of their mechanism of action. Compounds 1, 2,
17, 27 and 30, representing various potencies and selectivity for MDAꢀMBꢀ453 cells, were
evaluated for their effects on cell cycle distribution of MDAꢀMBꢀ453 cells after 18 h of
treatment. A range of concentrations spanning the GI to LC values for each compound were
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50
tested, but no significant changes in cell cycle distribution were observed for any of the
compounds (data not shown). Accumulation of a subꢀG cell population was observed with
1
compounds 17, 27 and 30, consistent with the cytotoxicity of these compounds. Next the effects
of compound 17 were evaluated using a fluorescenceꢀbased intracellular signaling array (data not
shown). These data suggested that 17 affected PI3K/Akt/mTORC1 signaling in MDAꢀMBꢀ453
cells, so further studies were conducted to evaluate the effects of the compounds on this
signaling pathway.
The mechanisms of action of 30 were investigated because it shows the highest degree of
selectivity for MDAꢀMBꢀ453 cells. Interestingly, Lehmann and Bauer et al initially
demonstrated that LAR cells are not only sensitive to antiandrogens, but are also more sensitive
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23
to PI3K inhibitors than cells representing other TNBC subtypes in vitro and in vivo.
Additionally, we previously reported that LAR cells are particularly sensitive to inhibition of
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mTORC1 signaling. Based on this knowledge and our preliminary mechanistic data, we
hypothesized that the highly selective activity of 30 against LAR cells results from inhibition of
mTORC1 signaling. An immunoblotting timeꢀcourse experiment was conducted to measure the
activation of Akt, an upstream mediator of mTORC1 signaling, after treatment with 30 (Figure
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). Akt is phosphorylated at T308 by PDK1, which is phosphorylated by PI3Ks. Akt is also
phosphorylated at S473 by mTORC2. Treatment of MDAꢀMBꢀ453 cells with 30 resulted in a
nearly twoꢀfold increase in phosphorylation of Akt at T308, which was noticeable 1 – 6 h after
treatment. No significant differences in Akt phosphorylation at S473 were observed relative to
vehicleꢀtreated cells over the same timeꢀcourse. These data suggest that 30 caused an increase in
upstream signaling from PI3K/PDK1 to Akt. Signaling downstream of Akt was then evaluated to
investigate the effects of 30 on the entire PI3K/PDK1/Akt/mTORC1 signaling pathway in MDAꢀ
MBꢀ453 cells. At one hour postꢀtreatment, we observed an approximate 49% decrease in the
relative phosphorylation of mTOR at S2448 in compound 30ꢀtreated cells compared to vehicleꢀ
treated controls. This effect was transient and phosphorylation at S2448 increased over the next 5
h and by 6 h postꢀtreatment the relative phosphorylation at S2448 was approximately 60% higher
than in vehicleꢀtreated controls. mTOR is phosphorylated at S2448 via PI3K/Akt signaling, so
these data suggest that 30 causes an initial inhibition of mTORC1 activation. We then
investigated the activity of signaling proteins downstream of mTORC1. Interestingly, treatment
with 30 resulted in complete loss of S6 kinase (S6K) phosphorylation at T389, as well as loss of
S6 phosphorylation at S236/236. Collectively, these results suggested that 30 inhibits
downstream signaling through mTORC1. Although the precise point of inhibition could not be
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established from these results, it is likely the observed increase in phosphorylation of Akt at
T308 is a result of compensatory upstream activation of PI3Kꢀmediated signaling, which can
24
occur as a result of mTORC1 inhibition.
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CONCLUSION
Evidence suggests that the molecular characteristics of the LAR subtype are significantly
different than those of other TNBC subtypes and these tumors respond poorly to current
chemotherapeutic agents. Masuda et al found that patients with the LAR subtype had one of the
poorest rates of pathological complete response (10%) to neoadjuvant chemotherapy with
taxanes and anthracyclines, suggesting that this subtype may be inherently resistant to these
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agents. These findings highlight the need for the development of better, more rationally
targeted therapies for ARꢀexpressing breast cancers.
Our results, obtained by initially evaluating extracts of Texasꢀnative plants for selective
activity against TNBC molecular subtypes, identified a new class of syntheticallyꢀtractable,
potent molecules with selective effects against cells modeling the LAR subtype of TNBC.
Although the natural products that we initially procured had low potency and only moderate
selectivity, SAR studies and medicinal chemistry efforts led to the identification of substantially
more potent and selective analogs with improved physicochemical properties. Many of the
compounds described herein are new chemical structures that may be useful scaffolds for
developing additional compounds with efficacy against ARꢀexpressing breast cancer cells, or
form the basis for the development of new chemical tools to be used for mechanism of action
studies. Additionally, these studies identified lead molecules such as compound 30 with more
favorable drugꢀlike physicochemical properties, which can be used to evaluate the potential in
vivo antitumor efficacy of this class of molecules in ARꢀexpressing TNBC models.
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EXPERIMENTAL SECTION
Materials and Instrumentation. All compounds for cell treatments were dissolved in DMSO
SigmaꢀAldrich) and stored at ꢀ20°C. Absorbance (A ) of SRB solutions was measured with a
(
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Spectramax 384 Plus spectrophotometer (Molecular Devices). NMR spectra were collected on a
Varian 400 MHz NMR spectrometer. LCESIMS data were obtained on a Shimadzu LCꢀMS 2020
system (ESI quadrupole) coupled to a photodiode array detector, with a Phenomenex Kintex
column (2.6 µm C column, 100 Å, 75 × 3.0 mm). The preparative HPLC system utilized SCLꢀ
18
1
0A VP pumps and system controller with Luna 5 ꢀm C columns (110 Å, 250 × 21.2 mm, 10
18
mL/min). The analytical and semiꢀpreparative HPLC system utilized 1525 binary pumps
(Waters) with 2998 photodiode array detectors (Waters), and Luna 5 ꢀm C columns (110 Å,
18
250 × 4.6 mm, 1 mL/min and 110 Å, 250 × 10 mm, 4 mL/min). All solvents were of ACS grade
or better.
Compound Isolation. A supercritical CO extract (5.0 g) was obtained from approximately 106
2
g of lyophilized Amyris texana (collected at San Antonio Botanical Gardens). A 5 mg sample of
HP20ss and C18 processed crude extract was applied to a HPLCꢀbased microtiter plate
26
fractionation process, as previously described. Fractions F2 and F3 were identified as having
selective activity and became our target fractions. To generate greater quantities of the
compounds found in these fractions, the remaining crude extract was fractionated over silica gel
and eluted with hexaneꢀCH Cl ꢀMeOH (hexane, hexaneꢀCH Cl 50:50, CH Cl , CH Cl ꢀMeOH
2
2
2
2
2
2
2
2
90:10, MeOH). Fractions F2 and F3 contained the targeted chemical signatures (mass
spectrometric and UV profiles from LCMS) and these fractions were combined and applied to an
HP20ss column, and eluted with MeOHꢀH O. The fifth subfraction (100% MeOH) was further
2
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separated over C preparative HPLC (gradient elution with MeOHꢀH O from 30:70 to 100%
1
8
2
organic phase in 30 min.), followed by C semiꢀpreparative HPLC (MeOHꢀH O 90:10) to obtain
18
2
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(4.0 mg) and 2 (10.0 mg), which corresponded to the target peaks from fraction F2.
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Balsoxin,
Oꢀisopentenylꢀhalfordinol,
Texamine,
Uguenenazole,
and 3ꢀ[5ꢀ(3,4ꢀ
2
9
dimethoxyphenyl)ꢀ2ꢀoxazolyl]ꢀPyridine, which displayed similar UV profiles were purified
from the fourth subfraction (eluted with MeOHꢀH O 90:10) of HP20ss column via C
2
18
preparative HPLC (gradient elution with MeOHꢀH O from 30:70 to 100% organic phase in 30
2
min) followed by C18 semiꢀpreparative HPLC (MeCNꢀH O 75:25). These compounds were
2
obtained from fractions that did not demonstrate selective activity against MDAꢀMBꢀ453 cells
and their cytotoxic activities were not determined.
5ꢁ(4ꢁ((3ꢁmethylbutꢁ2ꢁenꢁ1ꢁyl)oxy)phenyl)ꢁ2ꢁphenyloxazole (1): White amorphous powder; UV
1
(
from HPLC PDA detector) λmax 200, 314 nm; H NMR (CDCl , 400 MHz): 8.02 (2H, dd, 7.9,
3
1.7), 7.64 (2H, brd, 8.7), 7.48 (3H, m), 7.32 (1H, s), 6.98 (2H, brd, 8.7), 5.51 (1H, t, 6.7), 4.56
1
3
(
2H, d, 6.7), 1.81 (3H, s), 1.77 (3H, s); C NMR (CDCl , 100 MHz): 160.9 , 159.1, 151.4, 138.6,
3
130.1, 128.8 (×2), 127.6, 126.1 (×2), 125.7 (×2), 121.9, 120.8, 119.3, 115.1 (×2), 64.9, 25.8,
+
18.2; ESIMS [M + H] m/z 306.
Chemistry Experimentals
General procedures. Unless otherwise indicated all reactions were conducted in standard
commercially available glassware using standard synthetic chemistry methods and setup. All airꢀ
and moistureꢀsensitive reactions were performed under nitrogen atmosphere with dried solvents
and glassware under anhydrous conditions. Starting materials and reagents were commercial
compounds of the highest purity available and were used without purification. Solvents used for
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reactions were indicated as of commercial dry or extraꢀdry or analytical grade. Analytical thin
layer chromatography was performed on aluminium plates coated with Merck Kieselgel 60F254
and visualized by UV irradiation (254 nm) or by staining with a solution of potassium
permanganate. Flash column chromatography was performed on Biotage Isolera One 2.2 using
commercial columns that were preꢀpacked with Merck Kieselgel 60 (230– 400 mesh) silica gel.
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1
Final compounds for biological testing are all ≥96% purity as determined by HPLCꢀMS and H
1
NMR. H NMR experiments were recorded on Agilent DD2 400MHz spectrometers at ambient
temperature. Samples were dissolved and prepared in deuterated solvents (CDCl , CD OD and
3
3
DMSOd ) with TMS used as the internal standard in all cases. All deuterated solvent peaks were
6
corrected to the standard chemical shifts (CDCl , d = 7.26 ppm; CD OD, d = 3.31 ppm;
3
H
3
H
DMSOꢀd , d = 2.50 ppm). Spectra were all manually integrated after automatic baseline
6
H
correction. Chemical shifts (d) are given in parts per million (ppm), and coupling constants (J)
are given in Hertz (Hz). The proton spectra are reported as follows: d (multiplicity, coupling
constant J, number of protons). The following abbreviations were used to explain the
multiplicities: app = apparent, b = broad, d = doublet, dd = doublet of doublets, ddd = doublet of
doublet of doublets, dddd = doublet of doublet of doublet of doublets, m = multiplet, s = singlet,
t = triplet. All samples were analyzed on Agilent 1290 series HPLC system comprised of binary
pumps, degasser and UV detector, equipped with an autoꢀsampler that is coupled with Agilent
6150 mass spectrometer. Purity was determined via UV detection with a bandwidth of 170nm in
the range from 230ꢀ400nm. The general LC parameters were as follows: Column ꢀ Zorbax
Eclipse Plus C18, size 2.1 X 50 mm; Solvent A: 0.10 % formic acid in water, Solvent B: 0.00 %
formic acid in CH CN; Flow rate – 0.7 mL/min; Gradient: 5 % B to 95 % B in 5 min and hold at
3
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5 % B for 2 min; UV detector – channel 1 = 254 nm, channel 2 = 254 nm. Mass detector
Agilent Jet Stream – Electron Ionization (AJSꢀES).
General Procedure for Pd/Cu Mediated Cross Coupling. In a dried microwave vial equipped
with a stir bar, oxazole (5.70 mmol), aryl bromide (11.25 mmol), anhydrous potassium carbonate
(1.58 g, 11.4 mmol), copper (I) iodide (1.08 g, 5.70 mmol), and palladium (II) acetate (65 mg,
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.29 mmol) were dissolved in DMF (7 mL). The vial was purged with N and the vial heated to
2
o
1
50 C for 13 minutes under microwave irradiation. The crude reaction was then filtered over a
pad of celete, pushing with DCM and concentrated under reduced pressure. The crude oil was
then diluted with EtOAc (100 mL) and extracted with water (70 mL x 2) and brine (70 mL x 3).
The organic layer was dried over sodium sulfate, filtered and concentrated under reduced
pressure. The crude mixtures were then purified on a silica gel column, eluting with
EtOAc/Hexanes.
General Phenol Alkylation Procedure. In a dried round bottom flask equipped with a stir bar,
sodium hydride (1.50 mmol., 60% dispersion in mineral oil) was added and the atmosphere
exchanged for N . The sodium hydride is then washed with Hexanes (3 mL) before the addition
2
o
of THF (3 mL) and cooled to 0 C. In a separate vial, the phenol (1.0 mmol) was dissolved in
DMF (2 mL) and added to the sodium hydride solution mixture slowly. The reaction was
allowed to stir for 5 minutes before the slow addition of the alkyl bromide (1.1 mmol). Once the
reaction was deemed complete by TLC, the reaction was quenched with saturated aqueous
sodium bicarbonate (5 mL), diluted with EtOAc (50 mL) and washed with water (30 mL) and
brine (30 mL x 3). The organic layer was dried over sodium sulfate, filtered and concentrated
under reduced pressure. The crude mixture was then purified on a silica column, eluting with
EtOAc/Hexanes.
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+
5
ꢁ(4ꢁPropoxyphenyl)oxazole. Brown solid (155 mg, 31% yield), LCꢀMS m/z 204.2 (APCI )
+
1
(
M+H) . H NMR (400 MHz, CDCl ): δ 7.87 (s, 1H), 7.57 (d, J = 9.2 Hz, 2H), 7.22 (s, 1H), 6.94
3
(d, J = 9.2 Hz, 2H), 3.96 (t, J = 6.4 Hz, 2H), 1.83 (sxt. J = 6.8 Hz, 2H), 1.05 (t, J = 7.2 Hz, 3H).
10
11
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16
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59
60
General Procedure for Phenol Deprotection. In a dry round bottom flask equipped with a stir
bar, the methoxy phenol (2.0 mmol) is dissolved in DCM (20 mL). The solution was then cooled
o
to ꢀ78 C and the headspace exchanged for N . Boron tribromide (12 mL, 12.0 mmol, 1.0 M in
2
DCM) was then added slowly. The reaction was then allowed to reach RT over 18 hours before
o
cooling to ꢀ78 C and slowly quenching with methanol (30 mL). The crude reaction mixture was
then warmed to RT and concentrated under reduced pressure. The crude mixture was then
dissolved EtOAc (300 mL) and extracted with brine (200 mL x 3). The organic layer was then
dried over sodium sulfate, filtered and concentrated under reduced pressure.
+
1
4
ꢁ(2ꢁPhenyloxazolꢁ5ꢁyl)phenol. Tan solid (423 mg, 91% yield), LCꢀMS m/z 328.1 (APCI ) H
NMR (500 MHz, d6ꢀDMSO): δ 9.89 (s, 1H), 8.08 (d, J = 6.3 Hz, 2H), 7.69 (d, J = 7.8 Hz, 2H),
7.62 (s, 1H), 7.56ꢀ7.54 (m, 3H), 6.93 (d, J = 7.8 Hz, 2H).
+
+
4
ꢁ(Oxazolꢁ5ꢁyl)phenol. Brown solid (777 mg, 96% yield), LCꢀMS m/z 162.1 (APCI ) (M+H) .
1
H NMR (500 MHz, d6ꢀDMSO): δ 9.82 (s, 1H), 8.34 (s, 1H), 7.55 (d, J = 7.3 Hz, 2H), 7.46 (s,
1H), 6.88 (d, J = 6.9, 2 H).
4ꢁ(2ꢁ(Pyrimidinꢁ5ꢁyl)oxazolꢁ5ꢁyl)phenol. Light yellow solid (42 mg, 95% yield), LCꢀMS m/z
+
+ 1
2
40.1 (APCI ) (M+H) . H NMR (400 MHz, d ꢀMeOD): δ 9.40 (s, 2H), 9.24 (s, 1H), 7.68 (d, J =
4
9.0 Hz, 2H), 7.54 (s, 1H), 6.90 (d, J = 8.0 Hz, 2H).
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ꢁ(4ꢁ[(3ꢁMethylbutꢁ2ꢁenꢁ1ꢁyl)oxy]phenyl)ꢁ2ꢁphenyloxazole (1). White Solid (296 mg, 81%
+
+ 1
yield), LCꢀMS m/z 306.2 (APCI ) (M+H) . H NMR (500 MHz, CDCl ): δ 8.07 (d, J = 7.4 Hz,
3
2
H), 7.60 (d, J = 8.3 Hz, 2H), 7.45ꢀ7.38 (m, 3H), 7.29 (s, 1H), 6.95 (d, J = 6.3 Hz, 2H), 5.48 (t, J
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
=
6.4 Hz, 1H), 4.51 (d, J = 6.9 Hz, 2H), 1.78 (s, 3H), 1.73 (s, 3H). C (500 MHz, CDCl ): δ
3
160.53, 159.18, 151.42, 138.46, 130.10, 128.81, 127.68, 126.16, 125.73, 121.97, 120.80, 119.48,
+
+
115.16, 64.93, 25.88, 18.28. HRMS (ESI ) Calculated m/z 306.1489, Found 306.1502 (M+H) .
(E)ꢁ5ꢁ(4ꢁ[(3,7ꢁDimethyloctaꢁ2,6ꢁdienꢁ1ꢁyl)oxy]phenyl)ꢁ2ꢁ(pyridinꢁ3ꢁyl)oxazole (2). Yellow oil
+
+ 1
(95 mg, 20%), LCꢀMS m/z 375.3 (APCI ) (M+H) . H NMR (500 MHz, CDCl ): δ 9.31 (s, 1H),
3
8
.66 (d, J = 4.4 Hz, 1H), 8.32 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.40 (dd, J = 6.8 Hz,
.8 Hz, 1 H), 7.34 (s, 1H), 6.98 (d, J = 8.8 Hz, 2H), 5.49 (t, J = 6.3 Hz, 1 H), 5.09 (t, J = 5.8 Hz,
4
1
3
1H), 4.58 (d, J = 6.3 Hz, 2H), 2.09ꢀ2.14 (m, 4H), 1.75 (s, 3H), 1.67 (s, 3H), 1.60 (s, 3H). C
(500 MHz, CDCl ): δ 159.60, 158.24, 152.34, 150.86, 147.58, 141.79, 133.34, 132.03, 126.02,
3
123.99, 123.89, 123.73, 122.25, 120.42, 119.28, 115.42, 65.20, 39.70, 26.44, 25.84, 17.87, 16.87.
+
+
HRMS (ESI ) Calculated m/z 375.2067, Found 375.2062 (M+H) .
5
ꢁ(4ꢁ[(3ꢁMethylbutꢁ2ꢁenꢁ1ꢁyl)oxy]phenyl)ꢁ2ꢁ(pyridinꢁ3ꢁyl)oxazole (3). Pale yellow solid (82 mg,
+
+ 1
4
0%), LCꢀMS m/z 307.2 (APCI ) (M+H) . H NMR (400 MHz, CDCl ): δ 9.32 (s, 1H), 8.68 (d,
3
J = 3.9 Hz, 1H), 8.35 (dt, J = 8.2 Hz, 2.0 Hz, 1H), 7.65 (d, J = 9.0 Hz, 2H), 7.42 (dd, J = 7.4 Hz,
4
.7 Hz, 1H), 7.36 (s, 1H), 6.99 (d, J = 9.0 Hz, 2H), 5.51 (t, J = 5.1 Hz, 1H), 5.50 (d, J = 1.6 Hz,
1
3
2
H), 1.82 (s, 3H), 1.77 (s, 3H). C NMR (400 MHz, CDCl ): δ 159.60, 158.28, 152.34, 150.90,
3
147.62, 138.81, 133.35, 126.05, 124.01, 123.73, 122.27, 120.46, 119.45, 65.11, 26.00, 18.41.
+
+
HRMS (ESI ) Calculated m/z 307.1441, Found 307.1442 (M+H) .
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(
E)ꢁ5ꢁ(4ꢁ[(3,7ꢁDimethyloctaꢁ2,6ꢁdienꢁ1ꢁyl)oxy]phenyl)ꢁ2ꢁphenyloxazole (4). White solid (152
+
+ 1
mg, 45% yield), LCꢀMS m/z 374.2 (APCI ) (M+H) . H NMR (500 MHz, CDCl ): δ 8.09 (d, J =
3
7
.8 Hz, 2H), 7.65 (d, J = 8.3 Hz, 2H), 7.49ꢀ7.43 (m, 3H), 7.32 (s, 1h), 6.99 (d, J = 8.8 Hz, 2H),
.50 (t, J = 6.4 Hz, 1H), 5.10 (t, J = 6.1 Hz, 1H), 4.59 (d, J = 6.3 Hz, 2H), 2.17ꢀ2.10 (m, 4H),
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
5
1
3
1.76 (s, 3H), 1.68 (s, 3H), 1.61 (s, 3H). C (500 MHz, CDCl ): δ 160.73, 159.35, 151.59,
3
1
41.77, 132.07, 130.27, 128. 97, 127.80, 127.80, 126.32, 125.90, 123.93, 122.10, 120.96, 119.38,
+
1
15.38, 65.21, 39.74, 26.48, 25.88, 17.90, 16.90. HRMS (ESI ) Calculated m/z 374.2115, Found
+
374.2121 (M+H) .
General Procedure for the Van Leusen Oxazole Synthesis. 5ꢁ(4ꢁMethoxyphenyl)oxazole (5).
In a round bottom flask equipped with a stir bar, pꢀtoluenesulfonylmethyl isocyanide (10.04 g,
51.41 mmol), anhydrous potassium carbonate (7.00 g, 51.4 mmol), and pꢀanisaldehyde (6.25 mL,
8
4.8 mmol) were dissolved in MeOH (257 mL). The solution was then heated to reflux,
monitoring by TLC. Once complete, the reaction was cooled to room temperature and
concentrated under reduced pressure. The crude mixture was then dissolved in EtOAc (200 mL)
and washed with water (100 mL x 3). The organic layer was dried over sodium sulfate, filtered
and concentrated under reduced pressure to yield 5ꢁ(4ꢁMethoxyphenyl)oxazole as a light yellow
+
1
solid (8.7 g, 97% yield). LCꢀMS: 176.1 m/z (APCI) (M+H) . H NMR (400 MHz, CDCl ): δ
3
7
.87 (s, 1H), 7.59 (d, J = 9.0 Hz, 2H), 7.23 (s, 1H), 6.96 (d, J = 8.6 Hz, 2H), 3.85 (s, 3H).
[
Coupling route] 5ꢁ(4ꢁMethoxyphenyl)ꢁ2ꢁphenyloxazole (6). Yellow solid (580 mg, 41% yield),
+
1
LCꢀMS m/z 252.2 (APCI) (M+H) . H NMR (400 MHz, CDCl ): δ 8.09 (d, J = 7.8 Hz, 2H), 7.64
3
(
d, J = 8.0 Hz, 2H), 7.41ꢀ7.50 (m, 3H), 7.31 (s, 1H), 6.97 (d, J = 8.0 Hz, 2H), 3.84 (s, 3H).
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[
Cyclization route] 5ꢁ(4ꢁmethoxyphenyl)ꢁ2ꢁphenyloxazole (6). In a round bottom flask equipped
with a stir bar, Nꢀ(2ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀoxoethyl)benzamide 9 was dissolved in acetic
anhydride (1 mL) followed by dropwise addition of conc. H SO (10 ꢀL, 0.18 mmol). The
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reaction was heated to 90°C and monitored by TLC. Upon completion, the reaction was cooled
to RT and diluted with 50 mL water and 25 mL DCM. The organic layer was then washed with
2
5 mL sat. sodium bicarbonate (x1), brine (x3), and then dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The crude mixture was purified on a silica gel column,
eluting with EtOAc/hexanes to yield 5ꢁ(4ꢁmethoxyphenyl)ꢁ2ꢁphenyloxazole as an offꢀyellow
+
1
solid (31.5 mg, 85% yield). LCꢀMS m/z 252.2 (APCI) (M+H) . H NMR (400 MHz, CDCl ): δ
3
8.09 (d, J = 7.8 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.41ꢀ7.50 (m, 3H), 7.31 (s, 1H), 6.97 (d, J =
8
.0 Hz, 2H), 3.84 (s, 3H).
Nꢁ(2ꢁ(4ꢁmethoxyphenyl)ꢁ2ꢁoxoethyl)benzamide (9). In a round bottom flask equipped with a stir
bar, benzoic acid (101.5 mg, 0.83 mmol), and HATU (381.5 mg, 1 mmol) were dissolved in
DMF and allowed to stir for ten minutes before slow addition of 2ꢀaminoꢀ4’ꢀ
methoxyacetophenone HCl 8 (177.8 mg, 0.91 mmol). Once homogenous, DIPEA (0.36 mL, 2
mmol) was added dropwise to the solution. The reaction was monitored by TLC and upon
completion, taken into 20 mL EtOAc and washed with 50 mL water (x2) and brine (x3). The
organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure.
The crude mixture was purified on a silica gel column, eluting with EtOAc/hexanes to yield Nꢀ
(2ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀoxoethyl)benzamide as a white solid (139.1 mg, 57% yield). LCꢀMS
+
+ 1
m/z 270.1 (APCI ) (M+H) . H NMR (400 MHz, Methanolꢀd ) δ 8.05 (dd, 2H), 7.90 (dd, J = 8.3,
4
1
.3 Hz, 2H), 7.59 – 7.45 (m, 3H), 7.06 (dd, 2H), 3.89 (s, 3H), 3.31 (s, 2H).
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Journal of Medicinal Chemistry
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8
6
ꢁ(4ꢁPropoxyphenyl)ꢁ2ꢁ(pyridinꢁ3ꢁyl)oxazole (10). Yellow solid (69 mg, 30% yield), LCꢀMS m/z
+
+ 1
81.2 (APCI ) (M+H) . H NMR (500 MHz, CDCl ): δ 9.32 (s, 1H), 8.67 (d, J = 3.4 Hz, 1H),
3
.34 (d, J = 7.8 Hz, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.41 (dd, J = 7.8 Hz, 5.1 Hz, 1H), 7.35 (s, 1H),
.97 (d, J = 8.8 Hz, 2H), 3.97 (t, J = 6.8 Hz, 2H), 1.84 (sxt, J = 7.3 Hz, 2H), 1.06 (t, J = 7.8 Hz,
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
+
+
3H). HRMS (ESI ) Calculated m/z 281.1285, Found 281.1293 (M+H) .
5
7
8
2
ꢁ(4ꢁMethoxyphenyl)ꢁ2ꢁ(6ꢁ(pyrrolidinꢁ1ꢁyl)pyridinꢁ3ꢁyl)oxazole (11). Yellow solid (148 mg,
+
+ 1
0% yield). LCꢀMS m/z 322.2 (APCI ) (M+H) . H NMR (500 MHz, CDCl ): δ 8.87 (s, 1H),
3
.05 (dd, J = 8.8 Hz, 1.5 Hz, 1H), 7.61 (d, J = 8.3 Hz, 2 H), 7.25 (s, 1 H), 6.95 (d, J = 8.3 Hz,
+
H), 6.41 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H), 3.53 (broad s, 4H), 2.02 (broad s, 4H). HRMS (ESI )
+
Calculated m/z 322.1550, Found 322.1551 (M+H) .
(
E)ꢁ5ꢁ(4ꢁ[(3,7ꢁdimethyloctaꢁ2,6ꢁdienꢁ1ꢁyl)oxy]phenyl)oxazole (12). Yellow solid (259 mg, 28%
+
+ 1
yield), LCꢀMS m/z 298.2 (APCI ) (M+H) . H NMR (400 MHz, CDCl ): δ 7.78 (s, 1H), 7.77 (d,
3
J = 8.7 Hz, 2H), 7.23 (s, 1H), 6.96 (d, J = 9.0 Hz, 2H), 5.49 (t, J = 5.4 Hz, 1H), 5.09 (t, J = 5.1
Hz, 1H), 4.57 (d, J = 6.3 Hz, 2H), 2.17ꢀ2.07 (m, 4H), 1.75 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H).
+
+
HRMS (ESI ) Calculated m/z 282.0873, Found 282.0874 (M+H) .
5
ꢁ(4ꢁPropoxyphenyl)ꢁ2ꢁ(pyrimidinꢁ5ꢁyl)oxazole (13). Yellow solid (6 mg, 15% yield), LCꢀMS
+
+ 1
m/z 282.2 (APCI ) (M+H) . H NMR (400 MHz, CDCl ): δ 9.40 (s, 2H), 9.28 (s, 1H), 7.65 (d, J
3
=
9.0 Hz, 2H), 7.39 (s, 1H), 6.99 (d, J = 8.6 Hz, 2H), 3.98 (t, J = 6.7 Hz, 2H), 1.84 (sxt, J = 6.7
+
Hz, 2H), 1.07 (t, J = 7.4 Hz, 3H). HRMS (ESI ) Calculated m/z 282.1237, Found 282.1243
+
(M+H) .
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2
2
2
2
2
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ꢁ(2ꢁ(Pyrimidinꢁ5ꢁyl)oxazolꢁ5ꢁyl)phenyl acetate (14). Pale yellow solid (11 mg, 22 % yield), LCꢀ
+
+ 1
MS m/z 282.1 (APCI ) (M+H) . H NMR (400 MHz, CDCl ): δ 9.38 (s, 2H), 9.27 (s, 1H), 7.73
3
(d, J = 9.0 Hz, 2H), 7.48 (s, 1H), 7.20 (d, J = 8.6 Hz, 2H), 2.14 (s, 3H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4ꢁ(2ꢁ(5ꢁmethylꢁ1Hꢁpyrazolꢁ4ꢁyl)oxazolꢁ5ꢁyl)phenyl acetate (15). Pale yellow solid (9 mg, 51%
+
+ 1
yield). LCꢀMS m/z 284.1 (APCI ) (M+H) . H NMR (400 MHz, CDCl ): δ 7.71 (d, J = 9.2 Hz,
3
2H), 7.68 (s, 1H), 7.31 (s, 1H), 7.17 (d, J = 8.0 Hz, 2H), 6.24 (s, 1H), 3.49 (s, 3H), 2.67 (s, 3H).
5ꢁ(4ꢁMethoxyphenyl)ꢁ2ꢁ(5ꢁmethylꢁ1Hꢁpyrazolꢁ4ꢁyl)oxazole (16). Pale yellow solid (8 mg, 20%
+
+ 1
yield). LCꢀMS m/z 256.2 (APCI ) (M+H) . H NMR (400 MHz, CDCl ): δ 7.67 (d, J = 1.6 Hz,
3
1H), 7.62 (d, J = 8.8 Hz, 2H), 7.21 (s, 1H), 6.95 (d, J = 9.2 Hz, 2H), 6.23 (s, 1H), 3.85 (s, 3H),
+
+
2
.66 (s, 3H). HRMS (ESI ) Calculated m/z 256.1081, Found 256.1081 (M+H) .
5
ꢁ(4ꢁ[(3ꢁMethylbutꢁ2ꢁenꢁ1ꢁyl)oxy]phenyl)ꢁ2ꢁ(5ꢁ(pyrrolidinꢁ1ꢁylmethyl)pyridinꢁ2ꢁyl)oxazole (17).
+
+
1
Yellow oil (65 mg, 49% yield). LCꢀMS m/z 390.3 (APCI ) (M+H) . H NMR (400 MHz,
CDCl ): δ 8.67 (d, J = 1.9 Hz, 1H), 8.10 (d, J = 8.3 Hz, 1H), 7.82 (dd, J = 8.2 Hz, 1.9 Hz, 1H),
3
7.71 (d, J = 9.0 Hz, 2H), 7.38 (s, 1H), 6.97 (d, J = 8.6 Hz, 2H), 5.50 (tt, J = 6.6 Hz, 1.1 Hz, 1H),
4.56 (d, J = 6.7 Hz, 2H), 3.73 (s, 1H), 3.70 (s, 1H), 2.54 (t, J = 5.1 Hz, 4H), 1.81 (broad s, 6H),
1.76 (broad s, 4H).
5ꢁ(4ꢁ[(3ꢁMethylbutꢁ2ꢁenꢁ1ꢁyl)oxy]phenyl)ꢁ2ꢁ(5ꢁ(pyrrolidinꢁ1ꢁylmethyl)pyridinꢁ2ꢁyl)oxazole
1
hydrochloride (17-HCl salt). Yellow solid (61 mg). LCꢀMS m/z 390.3 (APCI+) (M+H)+. H
NMR (400 MHz, CDCl3): δ 8.84 (s, 1H), 8.29 (d, J = 8.2 Hz, 1H), 8.16 (dd, 8.2 Hz, 2.3 Hz, 1H),
7
4
2
.80 (d, J = 9.0 Hz, 2H), 7.61 (s, 1H), 7.04 (d, J = 9.0 Hz, 2H), 5.48 (tt, J = 6.6 Hz, 1.6 Hz, 1H),
.60 (d, J = 6.7 Hz, 2H), 4.55 (s, 2H), 3.58 (broad s, 2H), 3.26 (broad s, 2H), 2.23 (broad s, 2H),
13
.05 (broad s, 2H), 1.80 (d, J = 1.2 Hz, 3H), 1.77 (d, J = 0.8 Hz, 3H). C (400 MHz, CDCl3): δ
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Journal of Medicinal Chemistry
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159.92, 158.33, 151.05, 146.38, 139.55, 137.66, 125.92, 121.96, 121.74, 199.68, 119.40, 115.01,
64.57, 54.71, 53.77, 24.41, 22.43, 17.83, 16.78. HRMS (ESI): Calculated: 390.2176 m/z, Found:
390.2193 m/z (M+H)+.
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ꢁ(4ꢁ[(3ꢁmethylbutꢁ2ꢁenꢁ1ꢁyl)oxy]phenyl)ꢁ2ꢁ(pyrimidinꢁ5ꢁyl)oxazole (18). Yellow Solid (6 mg,
+
+ 1
3% yield). LCꢀMS m/z 308.2 (APCI ) (M+H) . H NMR (400 MHz, CDCl ): δ 9.38 (s, 2H),
3
.28 (s, 1H), 7.65 (d, J = 9.0 Hz, 2H), 7.39 (s, 1H), 7.00 (d, J = 9.0 Hz, 2H), 5.51 (t, J = 6.6 Hz,
+
H), 4.57 (d, J = 7.1 Hz, 2H), 1.82 (s, 3H), 1.77 (s, 3H). HRMS (ESI ) Calculated m/z 308.1394,
+
Found 308.1394 (M+H) .
N,NꢁDimethylꢁ2ꢁ(4ꢁ(2ꢁ(pyrimidinꢁ5ꢁyl)oxazolꢁ5ꢁyl)phenoxy)ethanamine (19). Modification to the
general procedures as follows. In this reaction 3ꢀchloroꢀN,Nꢀdimethylpropanꢀ1ꢀamine (36 mg,
o
0
.25 mmol) and sodium iodide (4 mg, 0.03 mmol) was used and the reaction was heated 100 C
After deemed complete by TLC, the reaction was diluted with DCM (5 mL) and extracted with
NaOH (1.0 M, 5 mL x2). The organic layer was then extracted with HCl (1.0 M, 5 mL x 2). The
combined acidic aqueous extracts were extracted with DCM (5 mL x 2). The combined DCM
layers were combined, dried over sodium sulfate, filtered and concentrated under reduced
+
+ 1
pressure. Amber oil (14 mg, 36% yield), LCꢀMS m/z 311.2 (APCI ) (M+H) . H NMR (400
MHz, d ꢀMeOD): δ 9.42 (s, 2H), 9.27 (s, 1H), 7.79 (d, J = 9.0 Hz, 2H), 7.61 (s, 1H), 7.09 (d, J =
4
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.0 Hz, 2H), 3.35 (t, J = 1.6 Hz, 2H), 2.84 (t, J = 5.5 Hz, 2H), 2.40 (s, 6H).
N,NꢁDimethylꢁ2ꢁ(4ꢁ(2ꢁ(pyrimidinꢁ5ꢁyl)oxazolꢁ5ꢁyl)phenoxy)ethanamine hydrochloride (19-HCl
+
+ 1
salt). Yellow solid (6 mg, Quant.). LCꢀMS m/z 311.2 (APCI ) (M+H) . H NMR (400 MHz, d ꢀ
4
MeOD): δ 9.42 (s, 2H), 9.26 (s, 1H), 7.85 (d, J = 9.0 Hz, 2H), 7.64 (s, 1H), 7.18 (d, J = 9.0 Hz,
2
H), 4.44 (t, J = 5.1 Hz, 2H), 3.64 (t, J = 5.1 Hz, 2H), 3.01 (s, 6H).
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