Development of posaconazole-based analogues as hedgehog signaling pathway inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.11.056

Inhibition of the hedgehog (Hh) signaling pathway has been validated as a therapeutic strategy to treat basal cell carcinoma and holds potential for several other forms of human cancer. Itraconazole and posaconazole are clinically useful triazole anti-fungals that are being repurposed as anti-cancer agents based on their ability to inhibit the Hh pathway. We have previously demonstrated that removal of the triazole from itraconazole does not affect its ability to inhibit the Hh pathway while abolishing its primary side effect, potent inhibition of Cyp3A4. To develop structure-activity relationships for the related posaconazole scaffold, we synthesized and evaluated a series of des-triazole analogues designed through both ligand- and structure-based methods. These compounds demonstrated improved anti-Hh properties compared to posaconazole and enhanced stability without inhibiting Cyp3A4. In addition, we utilized a series of molecular dynamics and binding energy studies to probe specific interactions between the compounds and their proposed binding site on Smoothened. These studies strongly suggest that the tetrahydrofuran region of the scaffold projects out of the binding site and that π-π interactions between the compound and Smoothened play a key role in stabilizing the bound analogues.

Full text of DOI:10.1016/j.ejmech.2018.11.056

Accepted Manuscript
Development of posaconazole-based analogues as hedgehog signaling pathway
inhibitors
Kelly A. Teske, Radha Charan Dash, Shana R. Morel, Lianne Q. Chou, Robert J.
Wechsler-Reya, M. Kyle Hadden
PII:
S0223-5234(18)31015-8
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.056
EJMECH 10914
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 September 2018
Revised Date: 13 November 2018
Accepted Date: 22 November 2018
Please cite this article as: K.A. Teske, R.C. Dash, S.R. Morel, L.Q. Chou, R.J. Wechsler-Reya, M.K.
Hadden, Development of posaconazole-based analogues as hedgehog signaling pathway inhibitors,
European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.11.056.
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ACCEPTED MANUSCRIPT
Development of Posaconazole-based Analogues as Hedgehog Signaling
Pathway Inhibitors
Kelly A. Teske1,#, Radha Charan Dash1, Shana R. Morel1 Lianne Q. Chou2, Robert J. Wechsler-
Reya2, and M. Kyle Hadden1,*
1Department of Pharmaceutical Sciences, University of Connecticut, 69 N Eagleville Rd, Unit
3092, Storrs, CT, USA 06269-3092
2Tumor 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
*Corresponding author: kyle.hadden@uconn.edu, Ph: 1-860-486-8446.
#Current address: Department of Chemistry, Western Michigan University, Email:
Kelly.teske@wmich.edu.

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ABSTRACT
Inhibition of the hedgehog (Hh) signaling pathway has been validated as a therapeutic
strategy to treat basal cell carcinoma and holds potential for several other forms of human
cancer. Itraconazole and posaconazole are clinically useful triazole anti-fungals that are being
repurposed as anti-cancer agents based on their ability to inhibit the Hh pathway. We have
previously demonstrated that removal of the triazole from itraconazole does not affect its ability
to inhibit the Hh pathway while abolishing its primary side effect, potent inhibition of Cyp3A4.
To develop structure-activity relationships for the related posaconazole scaffold, we synthesized
and evaluated a series of des-triazole analogues designed through both ligand- and structure-
based methods. These compounds demonstrated improved anti-Hh properties compared to
posaconazole and enhanced stability without inhibiting Cyp3A4. In addition, we utilized a series
of molecular dynamics and binding energy studies to probe specific interactions between the
compounds and their proposed binding site on Smoothened. These studies strongly suggest that
the tetrahydrofuran region of the scaffold projects out of the binding site and that π-π interactions
between the compound and Smoothened play a key role in stabilizing the bound analogues.

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1. Introduction
The hedgehog (Hh) signaling pathway plays an essential role in embryonic development by
regulating the proliferation and differentiation of cells in a time-dependent manner [1-3]. Hh
signaling is significantly less active in adults with the exception of maintaining stem cell
homeostasis, predominately in the skin and central nervous system [2]. Aberrant activation of the
Hh signaling pathway has been implicated as a driving force in several cancers; most notably,
basal cell carcinoma (BCC) and medulloblastoma (MB) [4-5]. Constitutive activation of the Hh
pathway in these cancers is primarily caused by mutations in several key members of the Hh
signal cascade, including Patched (Ptch), Smoothened (Smo), and Suppressor of fused (Sufu) [6].
Current FDA approved drugs that target the Hh pathway (vismodegib and sonidegib) function
through direct antagonism of Smo; however, resistance due to point mutations in the binding
pocket of these drugs on Smo has been identified in both BCC and MB patients who used these
drugs for initial treatment [7-11]. The need to develop potent inhibitors that overcome the
resistance observed with vismodegib and sonidegib has led to the identification of multiple
small-molecule scaffolds that target different points along the Hh signaling pathway. [12-16]
Posaconazole (PSZ) and itraconazole (ITZ) are clinically approved azole antifungals that
have been identified as Hh pathway inhibitors in vitro and in vivo [17-20]. Previous studies
strongly suggest that both of these drugs inhibit the Hh pathway by directly binding Smo [17-18,
21-22]. PSZ and ITZ retain their anti-Hh activity in the presence of mutant forms of Smo that
confer resistance to vismodegib and sonidegib, highlighting their potential as improved anti-
cancer drugs targeting the Hh pathway. This activity is presumably a result of their ability to bind
Smo in a manner distinct from the clinically approved drugs. We have undertaken a series of
studies to more fully explore the anti-cancer potential of the PSZ scaffold through targeting the

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Hh pathway. Herein, we report the synthesis and evaluation of PSZ analogues designed to
enhance both their Hh inhibitory activity and their drug-like properties. In addition, we have
generated a homology model of Smo that was utilized in an extensive series of computational
studies to probe potential intermolecular interactions between PSZ and Smo and to examine the
conformational changes in Smo that drive its inactivation following antagonist binding.
2. Chemistry
2.1. PSZ Analogue Design
Multiple strategies were employed to rationally design improved analogues of PSZ for
evaluation as anti-cancer agents that target the Hh signaling pathway. Our previous studies with
the ITZ scaffold demonstrated that the triazole moiety, which is responsible for the potent
inhibition of Cyp3A4 associated with the ITZ scaffold, is not required for inhibition of Hh
signaling [17]. With this in mind, we designed an initial proof-of-concept des-triazole PSZ
analogue, 1, that removed the triazole while also incorporating a simplified alkyl side chain
present in our most potent ITZ analogues (Figure 1, top). Based on our preliminary docking
studies for PSZ in complex with Smo (vide infra), we incorporated modified side chains that
contained either a primary alcohol or carboxylic acid to take advantage of enhance
intermolecular interactions with the PSZ binding site on Smo (Figure 1, middle). Finally, we
prepared several simplified PSZ analogues that replace the triazolone/side chain region with an
aromatic moiety tethered to the central region of the scaffold through an amide bond (Figure 1,
bottom). These compounds were prepared based on recent SAR results for the ITZ scaffold
generated in our group [20].

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Figure 1. Structure of PSZ and design strategies for PSZ analogues.
2.2. Compound Synthesis
PSZ analogue 1 was prepared through modification of a previously published synthesis of
PSZ (Scheme 1) [23]. Friedel-Craft acylation of 1,3-difluorobenzene (2) with succinic anhydride
(3), followed by conversion of the resulting ketone to the alkene through standard Wittig
conditions provided carboxylic acid 5. Amidation of 5 with (4R)-(+)-4-benzyl-2-oxazolidinone
(OXZ) affords OXZ-protected intermediate 6, which undergoes selective hydroxymethylation at
the opposite face in the presence of titanium tetrachloride to form intermediate 7. Iodocyclization
of 7 results in selective formation of the 2S,4R conformation around the intact tetrahydrofuran
(THF) moiety (8). Reduction of 8 with sodium borohydride removes both the iodine and reduces
the OXZ to the primary alcohol, which is subsequently tosylated to provide key des-triazole THF
intermediate 10. Intermediate 11 was prepared as described previously [17] and coupled directly to
10 to afford desired analogue 1 with the trans conformation in 5% overall yield.

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Scheme 1. Synthesis of THF Ring and final coupling to make Des-PSZ Analogue 1. Reagents
and conditions: (a) AlCl3, DCM, 45 ⁰C, 3-4 h, 65%; (b) NaOtBu, MePPh3Br, THF, 40 ⁰C, 12 h,
100%; (C) i. DCM, TEA, pivaloyl chloride, ii. OXZ, DMAP, DMF, 45-50 ⁰C, 12 h, 68%; (d) i.
DCM, TiCl4, DIPEA, 0 ⁰C, 1 h, ii. s-trioxane, TiCl4, DCM, 0 ⁰C, 12 h, 50%; (e) I2, pyridine,
CH3CN, R.T., 20 h, 61%; (f) NaBH4, DMSO, 110⁰C, 12 h, 62%; (g) Pyridine, TsCl, R.T., 12 h,
100%; (h) Cs2CO3, DMSO, 90 ⁰C, 12 h, 63%.
Analogues incorporating either a carboxylic acid or primary alcohol in the side chain
region of the PSZ scaffold were prepared by coupling either ethyl 3-bromopropanoate or ethyl
bromoacetate to well-characterized intermediate 12 [17] under basic conditions, followed by
demethylation with boron tribromide to afford esters 15 and 16, respectively (Scheme 2).
Subsequent coupling of the tosylated THF intermediate 10 to both esters resulted in 17 and 18,
which had been hydrolyzed to the carboxylic acid, presumably due to the basic conditions and
elevated temperature required for the coupling. Alcohol 19 was prepared through reduction of 17
with lithium aluminum hydride. Surprisingly, attempts to reduce 18 to the corresponding alcohol
were unsuccessful, resulting in multiple products that were difficult to characterize. Because 18

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was significantly less active than 17 against the Hh signaling pathway (Table 2 below), we did
not optimize the conditions necessary to reduce 18 to the corresponding alcohol. A similar
synthetic route was also utilized to synthesize an ITZ analogue incorporating the propanoic acid
side chain (21) to explore whether incorporation of this moiety affected the activity of the des-
triazole ITZ scaffold (Scheme 3).
Scheme 2.Synthesis of Hydroxylated Des-triazole PSZ Analogues. Reagents and conditions: (a)
ethyl 3-bromopropanoate or ethyl bromoacetate, Cs2CO3, DMF, 60 °C – 100 °C, 12 h, quant; (b)
BBr3 (1M), DCM, 0 °C - R.T., 4 h, 29-35%; (c) Cs2CO3, DMSO, 90 °C, 12 h, 36%; (d) LAH,
THF, 0 °C to R.T., 63%.
Scheme 3. Synthesis of Hydroxylated Side Chain Des-triazole PSZ Analogues. Reagents and
conditions: (a) Cs2CO3, DMSO, 90 °C, 12 h, 54%.
A series of ‘right side’ analogues based on previous ITZ SAR conducted in our lab were
also synthesized (Scheme 4). They were prepared by coupling THF region 10 and the linker
region precursor 22, which provides the nitro-containing intermediate 23. The nitro intermediate

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was reduced in the presence of 10% palladium on carbon and hydrazine monohydrate to produce
aniline 24. The carbamate 25 was generated through the addition of phenyl chloroformate to
aniline 24 and subsequent treatment with hydrazide monohydrate provided carboxamide
intermediate 26. The final unsubstituted triazolone 27 was generated by heating carboxamide 26
with formamidine acetate. Finally, three analogues that replaced the triazolone side chain with an
aromatic amide were prepared by directly coupling of aniline 24 to the requisite aromatic
carboxylic acid (28-30, Scheme 5).
Scheme 4. Synthesis of ‘Right-Side Analogues’− Linker Precursors. Reagents and conditions:
(a) Cs2CO3, DMSO, 90 °C, 12 h, 67%; (b) 10% Pd/C, EtOH, NH2NH2, reflux, 12 h, 90%; (c)
Pyr, ClCOOPh, 3 h, 40%; (d) NH2NH2-H2O, reflux, 3h, 30%; (e) formamidine acetate, acetic
acid, reflux, 3 h, 46%.
Scheme 5. Synthesis of ‘Right-Side Analogues’− Amide Linkage. Reagents and conditions: (a)
EDCI, DMAP, DCM, RT, 12 h, 27 - 63% or HATU, NMM, DMF, RT, 12h, 80%.

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3. Initial Biological and Computational Studies for PSZ and Analogue 1.
3.1. Hh inhibitory activity for PSZ and 1.
To determine whether removal of the triazole moiety had any effect on the ability of the PSZ
scaffold to inhibit Hh signaling, we evaluated whether 1 could down-regulate mRNA expression
of the Hh-dependent target gene Gli1 in ASZ cells, a well-characterized Hh-dependent murine
BCC cell line [24]. Interestingly, des-triazole analogue 1 was approximately 2.5-fold more potent
than PSZ (IC50 values 0.19 and 0.50 µM, respectively; Table 1). A similar trend was observed
with regards to the ability of 1 to inhibit the growth of a primary Hh-dependent MB derived from
a conditional Patched knockout (Math1-Cre-ER:Ptchfl/fl, termed Ptch-CKO) mouse [25].
Table 1. In Vitro and Pharmacokinetic Activity of PSZ and initial Des-triazole PSZ Analogue.
Ptch-CKO
CYP3A4
P-gp
T1/2
Clint
Cmpd
IC50 (µM)a
GI50 (µM)b
IC50 (µM)c Substratec
(min)c
(µL/min/mg)c
PSZ
1.5 ± 0.3
0.15
>20
Yes
No
108
21.5
37.4
0.50 ± 0.1
0.19 ± 0.03
0.61 ± 0.12
61.8
1
aIC50 values from ASZ cells represent the Mean ± SEM of at least two separate experiments performed in triplicate
following a 48 hr incubation.
bValues represent the Mean ± SEM of at least two separate experiments performed in triplicate.
cPK data generated by Pharmaron or Cyprotex as a CRO service via their standard protocols.
3.2. Computational Modeling of the Smoothened Receptor.
Several distinct lines of evidence strongly suggest that both PSZ and ITZ bind to the 7-
TM domain of Smo in the same region where other Smo agonists and antagonists bind [17-18,
21-22]. Both analogues were active in Ptch-/- knockout mouse embryonic fibroblasts (MEFs), but
neither compound was inhibited Hh signaling in the presence of a constitutively active Smo
mutant, indicating that inhibition of pathway signaling as at the level of Smo [17,21]. In addition,
ITZ significantly displaced a tritiated Smo antagonist from HEK293 cells that stably express
human Smo [22]. Both azoles retain in vitro and in vivo activity in the presence of several

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mutant forms of Smo resistant to other Smo antagonists, suggesting that the PSZ/ITZ scaffold
may adopt a distinct binding conformation when in complex with Smo [17, 21]. With this in
mind, we had three primary goals for our computational studies. First, we sought to predict the
conformation these compounds adopt when binding to Smo to determine how they maintain
potent anti-Hh activity in the presence of mutant forms of Smo. Second, we sought to correlate
inhibition of the Hh pathway with binding energy calculations. Finally, we explored the
conformational changes that occur in Smo following compound binding to validate the recent
models predicting whether a Smo ligand is an agonist or antagonist [26, 27]. Taken together, this
information could aid in the future design of PSZ/ITZ analogues as improved Hh pathway
inhibitors. Our first step in this process was to generate a homology model of SMO based on
previously published Smo structures in complex with known Smo antagonists (PDB IDs: 4JKV
[26], 5V56 [28], and 5L7I [5]). We chose to generate a new model rather than use the existing
structures because the complexes described above either removed or mutated multiple residues to
allow for improved crystallization. We replaced these altered residues with the natural amino
acids and refined the overall structure through homology modeling (Modeller 9.16).
The three-dimensional structure of the SMO receptor generated through our homology
modeling process is analogous to the experimentally-derived structures previously disclosed.
Smo contains an extracellular domain (ECD), which is composed of the well-characterized
cysteine rich domain (CRD) and a linker domain (ELD) (Figure 2A). Other key regions of the
Smo receptor include the seven transmembrane domain (7TM), extracellular loop 2 (ECL2) that
connects TM4 and TM5, and the KTXXXW motif (K539-T553) located in helix VIII of the
intracellular domain. ECL2 contains a β-hairpin that encompasses the primary binding pocket for
SMO ligands. Orientation of the intracellular KTXXXW motif is important for both stabilizing

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the entire Smo receptor in an inactive state and inducing a conformational change that results in
Smo activation [28]. Stability of the KTXXXW motif in the inactive state is primarily governed
by molecular interactions of T541, I544 and W545 in helix VIII with T251 and A254 in helix I.
Most notably, the side chain hydroxyl of T541 forms a hydrogen bond with the backbone
carbonyl of V536 in the intracellular end of helix VII and the indole hydrogen of W545 forms a
hydrogen bond with the hydroxyl in the side chain of T251 in helix I (Supplemental Figure 1).
3.3. Binding modes for PSZ, 1, and 19
In order to explore the binding interaction of PSZ and 1 with Smo, a grid box was generated
around the well-characterized Smo binding site. Both PSZ and 1 were docked into this grid on
Smo utilizing the XP mode in Schrödinger Glide. The putative ligand binding site for these
compounds is located at the extracellular surface of the β-hairpin in ECL2 and the α-helical
extension of TM6 of the 7-TM domain. As shown in Figure 2, both PSZ and 1 adopt similar
orientations and binding interactions inside the binding pocket. For both compounds, the triazole
region is oriented towards the extracellular surface with the linker/side chain region penetrating
deeply into the Smo binding pocket. The primary difference in the binding modes of the two
compounds is in regards to the triazole ring of PSZ, which orients towards the solvent accessible
surface of the binding pocket resulting in unfavorable polar binding interactions (Figure 2B and
2C). By contrast, removal of the triazole in analogue 1 results in reduced interactions with the
water molecules present at the pocket surface, which may be a primary reason why analogue 1
demonstrates enhanced anti-Hh activity. Additional intermolecular interactions with Smo common
to both compounds include a network of π–π and cation–π interactions between the phenyl ring
adjacent to the ether and Y207, K395, R485, and/or F484 (Figure 3). The triazolone/side chain

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region of the scaffold forms two hydrogen bonds with polar residues (D384 and N219) while
also exhibiting favorable non-polar interactions between the aliphatic side chain and several
hydrophobic residues (Y394, E518 and P513).
Figure 2. Binding mode of PSZ and 1 in complex with Smo. (A) The binding orientations of
PSZ (yellow) and 1 (blue) inside the binding pocket on Smo. The opening of the binding site is
sandwiched between the ELD and ECL2. (B) The furan region of PSZ and 1 orient out of the
binding pocket towards the ELD. (C) Compared to PSZ, 1 penetrates more deeply inside the
binding pocket.
Based on these predicted binding modes for PSZ and 1, we sought to design an improved
analogue that focused on exploring whether an analogue of 1 that replaced the sec-butyl with a
simplified hydrophilic side chain would maintain potent anti-Hh activity. In addition, the

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incorporation of the terminal alcohol could potentially improve the solubility of the scaffold.
Prior to the synthesis of this analogue (19), the compound was docked into our Smo model.
Overall, the orientation and binding mode of 19 was comparable to both PSZ and 1 (Figure 3).
The difluorophenyl ring and tetrahydrofuran are oriented at the solvent accessible surface, while
the triazolone/side chain penetrated deeply into the pocket. The primary alcohol on the side chain
formed a hydrogen bond with D384, mimicking the interaction between this amino acid residue
and the secondary alcohol in the side chain of PSZ (Figure 3). Finally, neither PSZ, 1, nor 19
demonstrated specific intermolecular interactions with the primary residue mutations that have
been linked to vismodegib (D477G and E522K) and sonidegib resistance (N223D, L225R, and
G457S), providing evidence as to why these compounds maintain activity in the presence of
mutated forms of Smo.
Figure 3. Two-dimensional Smo:ligand contact maps for PSZ (A), 1 (B), and 19 (C) in complex
with our Smo structural model. Color code is as follows: red residue, negatively charged residue;
purple, positively charged residue; green, hydrophobic residue; light blue, polar residue; gray,
solvent exposed area; pink arrow, hydrogen bond; green line, pi-pi interactions.

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4. Biological Activity of Second Generation PSZ Analogues.
4.1. Anti-Hh activity of PSZ analogues.
Preliminary docking studies with our Smo model encouraged the synthesis of 19 as well as two
additional hydroxylated analogues, 17 and 18. When evaluated in ASZ cells for Hh inhibition,
19, which incorporates a propanol side chain, was most active amongst the hydroxylated
analogues (IC50 = 0.020
μ
M). Overall, 17
-
19 had significantly increased anti-Hh activity when
, highlighting the potential of
compared to PSZ and our initial proof-of-concept analogue
1
introducing a hydrophilic side chain in addition to the removal of the triazole ring when
improving this scaffold. Curious about the impact a hydroxylated side chain would have on the
Hh activity of the ITZ scaffold, the non-stereochemically defined propanoic acid side chain
analogue, 21, was synthesized. Interestingly, 21 was more active than 19 by approximately 2-
fold.
Based on our recent SAR for the ITZ scaffold [20], PSZ analogues 23
incorporating modifications to the triazolone/side chain region or ‘right-side’ of the scaffold.
Analogues 23 27 integrated the des-triazole THF region with the linker intermediates leading to
the un-substituted triazolone ring while an amide linkage in analogues 28 30 introduced phenyl
rings with hydrogen bonding potential. Evaluation in ASZ cells revealed the truncated nitro (23
and aniline (24) PSZ analogues had weakened Hh inhibition compared to indicating an extended
scaffold is required for optimal activity. The carbamate (25), carboxamide (26), and triazolone
27) PSZ analogues had similar Hh activity as their trans-ITZ counterparts previously published
from our group [20]. When investigating the amide linkage analogues, we found that the addition
of a meta-acetyl phenyl ring (28), increased anti-Hh activity (IC50 = 0.024 M) that was
-30 focused on
-
-
)
1
(
μ

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comparable to the hydroxylated analogues (17-19). A meta-hydroxylated phenyl ring (29
)
maintained Hh activity similar to
inhibition in ASZ cells.
1 while the addition of a meta-pyridyl group (30) abolished Hh
Table 2. Hh Inhibition of PSZ Analogues in ASZ cells.
Cmpd
IC50 (µ
M)a, b
0.50 ± 0.1
0.19 ± 0.03
0.032 ± 0.01
0.15 ± 0.02
0.020 ± 0.01
0.012 ± 0.005
0.74 ± 0.15
0.33 ± 0.12
>10
0.22 ± 0.08
0.19 ± 0.10
0.024 ± 0.009
0.19 ± 0.03
>10
PSZ
1
17
18
19
21
23
24
25
26
27
28
29
30
aIC50 values represent the Mean ± SEM of at least two separate experiments performed in triplicate.
bAll analogues evaluated following 48 h incubation.
4.2. Preliminary pharmacokinetic profiles of PSZ and analogues.
Based on the promising and improved potency of the PSZ analogues, we evaluated several
pharmacokinetic parameters of representative compounds (Tables 1 and 3). All of the
compounds evaluated were minimally soluble in PBS at pH 7.4. As expected, removal of the
triazole moiety completely abolished the ability of PSZ analogues (
1
,
21, and 28) to inhibit
was
Cyp3A4. Interestingly, while PSZ was identified as a substrate for P-glycoprotein (Pgp),
1
not a substrate, suggesting the triazole also plays a role in efflux pump recognition for the PSZ
scaffold (Table 1). In addition, none of the compounds inhibited the hERG channel at
concentrations up to 25 µM. Overall, PSZ was more stable than ITZ; however, removal of the
triazole from PSZ resulted in a significant decrease in metabolic stability for analogue
1 (T1/2 =

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108 and 61.8 min, respectively). By contrast, analogues 28
-30 had improved metabolic stability
compared to both PSZ and as evidenced by T1/2 values ranging from 115 to >180 min and
1
internal clearance rates as low at 2.23 µl/min/mg.
Table 3. Preliminary Pharmacokinetic Activity of PSZ and Analogues.
Solubility cCYP3A4
hERG
T1/2
(min)c
108 ± 5.3
27.0 ± 6.2
61.8 ± 2.1
---
---
115 ± 9.8
> 180
Clint
(µL/min/mg)c
Cmpd
(µ
M)a
IC50 (
µ
M) Inhibition (%)b
0.781
0.8
---
1.56
0.781
3.13
3.13
1.56
0.15
0.04
>20
---
>20
>20
---
14.7
< 1
---
21.5
---
37.4
---
PSZ
ITZ
1
19
21
28
29
30
---
3.6
4.2
---
---
20.0
2.23
7.15
---
---
> 180
aKinetic solubility determined in PBS at pH 7.4.
bPercent inhibition of the hERG channel at 25 µM.
cT1/2 and Clint were measured in human liver microsomes at 1 µM.
--- indicates values were not determined.
4.3. Molecular dynamics studies for PSZ, 1, and 19.
4.3.1. Structural conformation studies. The molecular docking described above represents a
single snapshot of the binding complex between Smo and our PSZ-based ligands. In order to
more closely probe the binding interactions and conformational changes generated when PSZ or
its analogues bind with Smo, we performed additional molecular dynamics (MD, 50 ns) studies
on the Smo:PSZ, Smo:1, and Smo:19 docked structures. First, we utilized the root mean square
deviations (RMSDs) of the heavy atoms in the Smo:ligand complexes to evaluate the dynamic
structural changes that occurred during the MD simulations (Figure 4). The overall Smo:ligand
complex was more stable for analogues
1 and 19 compared to PSZ, which suggests a tighter
binding affinity (Table 3). A similar trend was observed for the RMSD values of the ligand,
further highlighting that the two analogues are more stable in complex with Smo. The RMSD

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values of the 7-TM domain and the KTXXXW motif suggest that these regions of Smo were in a
stable conformation throughout the simulation. By contrast, the CRD is more flexible, most
likely due to fluctuation of the ELD. The KTXXXW motif, which drives downstream activation
of the pathway following a conformational change [27], demonstrated stable RMSD values for
each complex providing strong evidence that Smo adopts its inactive conformation upon binding
to the three small molecules (Table 3). To further probe the activation/inactivation state of Smo
in complex with our analogues, we evaluated the conformational stability of helix VI/TM6 by
RMSD analysis of the backbone atoms in this region. The α-helical extension of TM6 remained
stable throughout the 50 ns simulation time for two primary reasons. First, a cation-π interaction
between R451 of TM6 and W535 in TM7 was maintained throughout the simulation. This
specific interaction is known to play a critical role in stabilizing Smo in an inactive conformation
[27]. Second, a network of hydrogen bonds between several water molecules and amino residues
475-479 stabilize the non-proline kink of the α-helical extension and TM6.

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Figure 4. RMSD for heavy atoms of the Smo:PSZ (A), Smo:1 (B), and SMO:19 complexes. 7-
TM (black), ligand (blue), CRD (red), KTXXXW motif (green), TM6 (magenta), and R451-
W535 (gray) with respect to the equilibrated structure are plotted as a function of MD simulation
time.
Table 3. RMSD comparisons for different domains of the Smo:PSZ, Smo:1, and Smo:19
complexes.
Smo:PSZ
Smo:1
Smo:19
Ligand
Complex
CRD
3.13 ± 0.5 1.95 ± 0.3 1.98 ± 0.4
8.85 ± 1.4 5.88 ± 1.3 4.48 ± 0.7
4.30 ± 0.5 5.41 ± 1.6 4.24 ± 0.7
3.30 ± 0.6 2.96 ± 0.3 2.89 ± 0.4
2.97 ± 0.2 2.53 ± 0.2 2.51 ± 0.4
1.43 ± 0.1 1.62 ± 0.2 1.59 ± 0.1
7-TM
KTXXXW
TM6
R451-W535 0.74 ± 0.2 0.70 ± 0.2 0.66 ± 0.2
4.3.2. Binding energy studies. The RMSD values for all the heavy atoms of the ligands and the
7-TM domain of the Smo:PSZ and Smo:1 complex converged and equilibrated after 2 ns, which
suggests that these binding conformations are appropriate to utilize for analysis of the binding

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energies of the complexes. By contrast, convergence and equilibration of the Smo:19 complex
was achieved after 10 ns; therefore, the stable trajectories of the last 45 ns of the simulation were
employed to calculate the average binding energies for the three complexes using the
MM/GBSA method. The predicted binding energies of PSZ, 1, and 19 with Smo are in general
agreement with experimental inhibition of Hh inhibition described above, with the ∆Gbinding for
the Smo:1 and Smo:19 complexes (-39.41 and -36.07 kcal/mol, respectively) significantly lower
than the Smo:PSZ complex (-27.90 kcal/mol) (Table 4). To gain a clearer understanding of
which individual energy terms have the greatest impact on the calculated binding affinities, four
individual energy components were calculated and compared. Overall, the van der Waals (∆Evdw
)
interactions served as the major contributor to the binding energies. The polar contributions
(∆Gpolar) negatively affected compound binding, primarily through polar solvation effects that
antagonized binding affinity.
Table 4. MM/PBSA binding energies (kcal/mol) for Smo:ligand complexes.
PSZ
1
19
∆Gbinding
∆Gvdw
-27.90 ± 4.9a -39.41 ± 4.3 -36.07 ± 6.5
-57.96 ± 5.2 -67.79 ± 3.3 -68.13 ± 4.2
-11.07 ± 3.2 -8.08 ± 2.8 -18.64 ± 3.2
∆Gelect
∆Gpolar
47.67 ± 7.1
-6.54 ± 0.4
43.95 ± 7.5 58.11 ± 8.7
-7.48 ± 0.2 -7.41 ± 0.3
∆Gnonpolar
aAll values are kcal/mol.
To evaluate the structural and energetic convergence of the complexes, averages of the
MM/PBSA ∆Gbinding values were plotted as a function of simulation time (Figure 5). These plots
allowed us to determine the most stable structure across the entire simulation. For Smo:PSZ, the
most energetically stable conformation occurred at 21 ns. In this complex, the difluorophenyl
ring and the triazole moiety of PSZ are located between the ELD and ECL2 of Smo and have

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minimal interactions with the residues present at the surface groove adjacent to the binding site
(Figure 6A). The central piperazine adopts a pseudo chair conformation within the binding site
that is stabilized by two strong π –π interactions between the phenyl ring adjacent to the furan
and Y207 and the phenyl ring adjacent to the triazolone ring with F484. In addition, the
piperazine demonstrates strong hydrophobic interactions with multiple residues in the binding
site (M301, L303, Y394, K395, E481, and R485). The triazolone adopts a unique orientation
such that the carbonyl forms a hydrogen bond (52% occupancy, interatomic distance 2.37Å) with
the backbone of S385. The hydroxyl group on the propyl side chain forms a hydrogen bond with
the side chain of D384 (40% occupancy rate, interatomic distance 2.63Å) while the aliphatic
regions of the side chain are stabilized by multiple van der Waals contacts.
Time (ns)
10
20
30
40
50
PSZ
1
19
0
-10
-20
-30
-40
-50
Figure 5. Binding energies for Smo:ligand complexes over time. The lowest energy
conformation was utilized to explore intermolecular interactions between Smo and PSZ (blue), 1
(red), and 19 (green).
In a similar manner, the binding conformation of Smo:1 at 35 ns was used to explore the
key interactions between the ligand and receptor (Figure 6B). When the triazole moiety is

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removed, the conformation of the THF region of the scaffold reorients such that the
difluorophenyl ring of 1 interacts with the same region previously occupied by the triazole of
PSZ. The van der Waals contacts between the difluorophenyl ring and multiple residues at the
surface of the binding site (L303, R296, K204, S205 and I215) are stronger than those for the
triazole in PSZ, which provides insight into the improved activity of the des-triazole analogues.
The biaryl piperazine in 1 also adopts a chair conformation that is stabilized by π–π and π–cation
interactions between the phenyl ring adjacent to the furan and F484 and K395, respectively.
Interestingly, in this structure, N1 of the triazolone moiety is protonated, resulting in the
formation of a salt bridge with the carboxylate side chain of D384 (49% occupancy, interatomic
distance 1.81Å). Additional stabilization of the triazolone ring is provided by a π–π interaction
with Y394 and a hydrogen bond with the side chain of N219. Finally, the highly flexible
aliphatic side chain forms multiple hydrophobic contacts with several amino acids at the bottom
of the binding pocket.
The most energetically stable conformation of Smo:19 (20 ns) demonstrated that
analogue 19 penetrates deeper into the binding site than 1, allowing for a π–cation interaction
between the difluorophenyl ring and K395 that is not present for the other two compounds
(Figure 6C). The slightly deeper positioning of 19 also allows for the phenoxy ether to form a
hydrogen bond with the backbone of E208 (36% occupancy, interatomic distance 1.81Å). The
central diaryl piperazine of 19 is stabilized in a chair orientation by the same π–π interactions
between the two flanking phenyl rings and Y207 and F484 seen in the Smo:PSZ structure.
Finally, the primary hydroxyl of the propanol side chain forms a hydrogen bond with D384 (58%
occupancy with interatomic distance 1.85Å).

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5. CONCLUSION
We have broadened our understanding of the optimal structural motifs required for potent
inhibition of the Hh signaling pathway for small molecule analogues based on the general PSZ
and ITZ scaffolds. Our studies continue to demonstrate that the triazole moiety present in the
parent scaffold is not required for potent anti-Hh activity and that its removal abrogates the
primary off-target side effect associated with PSZ/ITZ administration. Interestingly, removal of
the triazole in the PSZ scaffold results in a significant increase in Hh pathway inhibition and
additional modifications to the ‘right-side’ of the scaffold have resulted in analogues with low
nanomolar IC50 values in Hh-dependent cell lines. To more fully understand our experimental
SAR, we performed extensive computational studies, the results of which provide important
context for further analogue preparation. The THF region extends out of the binding pocket and
removal of the triazole decreases unfavorable polar interactions with water molecules at the
binding surface, which supports the improved activity of the des-triazole analogues. Finally, the
incorporation of a hydrophilic moiety in the triazolone/side chain region of the scaffold can
increase activity through improved hydrogen bond interactions with key binding site residues.

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Figure 6. Energetically stabilized structures of Smo in complex with PSZ (A), 1 (B), and 19 (C).
Intermolecular interactions between Smo and the ligand are represented as dotted lines. Yellow,
hydrogen bond; Dark blue, π-π interaction; Red, π-cation interaction.
6. Experimental section
6.1. General Information.
Starting materials and solvents were purchased from Sigma-Aldrich or Fisher Scientific.
All reactions were performed under an argon atmosphere. Intermediates 11 and 20 were prepared
as previously described [17]. NMR data was collected on a Bruker AVANCE 500 MHz

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spectrometer and analysis performed using MestReNova. HRMS data was obtained at the Mass
Spectrometry Facility at the University of Connecticut. FT-IR analysis was performed on a
Bruker Alpha Platinum ATR instrument using OPUS software (v 7.2). All PSZ analogues
evaluated in the biological assays were greater than 95% purity. Purity was determined by
injecting 25 µL of a 1 mg/mL solution each PSZ analogue dissolved in HPLC-grade MeCN 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 12 min and purity was assessed at 254 nm.
6.2. Chemical synthesis.
6.2.1. Synthesis of des-triazole intermediates.
6.2.1.1. ((3S,5R)-5-(2,4-difluorophenyl)-5-methyltetrahydrofuran-3-yl)methanol (9). To a
solution of 8 (150 mg, 0.3 mmol) in anhydrous DMSO, NaBH4 (43 mg, 1.1 mmol, 4 equiv.) was
added. The reaction was stirred at 100 C overnight. The reaction is cooled to 0 C before
being quenched by the dropwise addition of NH4Cl (aqueous, saturated). Once bubbling ceased,
the quenched reaction is extracted using EtOAc (4x). The organic layers were combined, dried
over Na2SO4, filtered and concentrated. The crude was purified by column chromatography
(SiO2, 10-30% EtOAc in Hexanes). The fractions containing product were collected and dried by
1
rotary evaportation to obtain a clear, colorless oil (Yield = 40 mg, 62%, 40 mg). HNMR (500
MHz, CDCl3) δ 7.57 (ddd, J = 34.4, 15.6, 8.8 Hz, 1H), 6.91 – 6.78 (m, 2H), 4.23 – 4.18 (m, 1H),
4.04 (t, J = 8.1 Hz, 1H), 3.91 – 3.86 (m, 1H), 3.75 – 3.66 (m, 2H), 2.72 (dt, J = 14.6, 7.4 Hz,
1H), 2.58 – 2.51 (m, 1H), 2.48 – 2.35 (m, 1H), 1.87 (dd, J = 12.7, 8.4 Hz, 1H), 1.62 (s, 2H), 1.55

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13
(s, 1H). CNMR (126 MHz, CDCl3) δ 163.04, 162.94, 161.07, 159.96, 158.08, 157.99, 127.68,
127.63, 127.60, 127.56, 110.77, 110.58, 104.55, 104.34, 104.13, 82.80, 82.77, 77.30, 77.04,
76.79, 69.89, 64.76, 42.31, 42.03, 41.86, 41.83, 41.59, 28.29. DART-HRMS: m/z [M+H]+ calcd.
for [C12H13F2O2]+, 227.0883; found 227.0899.
6.2.1.2.
((3S,5R)-5-(2,4-difluorophenyl)-5-methyltetrahydrofuran-3-yl)methyl
4-
methylbenzenesulfonate (10). Alcohol 9 (33 mg, 0.145 mmol, 1 equiv.), trimethylamine (0.29
mmol, 0.032 mL, 2 equiv.) and 4-DMAP (18 mg, 0.145 mmol, 1 equiv.) were dissolved in DCM
at room temperature. Next, TsCl was added slowly. The reaction was stirred for 2 hours at room
temperature. Upon completion, the reaction was diluted with EtOAc and washed with H2O and
brine. The organic layer was collected, dried over Na2SO4, filtered and concentrated. The crude
was purified by column chromatography (SiO2, 10-25% EtOAc in Hexanes). The fractions
containing product were collected and dried by rotary evaportation to obtain a clear, colorless oil
(Yield = 55 mg, ~100%). 1HNMR (500 MHz, CDCl3) δ 7.82 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 8.1
Hz, 1H), 7.49 – 7.45 (m, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 6.88 – 6.75 (m,
2H), 4.15 – 3.60 (m, 5H), 2.50 (m, 4H), 2.50 (m, 1H, hidden by CH3) 2.41 – 2.37 (m, 1H), 1.91 –
13
1.77 (m, 1H), 1.55 (s, 2H), 1.50 (s, 1H). CNMR (126 MHz, CDCl3) δ 163.07, 161.11, 159.91,
158.04, 145.02, 132.88, 129.96, 129.89, 129.60, 127.92, 127.87, 127.53, 127.45, 127.41, 127.07,
110.89, 110.72, 110.01, 109.98, 104.63, 104.42, 104.21, 82.74, 77.31, 77.05, 76.80, 71.29, 70.98,
69.75, 69.18, 42.02, 41.49, 41.46, 39.19, 39.00, 29.72, 28.26, 21.66
DART-HRMS: m/z [M+H]+
.
cald. for [C19H21F2O4S]+, 383.1050; found 383.1117. Also found [M–CH3]+ = 367.0803 and
[M+NH4]+ = 400.1388.

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6.2.2. Synthesis of proof-of-concept analogue 1.
6.2.2.1. General synthetic procedure for coupling the des-triazole THF region to linker
intermediate. Cs2CO3 (10 equiv.) and the requisite linker (1 equiv.) were diluted in anhydrous
DMSO and stirred for 30 minutes at room temperature before addition of the des-triazole THF
region in DMSO. The mixture was heated to 90 ⁰C and stirred for 12 h. The reaction was
quenched with water and extracted using EtOAc (2x) and DCM (2x). For 17 and 18, the water
layer was acidified to pH = 2 to ensure complete extraction of the carboxylic acid into the
organic phase. The organic layers were combined, dried over Na2SO4, filtered and concentrated.
The crude analogue was purified by column chromatography (SiO2, 10-30% Acetone in
Hexanes).
6.2.2.2. 2-((R)-sec-butyl)-4-(4-(4-(4-(((3R,5R)-5-(2,4-difluorophenyl)-5-methyltetrahydrofuran-
3-yl)methoxy)phenyl)-piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (1). Analogue 1
was prepared by coupling THF region intermediate 10 and linker/side chain intermediate 11 as
1
described above. Dark grey solid. (Yield = 63%). HNMR (500 MHz, CDCl3) δ 7.66 (s, 1H),
7.59 – 7.54 (dd, J = 16.2, 8.2 Hz, 1H), 7.47 (d, J = 8.5 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H), 6.98
(m, 1H), 6.89 (d, J = 7.6 Hz, 3H), 6.85 – 6.80 (m, 1H), 4.34 (m, 1H), 4.14 – 4.11 (t, J = 7.7 Hz,
1H), 3.97 – 3.96 (m, 3H), 3.85 – 3.74 (m, 1H), 3.41 (m, 4H), 3.28 (m, 4H), 2.64 – 2.62 (m, 2H),
2.10 – 1.87 (m, 2H), 1.79 – 1.75 (m, 1H), 1.65 (s, 3H), 1.43 (d, J = 6.7 Hz, 3H), 0.95 (t, J = 7.3
13
Hz, 3H). CNMR (126 MHz, CDCl3) δ 152.08, 150.59, 145.72, 127.74, 127.66, 125.98, 123.59,
118.57, 116.70, 115.32, 104.50, 104.36, 104.15, 82.80, 70.16, 52.72, 50.75, 49.27, 42.07, 39.54,
29.72, 28.47, 28.39, 19.27, 10.81. DART-HRMS: m/z [M+H]+ cald. for [C34H40F2N5O3]+,

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604.3021; found 604.3085. IR (solid) vmax: 2932, 2842, 1702, 1513, 1230, 1041, 966, 823.
Purity = 95.1%.
6.2.3. Synthesis of des-triazole analogues containing a modified side chain.
6.2.3.1. ethyl 3-(4-(4-(4-(4-methoxyphenyl)piperazin-1-yl)phenyl)-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl)propanoate (13). Ethyl-3-bromoproponate was coupled with linker region
intermediate 10 via the general procedure described in 5.2.2.1. above to afford 13. Light tan
solid. (Yield = quantitative) 1HNMR (500 MHz, CDCl3) δ 7.64 (s, 1H), 7.42 (d, J = 8.9 Hz, 2H),
7.05 (d, J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz, 3H), 6.90 (d, J = 9.0 Hz, 3H), 4.21 – 4.19 (m, 4H),
3.81 (s, 3H), 3.40 – 3.38 (m, 4H), 3.26 – 3.24 (m, 4H), 2.87 – 2.84 (t, J = 7.1 Hz, 2H), 1.31 –
1.28 (t, J = 7.1 Hz, 3H). 13CNMR (126 MHz, CDCl3) δ 170.83, 154.22, 151.99, 150.72, 145.49,
134.42, 125.60, 123.63, 121.26, 118.62, 116.78, 116.60, 114.56, 110.01, 109.98, 77.40, 77.15,
76.89, 60.81, 55.59, 50.78, 49.87, 49.18, 41.36, 33.36, 29.97, 14.20. DART-HRMS: m/z [M+H]+
cald. for [C24H30N5O4]+, 452.2220; found 452.2326.
6.2.3.2. ethyl 2-(4-(4-(4-(4-methoxyphenyl)piperazin-1-yl)phenyl)-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl)acetate (14). 3-bromoacetate was coupled with linker region intermediate 10 via the
general procedure described in 5.2.2.1. above to afford 14. Light tan to white solid. (Yield =
1
quantitative). HNMR (500 MHz, CDCl3) δ 7.71 (s, 1H), 7.46 (d, J = 9.0 Hz, 2H), 7.07 (d, J =
9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 4.67 (s, 2H), 4.47 – 4.28 (dd, J =
15.9, 8.8 Hz, 3H), 3.83 (s, 3H), 3.43 – 3.41 (m, 4H), 3.29 – 3.27 (m, 4H), 1.36 – 1.33 (t, J = 7.1
Hz, 3H). 13CNMR (126 MHz, CDCl3) δ 167.58, 167.05, 154.57, 154.27, 152.54, 150.78, 135.11,

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125.94, 125.50, 123.67, 118.66, 116.61, 114.56, 110.01, 109.98, 77.34, 77.09, 76.83, 63.88,
61.83, 61.69, 55.60, 50.83, 49.16, 46.81, 14.16, 14.10. DART-HRMS: m/z [M+H]+ cald. for
[C23H28N5O4]+, 437.2063; found 438.2149.
6.2.3.3. ethyl 3-(4-(4-(4-(4-hydroxyphenyl)piperazin-1-yl)phenyl)-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl)propanoate (15). To a solution of 13 (1 equiv.) in anhydrous DCM as 0
⁰C was
added 1M BBr3 in DCM (4-5 equiv.) dropwise. The mixture was stirred for 30 min at 0
⁰C
before warming to RT and stirred for an additional 3 h. The reaction was quenched by pouring
the mixture over ice, followed by washing with DCM (2x) and EtOAc (2x). The organic layers
were combined, dried over Na2SO4, filtered and concentrated. Column chromatography (SiO2, 1-
5% MeOH in DCM) afforded 15 as a light tan solid (Yield = 28%). 1HNMR (500 MHz, CDCl3)
δ 7.64 (s, 1H), 7.40 (d, J = 8.9 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.75
(d, J = 8.8 Hz, 2H), 4.25 – 4.22 (m, 4H), 3.40 – 3.38 (m, 4H), 3.25 – 3.13 (m, 4H), 2.89 – 2.86 (t,
13
J = 7.1 Hz, 2H), 1.32 – 1.30 (t, J = 7.1 Hz, 3H). CNMR (126 MHz, CDCl3) δ 170.87, 152.24,
150.90, 150.69, 145.04, 134.66, 125.24, 124.16, 118.84, 116.53, 116.00, 110.01, 109.98, 77.31,
77.05, 76.80, 60.90, 51.01, 49.10, 41.46, 33.35, 14.18. DART-HRMS: m/z [M+H]+ cald. for
[C23H28N5O4]+, 438.2063; found 438.2150.
6.2.3.4. ethyl 2-(4-(4-(4-(4-hydroxyphenyl)piperazin-1-yl)phenyl)-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl)acetate (16). Alcohol 16 was prepared using precursor 14 and BBr3 as described
1
above. Tan solid. (Yield = 23%). HNMR (500 MHz, CDCl3) δ 8.41 (s, 1H), 7.71 (s, 1H), 7.42
(d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.04 – 6.99(m, 2H), 6.93 – 6.90 (t, J = 9.1 Hz, 2H),
6.82 – 6.77 (m, 2H), 4.69 (d, J = 8.7 Hz, 1H), 4.47 (s, 1H), 4.30 – 4.22 (m, 2H), 3.38 –3.24 (m,
8H), 1.35 – 1.31 (m, 3H). 13CNMR (126 MHz, CDCl3) δ 168.51, 162.68, 150.85, 150.56, 150.10,

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150.07, 145.56, 135.14, 132.85, 125.96, 123.79, 118.84, 116.70, 116.61, 115.96, 114.58, 61.86,
61.50, 55.61, 52.65, 50.89, 49.23, 49.17, 47.79, 46.83, 46.70, 40.99, 29.71, 14.14. DART-
HRMS: m/z [M+H]+ cald. for [C22H26N5O4]+, 424.1907; found 424.1958.
6.2.3.5.
3-(4-(4-(4-(4-(((3R,5R)-5-(2,4-difluorophenyl)-5-methyltetrahydrofuran-3-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)propanoic
acid (17). Analogue 17 was prepared by coupling des-triazole intermediate 10 and linker region
1
intermediate 15 as described above. White solid. (Yield = 42%) HNMR (500 MHz, CDCl3) δ
7.63 (s, 1H), 7.59 – 7.51 (m, 2H), 7.44 (d, J = 8.9 Hz, 1H), 7.07 (d, J = 9.0 Hz, 2H), 7.00 – 6.97
(m, 3H), 6.90 – 6.79 (m, 5H), 4.29 – 4.21 (m, 1H), 4.14 – 4.07 (m, 1H), 4.03 – 3.91 (m, 4H), 3.8
– 3.72 (m, 1H), 3.42 – 3.40 (m, 3H), 3.32 – 3.31 (m, 1H), 3.28 – 3.27 (m, 4H), 2.71 – 2.51 (m,
13
3H), 2.11 – 1.97 (m, 2H), 1.66 (s, 1H), 1.65 (s, 2H). CNMR (126 MHz, CDCl3) δ 176.37,
162.98, 161.01, 153.50, 152.29, 150.77, 145.57, 134.42, 127.72, 125.49, 123.73, 118.69, 118.60,
118.04, 116.67, 116.50, 115.31, 110.79, 110.63, 110.01, 109.98, 104.57, 104.36, 104.16, 82.80,
77.32, 77.06, 76.81, 70.43, 70.16, 70.10, 50.77, 50.21, 49.12, 48.00, 42.99, 42.04, 39.51, 39.42,
36.79, 29.71, 28.36, 20.87. DART-HRMS: m/z [M+H]+ cald. for [C33H36F2N5O5]+, 620.2060;
found [M – 114.9653]+, 505.2407 m/z. IR (solid) vmax: 2931, 1705, 1614, 1512, 1231.6, 1039,
966, 822. Purity = 95.1%.
6.2.3.6.
2-(4-(4-(4-(4-(((3R,5R)-5-(2,4-difluorophenyl)-5-methyltetrahydrofuran-3-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetic acid
(18). Analogue 18 was prepared by coupling des-triazole intermediate 10 and linker region
intermediate 16 as described above. Tan solid. (Yield = 27%) 1HNMR (500 MHz, CDCl3) δ