Dimeric isoxazolyl-1,4-dihydropyridines have enhanced binding at the multi-drug resistance transporter

Article abstract of DOI:10.1016/j.bmc.2017.04.008

A series of dimeric isoxazolyl-1,4-dihydropyridines (IDHPs) were prepared by click chemistry and examined for their ability to bind the multi-drug resistance transporter (MDR-1), a member of the ATP-binding cassette superfamily (ABC). Eight compounds in t

Full text of DOI:10.1016/j.bmc.2017.04.008

Accepted Manuscript
Dimeric isoxazolyl-1,4-dihydropyridines have enhanced binding at the multi-
drug resistance transporter
Scott A. Steiger, Chun Li, Donald S. Backos, Philip Reigan, N.R. Natale
PII:
S0968-0896(17)30401-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.008
BMC 13678
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
27 February 2017
3 April 2017
5 April 2017
Please cite this article as: Steiger, S.A., Li, C., Backos, D.S., Reigan, P., Natale, N.R., Dimeric isoxazolyl-1,4-
dihydropyridines have enhanced binding at the multi-drug resistance transporter, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.04.008
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Steiger, et al
IDHP dimers @ VGCC, MDR-1 & 7TM
Dimeric isoxazolyl-1,4-dihydropyridines have enhanced binding at the multi-drug resistance
transporter
Scott A. Steiger,a Chun Li,b Donald S. Backos,c Philip Reigan,c and N.R. Natale *a
a Department of Biomedical and Pharmaceutical Science, University of Montana
Missoula, MT 59812
b Department of Chemistry, Ithaca College, 953 Danby Road, Ithaca NY 14850
c University of Colorado Anschutz Medical Campus, Skaggs School of Pharmacy, Aurora, CO
80045
* Corresponding author: nicholas.natale@umontana.edu
Abstract - A series of dimeric isoxazolyl-1,4-dihydropyridines (IDHPs) were prepared by click
chemistry and examined for their ability to bind the multi-drug resistance transporter (MDR-1), a
member of the ATP-binding cassette superfamily (ABC). Eight compounds in the present study
exhibited single digit micromolar binding to this efflux transporter. One monomeric IDHP m-Br-
1c, possessed submicromolar binding of 510 nM at MDR-1. Three of the dimeric IDHPs
possessed <1.5 µM activity, and 4b and 4c were observed to have superior binding selectivity
compared to their corresponding monomers verses the voltage gated calcium channel (VGCC).
The dimer with the best combination of activity and selectivity for MDR-1 was analog 4c
containing an m-Br phenyl moiety in the 3- position of the isoxazole, and a tether with five
ethyleneoxy units, referred to herein as Isoxaquidar. Two important controls, mono-triazole 5
and pyridine 6, also were examined, indicating that the triazole - incorporated as part of the click
assembly as a spacer - contributes to MDR-1 binding. Compounds were also assayed at the
allosteric site of the mGluR5 receptor, as a GPCR 7TM control, indicating that the p-Br IDHPs
4d, 4e and 4. with tethers of from n= 2 to 5 ethylenedioxy units, had sub-micromolar affinities
with 4d being the most efficacious at 193 nM at mGluR5. The results are interpreted using a
docking study using a human ABC as our current working hypothesis, and suggest that the
distinct SARs emerging for these three divergent classes of biomolecular targets may be tunable,
and amenable to the development of further selectivity.
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IDHP dimers @ VGCC, MDR-1 & 7TM
Dimeric isoxazolyl-1,4-dihydropyridines have enhanced binding at the multi-drug resistance
transporter
Scott A. Steiger,a Chun Li,b Donald S. Backos,c Philip Reigan,c and N.R. Natale *a
a Department of Biomedical and Pharmaceutical Science, University of Montana
Missoula, MT 59812
b Department of Chemistry, Ithaca College, 953 Danby Road, Ithaca NY 14850
c University of Colorado Anschutz Medical Campus, Skaggs School of Pharmacy, Aurora, CO
80045
* Corresponding author: nicholas.natale@umontana.edu
Molecules which reverse the effect of the multidrug resistance transporter (MDR-1, also
known as P-glycoprotein or P-gp1-5), exemplified by Tariquidar,6 Laniquidar7 and
Zosuquidar8(Figure 1) are the subject of clinical trials as adjuvants to cancer chemotherapy.
Dihydropyridines (DHPs) have been under intense study for many years for their ability to
inhibit the multidrug transporter.9,10 More recently we have reported that 4-isoxazolyl-1,4-
dihydropyridines (IDHPs) have significant binding at this target,11-13 and a unique structure
activity relationship (SAR) which diverges from their voltage gated calcium channel (VGCC)
activity. In our initial study, we found that branching at the C(5) of the isoxazole gave rise to
three of the four most efficacious IDHPs at MDR-1.11 In a follow-up study, attachment of a
fluorophore also enhanced activity. This suggested additional binding opportunities in proximity
to the putative nucleotide binding domain (NBD) of IDHPs (vide infra).12,13
Figure 1. Selective MDR-1 inhibitors Tariquidar (XR9576), Laniquidar, and Zosuquidar have
been investigated, alone and in combination with other chemotherapeutic agents, in clinical
trials.
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IDHP dimers @ VGCC, MDR-1 & 7TM
There have been previous indications that dimeric ligands enhance MDR-1 binding. The groups
of Andrus14 and Chmielewski and Hrycyna15-17 established that there was a relationship between
tether type, length and binding affinity. In contrast, during his study of dimeric tethered
dihydropyridines at the VGCC, Triggle found that monomeric analogs lacking a second 1,4-DHP
nucleus or possessing a second inactive pyridine were no more active,18 leading to the conclusion
that such dimers likely do not bridge adjacent DHP receptor motifs at the VGCC. We reasoned
we could use these observations, together with other known VGCC SAR, to design greater
selectivity for MDR-1 into the IDHPs.
Our hypothesis is that a bivalent inhibitor could bind more tightly to MDR1 and thus be a more
effective inhibitor than the corresponding monomer. Taking into account previous research the
tether’s length is critical, however, the MDR undergoes significant conformational
reorganization during the ATP driven efflux. In the apo state used in our previous homology
model12,13 the NBDs reside over 40 Å apart using the mouse P-gp data (pdb accession number
3G5U) (Figure 2, below), while in the ATP bound state exemplified by Sav1866 (pdb accession
number 2ONJ), that distance is reported as 4 Å. In our previous study of IDHPs tethered to
fluorophores,12 we used hydrocarbon tethers, and we were concerned that such groups could
suffer hydrophobic collapse during interaction with the target, given that such folded conformers
were evidenced in the NMR. We reasoned that oligo ethyleneoxy linkers (short PEGs), would be
more likely to adopt an extended conformation able to intercept the ABC at a stage intermediate
to the apo and outward facing ATP bound conformation. We then sought to synthesize such
small molecules to test our hypothesis.
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IDHP dimers @ VGCC, MDR-1 & 7TM
Figure 2: A. The SYBYL calculated distance between the Q-site predicted binding sites residing
proximal to the nucleotide binding domains in the non-ATP bound state (apo) of MDR-1, based
on the Mus musculus P-gp coordinates.12,13 B, C, D, calculated distances for n=2,3 and 5,
tethered dimers, respectively, in energy minimized conformations.
A
B.
C
D
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IDHP dimers @ VGCC, MDR-1 & 7TM
Chemistry
The monomeric IDHP 1a was prepared as previously described,19,20 The bromo IDHPs 1b -1d
were prepared in analogous fashion from the corresponding commercial aryl aldehydes, briefly,
by oxime formation, followed by oxidation to the oximidoyl chloride, nitrile oxide cycloaddition
to the isoxazole ester, lithium aluminum hydride reduction to the 4-carbinol (carefully
temperature controlled to avoid aryl debromination), chromium (VI) oxidation to the 4-
carbaldehyde, and completed by Hantzsch pyridine synthesis.21 The full experimental details and
characterization for all intermediates are detailed in the Supplementary Data. The dimers were
prepared by click chemistry22,23 by 1,3-dipolar cycloaddition of alkynyl- IDHPs 2 to bis-azide
PEGs 3, to produce the Dimeric IDHPs 4 (Scheme 1). We observed that mono-incorporation of
the alkyne moiety onto IDHPs was best accomplished by a modified two-component Hantzsch,
adding the Knoevenagel adducts 7, to alkynyl keto esters 9 to produce the click ready IDHP-
alkynes 2. The alkyne was incorporated onto ethylacetoacetate 8, using the excellent method of
Anand using boric acid trans-esterification to produce 9.24
Scheme 1. Synthesis of dimeric-IDHPs (4 a-f) and relevant controls. a. B(OH)3, prop-2-yne-1-ol,
toluene reflux. b. Aqueous Ammonia, EtOH, reflux. c. Cu(I)Br, 10 mole %.
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IDHP dimers @ VGCC, MDR-1 & 7TM
4 a. R = H; n = 5
b. R = o-Br, n = 5
c. R = m-Br, n = 5 Isoxaquidar
d. R = p-Br, n = 5
e. R = p-Br, n = 3
f. R = p-Br, n = 2
Single Crystal X-ray diffractometry
The conformations of DHPs are important to their structure activity relationships, and
IDHPs have particularly pronounced divergent topologies dependent on rotation about the bond
joining the heterocyclic rings.13 In the VGCC arena, several parameters have been found to
correlate with biological activity: the conformation of the esters at C-3 and C-5 of the DHP, the
sum of the six intra-ring torsion angle in the flattened boat conformation, and the forward
presentation of the C-4 aryl group of the DHP.25 We had earlier postulated that the heterocyclic
ring juncture could well adapt divergent conformations dependent on the biomolecular target.
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IDHP dimers @ VGCC, MDR-1 & 7TM
We have found single crystal x-ray diffractometry (Sc-Xrd) useful to help inform our
computational studies on rotational barriers and drug-receptor modeling, and Sc-Xrd of 1c. was
performed as previously described.26 In the event, IDHP monomer 1c. shows that both esters
adopt the syn- conformation in the solid state, the C-3 m-Br phenyl ring is oriented above the
DHP ring - so that the O of the isoxazole is exo-. The sum of the six intra-ring torsion angles 
is 66.33o and thus 1c adopts a very shallow boat conformer. The forward presentation of the 4-
isoxazolyl ring is reflected by the average magnitude of the C(2)-C(3)-C(4)-C(7) and C(6)-C(5)-
C(4)-C(7) torsion angles, designated 
o. These two variables are
not independent, and the biological activity at the VGCC has been found to correlate with both
parameters.
Important Intermolecular interactions. There is a plausible Halogen bond in the Sc-Xrd, as
defined by the Br1 to O3' distance of 3.363Å and halogen to nucleophile angle of 143.1o, which
are within the tolerances for a moderate strength halogen bond according to the criteria suggested
by Auffinger,27 and Wilcken.28
Figure 3. ORTEP of monomeric IDHP 1c, the unit cell reveals a plausible halogen bond (vide
infra).
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IDHP dimers @ VGCC, MDR-1 & 7TM
Table 1. Crystal data, data collection and refinement parameters for IDHP monomer 1c.
Crystal data
C23H25BrN2O5
Mr = 489.36
Dx = 1.459 Mg m−3
Melting point: ? K
Monoclinic, C2/c
a = 17.6211 (9) Å
b = 15.3041 (8) Å
c = 17.9370 (9) Å
β = 112.8770 (6)°
V = 4456.7 (4) Å3
Z = 8
radiation, λ = 0.71073 Å
Cell parameters from 323 reflections
θ = 3.4–28.4°
µ = 1.88 mm−1
T = 100 K
Prism, clear pale yellow
0.52 × 0.51 × 0.24 mm
F(000) = 2016
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IDHP dimers @ VGCC, MDR-1 & 7TM
Data collection
Bruker SMART BREEZE CCD
diffractometer
4567 independent reflections
4179 reflections with I > 2σ(I)
Rint = 0.021
Radiation source: Mo K
Detector resolution: 8.3333 pixels mm-1 θmax = 26.4°, θmin = 1.8°
Absorption correction: numerical
h = −22 21
SADABS V2012/1 (Bruker AXS Inc.)
Tmin = 0.41, Tmax = 0.66
k = −19 19
l = −22 22
24782 measured reflections
Refinement
Refinement on F2
Hydrogen site location: inferred from
neighbouring sites
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.035
wR(F2) = 0.098
H-atom parameters constrained
2
w = 1/[σ2(Fo ) + (0.0521P)2 + 9.9351P]
2
where P = (Fo + 2Fc2)/3
S = 1.07
(Δ/σ)max = 0.002
4567 reflections
285 parameters
80 restraints
Δρmax = 1.46 e Å−3
Δρmin = −0.45 e Å−3
Extinction correction: none
Tables containing the Fractional atomic coordinates and isotropic or equivalent isotropic
displacement parameters (Å2), Atomic displacement parameters (Å2), and Geometric parameters
(Å, °) are given in the Supplementary Data.
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IDHP dimers @ VGCC, MDR-1 & 7TM
MDR-1 Assay.
Screening of IDHPs was performed by the Psychoactive Drug Screening Program (PDSP) of
NIMH. The PDSP protocol utilizes live Caco-2 cells, which are derived from human colonic
epithelium cells which express MDR-1.29 The assay is based on the passive diffusion of calcein
acetoxymethyl ester (Calcein-AM), which is hydrolyzed inside the cell to calcein, which is both
fluorescent and negatively charged, and therefore trapped inside the cell. MDR-1 can transport
non-fluorescent Calcein-AM from cells, but not the hydrolysis product. The assay measures the
increase in calcein fluorescence as a function of time using a FlexStation II fluorimeter
– 3
pM) for 30 minutes, upon which time calcein-AM was added to a final concentration of 150 nM.
Fluorescence is monitored over 4 minutes, and each assay was performed in triplicate, with a
cyclosporin control. The value from untreated cells is 0% and the slope of the fluorescence is
normalized taking the value for cyclosporin as 100%.30 The results are shown in Table 2.
Insert Table 2
______________________________________________________________________________
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IDHP dimers @ VGCC, MDR-1 & 7TM
Table 2. Summary of biological evaluation of the Dimer-IDHPs (4) and relevant controls
Structure
R
n
PDSP #
VGCC
(IC50, nM)
13.9a
mGluR5
(nM)
MDR-1 (% vs.
Cyclosporin)b
48.9
MDR-1
(IC50, nM)
ND
H
0
0
0
0
5
5
5
5
3
2
0
0
21618
27215
27216
27217
27218
27219
27220
27221
27222
27223
27224
27225
ND
1a
1b
1c
1d
4a
4b
4c
4d
4e
4f
5
o-Br
m-Br
p-Br
H
909
208
1,524
>10,000
>10,000
>10,000
4,858
-12
101.9
58.4
>10,000
510
36
3,096
>10,000
2,862
1,296
1,394
3,231
1,449
1,643
>10,000
>10,000
>10,000
3,766
2,706
516
20.4
o-Br
m-Br
p-Br
p-Br
p-Br
p-Br
H
17.7
8,638
150.8
172.6
11.6
193
546
603
933
41.9
127
1,972
107.6
36.9
>10,000
>10,000
6
a reference 31.
b One dose inhibition compared to cyclosporin (150 nM) in the PDSP fluoresence assay (vide
infra). These one dose percent inhibition values represent the assay reported previously in
reference 11.
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IDHP dimers @ VGCC, MDR-1 & 7TM
Results and Discussion
As expected the new monomeric IDHPs 1b,1c and 1d exhibit robust calcium antagonist activity,
with the relationship of activity to halogen substitution at the 3-aryl-isoxazole position p- > m- >
o- as previously observed for the chloro-phenyl series,21 however, the range is more pronounced
for the Br series, with the p-Br 1d. with an IC50 of 36 nM (Figure 4), over 25 times more potent
than the o-Br 1b. However, to place this in perspective, the bromo-substituted phenyl IDHPs
monomers including click control 5, were all inferior at the VGCC to the unsubstituted 3-
isoxazole phenyl lead 1a (IC50 13.9 nM),31 and previously reported branched chiral32 and
lipophilic IDHPs,33 which were in the single digit nanomolar range.
Figure 4. Binding isotherms of nifedipine and IDHP monomer PDSP 27217 (1d) at the L-type
Voltage gated calcium Channel (VGCC).
The effect of dimerization on both binding and selectivity is dramatic. For the monomeric 1.b
compared to dimer 4b, MDR-1 activity appears to be improved, where in contrast VGCC is
essentially abolished for the dimer. The relationship for substitution at the isoxazole 3-aryl is
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IDHP dimers @ VGCC, MDR-1 & 7TM
maintained in both monomers and dimers, with m-Br > p-Br > o-Br > H, however, the m-Br 4.c
(Figure 5) and p-Br 4d dimers have significantly more binding potency at MDR-1.
Figure 5. IC50 of dimeric IDHP PDSP 27220 (4c). verses cyclosporine A at the multidrug
resistance transporter (MDR-1).
The isoxazolyl-pyridine 6 was envisioned as a control, as usually oxidation of DHPs abolished
VGCC activity.13 This was found to be the case, as 6 was essentially bereft of activity at both the
VGCC and mGluR5, however a small amount of binding at the MDR-1 was found in the one
dose transport inhibition assay (shown in the Supplementary Data).
G-Protein Coupled receptors are a significant class of receptors, with a large representation
among drugs in general medical practice.34 Furthermore, 7TM receptors have been reported
which bind DHPs.35 Therefore, we deemed that screening at a representative 7TM receptor,
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IDHP dimers @ VGCC, MDR-1 & 7TM
exemplified by the allosteric site of mGluR5, represented an important control. To our surprise,
while the monomeric IDHPs showed only modest binding at best, three dimeric IDHPs exhibited
sub-micromolar binding, most notably IDHP 4d at 193 nM, with selectivity for mGluR5 over the
VGCC and MDR-1. The putative 7TM allosteric binding site spans a large “big dipper” shaped
area,36 and could plausibly accommodate both IDHPs, if the PEG tether oriented outside of the
lipophilic cleft in the extracellular space. We are in the process of modeling 7TMs with IDHP
analogs, and further experimental and computational studies will be reported at a later date.
Docking and a new working hypothesis
Our previous ABCB1 homology model12,13 was based on the mouse P-gp structure,37 which has a
significantly different topology in its apo conformation than the recent human structure reported
by Carpenter's group (vide infra).38 Our calculation of the NBD distance in our previous human
homology model based on the M. musculus P-gp template was 42.5 Å, and the C. elegans
distance is 41 Å,2,38 which means that for our dimeric structures to be effective they would have
to intercept the efflux pump in a conformation intermediate at some point between apo and
Sav1866 nucleotide bound open-to-outside conformers. Calculations with SYBYL 8 and the
Molecular Mechanics forcefield using our mouse homology model gave highest scoring
solutions that were unsatisfying for the IDHP dimers, wherein the PEG tether collapsed inside
the ATP binding site, and both IDHP moieties were bound to the edge of the same NBD
(Supplementary Data).
In contrast, for the human ABC structure the distance observed between NBDs in the apo state is
17 Angstroms,38 and the tethers in our synthetic dimers are sufficiently long to bridge the NBD
to NBD interface in the human apo state, therefore we examined docking to Carpenter's ABC
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IDHP dimers @ VGCC, MDR-1 & 7TM
coordinates, PDB accession number 3ZDQ. The protein structure was typed with the CHARMM
forcefield39 and energy minimized with the smart minimizer protocol within Discovery Studio40
using the Generalized-Born with simple switching implicit solvent model to a root mean square
gradient (RMS) convergence <0.001 kcal/mol prior to use in the docking studies. Docking was
performed using the flexible docking protocol,41 which allows for flexibility in both the ligand
and the binding site residues. The final ranking of the docked poses was performed via consensus
scoring, combining the predicted binding energy with the Jain,42 PLP2,43 and Ludi344 scoring
functions. In general, the poses were much more reasonable using basic chemical intuition than
our previous Sybyl results. We initially examined the docking of a DHP with a reactive azide
incorporated that has been used for photo affinity labeling,13 which previously had
experimentally established that DHPs bind near the NBD. The best scored docking poses place
the azide moiety of azidopine proximal to the NBD, which we found encouraging. We then
examined the docking of the synthesized IDHP dimers, and almost all of the dimer poses bridged
the NBDs (Figure 3). The highest scoring pose was obtained for the RS-diastereomer of
Isoxaquidar 4c using the -CDocker scoring, and RR-4c had the highest calculated binding
energy. In the highest scored poses all of the salient functional groups of both DHP moieties of
the dimer are brought into interactions with critical moving parts of the ABC. The IDHP
interacts with critical residues in the ATP binding region, and channel helix 2, also referred to as
the sliding helix. In the interaction with the left NBD (as illustrated in Figure 6 for the RS-4c),
the isoxazole interacts with Ile578 by lipophilic interaction with the C(5) methyl group, and a π-
alkyl interaction with the isoxazole ring, and the C(3) phenyl has a π-cation interaction with
Arg362, which also bridges through a hydrogen bond with the C-3 ester carbonyl, and the m-Br
interacts with Tyr 501. Discovery Studio assigns this as a lipophilic interaction, although the
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IDHP dimers @ VGCC, MDR-1 & 7TM
geometry suggests the potential for a halogen bonding interaction (op cit). This array of multiple
interactions would prevent the sliding helix from performing its critical conformational change
which is necessary for the efflux of xenobiotics.
Similar significant interactions are observed for RR-4c, namely DHP N-H hydrogen bonding
with Asp658, and surprisingly since the click incorporated triazole was envisioned as a spacer,
there is a predicted π-alkyl interaction with Leu537, and an interaction of the polyethylene linker
with Glu576. The full complement of interactions are tabulated schematically in the LigPlot
diagram below (Figure 7). More detailed discussion of the binding interactions are illustrated in
the Supplementary Data.
Limitations of the computational model. Chang has noted that a flexible linker region
connecting the two halves of the MDR efflux pump is disordered and does not diffract, perhaps
owing to its conformational mobility.44 Interactions with this “missing linker” between the NBDs
could provide an important avenue for improving efficacy and selectivity of inhibitors.
Furthermore, Discovery Studio assigns the stereochemistry to the IDHP dimers, and the highest
calculated -CDocker Energy was predicted for RR-4c, while the highest binding energy was
predicted for specifically the RS-diastereomer of 4c., as well as the highest weighted average for
both scoring algorithms, assuming a statistical distribution of RR-/RS-/SS of 1:2:1. In our hands,
no separation of the diastereomers was observed during column or thin layer chromatography or
HPLC-MS, likely owing to the distance and conformational flexibility of the tether between the
DHP moieties of the dimers. Our recently reported organocatalytic asymmetric synthesis of
chiral IDHPs45 could plausibly allow us to prepare optically pure dimers, and this will be the
subject of future efforts.
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IDHP dimers @ VGCC, MDR-1 & 7TM
Conclusion
The 4-isoxazolyl-dihydropyridines (IDHPs) have complex polypharmacology analogous to their
4-aryl analogs, which is often observed for such so-called privileged scaffolds. Several IDHP
dimers described here have single digit micromolar binding to MDR-1, which is in the
approximate range for cyclosporin (800-900 nM), with dimer 4c (Isoxaquidar) exhibiting
selectivity for MDR-1 over both the VGCC and mGluR5. As a control for 7TM binding, it was
observed that the p-Br dimer 4d exhibited 193 nM binding, with selectivity for mGluR5 over the
VGCC and MDR-1, and further studies on 7TM binding will be the subject of future reports
from our laboratories. The position of the bromine substitution exerts a profound effect on
binding affinity, as well as selectivity, the question remains whether the critical contribution to
selectivity results mainly from lipophilic character or relates directly to halogen bonding.
However, the observation reported here that IDHPs exhibit selectivity between these three
classes of biomolecular targets suggest further SAR development could produce improved
selectivity. Such studies are part of an ongoing effort in our group, and our progress will be
reported in due course.
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IDHP dimers @ VGCC, MDR-1 & 7TM
Figure 6. Left, Docking of RS-IDHP dimer 4c Isoxaquidar (carbons green, nitrogens blue, and
oxygens red) at the human ABCB10 transporter (blue). The dimer 4c spans the interface between
the NBDs. Top right. Close up view of dimer 4c interacting with the left NBD, The isoxazole
moiety locks Ile578 by interacting with the C5 methyl and the isoxazole ring, Arg362 with the 3-
Ph, while the m-Bromo binds the Tyr501. Bottom right. Originally incorporated as a spacer, the
triazole appears to contribute interactions with Arg362.
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IDHP dimers @ VGCC, MDR-1 & 7TM
Figure 7. Top. Ligplot schematic representation of RR-IDHP dimer 4c interactions at the MDR-
1. Bottom Ligplot of RS-IDHP dimer 4c.
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IDHP dimers @ VGCC, MDR-1 & 7TM
Experimental
All reactions sensitive to air or moisture were run under an inert atmosphere of dry argon.
Chemicals were obtained from commercial sources and purified as needed. Purification of all
new compounds was performed using flash chromatography and/or preparative TLC. Melting
points were determined in open capillary tubes using a Melt-Temp instrument and are
uncorrected. NMR spectra were obtained using either a Bruker Avance III 400 MHz or a Varian
NMR systems 500 MHz spectrometer. NMR samples were run in and referenced to CDCl3,
unless stated otherwise. High-resolution mass spectrometry (HRMS) data were obtained using a
Waters/Micromass ESI/LCT-TOF instrument. Single crystal x-ray diffractometry was performed
on a Bruker SMART BREEZE CCD Diffractometer, data collection and refinement was
performed as previously described. The biological screening of IDHPs was performed by the
Psychoactive Drug Screening Program (PDSP) of NIMH. The known IDHP 1a was prepared as
previously described.19 The preparation of the isoxazole-4-carbaldehydes required for the
Hantzsch synthesis of the monomeric IDHPs 1b-d, IDHP alkynes 2, PEG-diazides 3,
Knoevenagel products 7, and 3-Oxo-butyric acid prop-2-ynyl ester 9 were prepared by standard
methods which are detailed in the Supplementary Data.
General Synthesis of 4-[3-(p-Bromo-phenyl)-5-methyl-isoxazol-4-yl]-2,6-dimethyl-1,4-
dihydro-pyridine-3,5-dicarboxylic acid diethyl ester (1d). An oven dried 100ml round
bottom was charged with 0.107g (0.405 mmol) of 3-(p-Bromo-phenyl)-5-methyl-isoxazole-
4-carbaldehyde, 0.11g (0.84 mmol) of ethyl acetoacatate, 50ml of benzene and a magnetic
stir bar. The solution was allowed to stir at room temperature. 0.10ml of 30% aqueous
ammonia was measured out along with 0.90ml of methanol, the mixture was then added to
the round bottom. The round bottom was then fitted with an oven dried condenser and the
solution was brought to reflux (70ºC). The reaction process was monitored via TLC. Once
complete the solution was then cooled to room temperature and the excess solvent was
removed via rotovap. The product was then purified via recrystallization from ethyl acetate
and hexanes to give (1d) as yellow crystals (0.084g, 0.0411 mmol, 42.92%).
1H NMR: (CDCl3) δ 7.55 (d, J = 8.4Hz, 2H), 7.37 (d, J = 8.7Hz, 2H), 5.06 (s, 1H), 5.02 (bs, 1H),
3.52 (s, 6H), 2.43 (s, 3H)
13C NMR: (CDCl3) δ167.27, 166.26, 162.71, 145.31, 143.76, 143.52, 131.24, 130.72, 130.01,
122.88, 119.46, 101.14, 59.85, 51.31, 29.29, 29.16, 19.35, 14.46, 11.46
MS: m/z = 489.15 (M+, 100% rel. intensity), 530.36([M+]+CH3CN, 70)
TLC: SiO2; Hexane:EtOAc 4:1 Rf =0.17
20

Steiger, et al
IDHP dimers @ VGCC, MDR-1 & 7TM
2,6-Dimethyl-4-(5-methyl-3-phenyl-isoxazol-4-yl)-1,4-dihydro-pyridine-3,5-dicarboxylic
acid diethyl ester (1a). The spectral data was in accord with that previously reported.19
1H NMR: (CDCl3) δ7.57 (d 2H J = 7.62Hz), 7.30 (d 2H J = 7.62Hz), 7.23 (s 1H), 4.98 (s 1H),
4.09 (q, J = 6.85Hz, 2H), 4.01 (q,J = 4.89Hz, 2H), 2.49 (s, 3H), 1.91 (s, 6H), 1.18 (t, J = 7.09Hz,
6H)
13C NMR: (CDCl3) δ 171.23, 167.44, 166.32, 165. 81, 163.92, 147.32, 130.25, 128.63, 125.77,
121.32, 101.12, 59.81, 31.65, 20.87, 14.51, 12.34
TLC: SiO2; Hexane:EtOAc 4:1 Rf =0.17
4-[3-(o-Bromo-phenyl)-5-methyl-isoxazol-4-yl]-2,6-dimethyl-1,4-dihydro-pyridine-3,5-
dicarboxylic acid diethyl ester (1b).
1H NMR: (CDCl3) δ 7.60 (d, J = 7.91Hz, 1H), 7.31 (d, J = 7.40Hz, 1H), 7.24 (d, J = 5.90Hz,
1H), 7.18 (d, J = 7.53Hz, 1H), 4.95 (s, 1H), 4.15 (q, J = 4.64Hz, 2H), 4.13 (q, J = 4.64Hz, 2H),
2.58 (s, 3H), 1.96 (s, 6H), 1.25 (t, J = 7.15Hz, 6H)
13C NMR: (CDCl3) δ 167.33, 165.60, 162.95, 144.54, 132.46, 132.25, 130.00, 126.24, 125.01,
118.27, 100.47, 59.79, 29.24, 19.61, 14.61, 11.54
HRMS: calculated for C23H26BrN2O5 489.1023 found 489.1025
MS: m/z = 489 (M+, 100)
TLC: SiO2; Hexane:EtOAc 4:1 Rf =0.07
4-[3-(m-Bromo-phenyl)-5-methyl-isoxazol-4-yl]-2,6-dimethyl-1,4-dihydro-pyridine-3,5-
dicarboxylic acid diethyl ester (1c)
1H NMR: (CDCl3) δ 7.59 (s, 1H), 7.56 (s, 1H), 7.55(s, 1H), 7.33 (s, 1H), 5.05 (s, 1H), 4.16 (q, J
= 3.76Hz, 2H), 4.09 (q, J = 3.76Hz, 2H), 2.54 (s, 3H), 2.06 (s, 6H), 1.25 (t, J = 7.03Hz, 6H)
13C NMR: (CDCl3) δ 167.27, 166.09, 162.48, 144.08, 133.18, 132.50, 129.21, 128.44, 121.64,
119.41, 101.00, 59.87, 29.41, 19.51, 14.51, 11.50
HRMS: calculated for C23H26BrN2O5 489.1028 found 489.1025
MS: m/z = 489 (M+, 100) 530 ([M+2]+CH3CN, 20)
TLC: SiO2; Hexane: EtOAc 4:1 Rf =0.26
General Synthesis of 4-[3-(p-Bromo-phenyl)-5-methyl-isoxazol-4-yl]-2,6-dimethyl-1,4-
dihydro-pyridine-3,5-dicarboxylic acid 3-ethyl ester 5-prop-2-ynyl ester, 2d, Scheme 1. An
oven dried 100ml round bottom was charged with 0.82g (2.17 mmol) of ethyl (2Z)-2-[(5-methyl-
3-p-bromophenyl-1,2-oxazol-4-yl)methylidene]-3-oxobutanoate, 0.305g (2.17 mmol) of prop-2-
yn-1-yl 3-oxobutanoate, 50 ml of absolute ethanol and a magnetic stir bar the vessel was sealed
and the solution was allowed to stirrer at room temperature. In a 10 ml graduated cylinder 0.47ml
of 30% ammonia was measured out along with 0.53ml of absolute ethanol, the mixture was then
added to the 100ml round bound bottom containing the reaction solution. The 100ml round
bottom was then placed on a heating mantel and fitted with a oven dried condenser. The solution
was then refluxed (78ºC) for 12 hours during which time the solution turned yellow. The solution
was then cooled to room temperature and the excess solvent was removed via rotovap. The
solution was then purified via recrystallized from ethyl acetate and hexanes to give off yellow
crystals. (0.355g, 0.713 mmol, 33%)
1H NMR: (CDCl3) δ 7.52 (d, J =7.83Hz, 2H), 7.29 (d, J = 8.31Hz, 2H), 5.01 (s, 1H), 4.98 (s,
2H) 4.56 (q, J = 9.78Hz, 2H), 4.09 (qq, J = 6.36Hz, 2H), 4.00 (qq, J = 6.85Hz, 2H), 2.50 (s,3H),
2.47 (s, 3H), 2.05 (s 6H), 1.21 (t, J = 7.03Hz, 3H)
21

Steiger, et al
IDHP dimers @ VGCC, MDR-1 & 7TM
13C NMR: (CDCl3) δ 167.28, 166.21, 162.70, 145.35, 143.78, 131.23, 130.72, 122.84, 119.47,
101.62, 101.12, 74.68, 59.85, 51.31, 29.69, 29.16, 19.35, 14.46, 11.46
MS: m/z = 499.35 (M+, 100% rel. intensity), 540.86 ([M+]+CH3CN,60)
TLC: SiO2; Hexane:EtOAc 4:1 Rf = 0.12
2,6-Dimethyl-4-(5-methyl-3-phenyl-isoxazol-4-yl)-1,4-dihydro-pyridine-3,5-dicarboxylic
acid 3-ethyl ester 5-prop-2-ynyl ester, 2a.
1H NMR: (CDCl3) δ 7.57 (d, J = 8.72Hz, 2H), 7.30 (d, J = 8.72Hz, 2H), 7.22 (s, 1H), 5.37 (s,
1H), 5.23 (s, 1H), 4.98 (s, 1H), 4.55 (q, J = 6.36Hz, 2H), 4.08 (q, J = 6.85Hz, 2H), 2.54 (s, 3H),
2.08 (s, 1H), 1.92 (s, 6H), 1.18 ( t, J = 7.09Hz, 3H)
13C NMR: (CDCl3) δ 171.18, 167.44, 166.41, 165.79, 163.81, 146.01, 144.39, 130.76, 129.52,
127.55, 119.43, 101.43, 74.59, 60.40, 29.20, 19.15, 14.47, 11.52
TLC: SiO2; Hexane:EtOAc 4:1 Rf = 0.18
4-[3-(o-Bromo-phenyl)-5-methyl-isoxazol-4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylic acid 3-ethyl ester 5-prop-2-ynyl ester, 2b.
1H NMR: (CDCl3) δ 7.58 (d, J = 8.66Hz, 1H), 7.31 (d, J = 6.53Hz, 1H), 7.24 (d, J = 7.65Hz,
1H), 7.18 (d, J = 7.53Hz, 1H), 4.95 (s, 1H), 4.67 (d, J = 3.04Hz, 2H), 4.15 (qq, J = 7.15Hz, 2H),
2.58 (s, 3H), 2.05 (s, 1H), 1.96 (s, 6H), 1.25 (t, J = 7.15Hz, 3H)
13C NMR: (CDCl3) δ 167.33, 165.50, 162.95, 144.54, 132.46, 132.25, 132.11, 130.00, 126.24,
125.01, 118.27, 100.47, 100.47, 59.79, 29.24, 19.61, 14.61, 11.54
TLC: SiO2; Hexane:EtOAc 4:1 Rf = 0.12
4-[3-(m-Bromo-phenyl)-5-methyl-isoxazol-4-yl]-2,6-dimethyl-1,4-dihydro-pyridine-3,5-
dicarboxylic acid 3-ethyl ester 5-prop-2-ynyl ester, 2c
1H NMR: (CDCl3) δ 7.59 (s, 1H), 7.56 (s, 1H), 7.55 (s, 1H), 7.33 (s, 1H), 5.05 (s, 1H), 4.65 (d, J
= 2.51Hz, 2H), 4.16 (qq, J = 7.15Hz, 2H), 2.54(s, 3H), 2.08 (s, 1H), 2.06 (s, 6H), 1.25 (t, J =
7.03Hz, 3H)
13C NMR: (CDCl3) δ 167.27, 166.09, 162.48, 144.08, 133.18, 132.50,131.51, 129.21, 128.44,
121.64, 119.41, 101.00, 59.87, 29.41, 19.51, 14.51, 11.50
TLC: SiO2; Hexane:EtOAc 4:1 Rf = 0.15
General Synthesis of 3-[1-(17-{4-[({4-[3-(p-bromophenyl)-5-methyl-1,2-oxazol-4-yl]-5-
(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridin-3-yl}carbonyloxy)methyl]-1H-1,2,3-
triazol-1-yl}-3,6,9,12,15-pentaoxaheptadecan-1-yl)-1H-1,2,3-triazol-4-yl]methyl 5-ethyl 4-
[3-(p-bromophenyl)-5-methyl-1,2-oxazol-4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate, 4d. An oven dried 100ml round bottom was charged with 0.164g (0.328 mmol)
of 4-[3-(4-Bromo-phenyl)-5-methyl-isoxazol-4-yl]-2,6-dimethyl-1,4-dihydro-pyridine-3,5-
dicarboxylic acid 3-ethyl ester 5-prop-2-ynyl ester, .034g of 1-Azido-2-[2-(2-{2-[2-(2-azido-
ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethane 3c, 0.010g of Copper(I) bromide, 0.010g of
anhydrous Copper sulfate and a magnetic stir bar. Freshly distilled THF (50ml)was then added
and the solution was allowed to stir at room temperature. Sodium ascorbate (0.003g) was then
added and the solution was allowed to stir, and the reaction progress was monitored by TLC,
once the reaction was completed excess solvent was removed via rotovap. The solution was then
purified via a silica column. The purification yielded 4d as a yellow oil. (0.0067g, 0.005 mmol,
15.34%)
22

Steiger, et al
IDHP dimers @ VGCC, MDR-1 & 7TM
1H NMR: (CDCl3) δ 7.65 (s, 2H), 7.49 (d, J = 8.28Hz, 4H), 7.22 (d, J = 8.28Hz, 4H), 4.99 (s,
2H), 4.52 (q, J = 4.52Hz, 4H), 4.13 (q, J = 7.28Hz, 2H), 4.07 (q, J = 6.78Hz, 2H), 3.89 (t, J =
5.52Hz, 3H), 3.75 (t, J = 6.02Hz, 4H), 3.65 (m, 16H), 2.33 (s, 6H), 2.05 (s, 9H), 1.19 (t, J
=7.03Hz, 6H)
13C NMR: (CDCl3) δ 168.74, 166.68, 159.89, 156.71, 135.10, 131.91, 131.22, 130.77, 129.04,
128.17, 127.45, 124.90, 124.24, 109.43, 71.36, 70.59, 69.35, 69.303, 61.72, 58.48, 42.76, 37.76,
31.94, 29.71, 23.16, 14.13, 13.63, 11.25
HRMS: calculated for C60H71Br2N10O15 1329.3467 found 1329.3462
MS: m/z = 1327 (M (79Br) 100% rel. intensity), 1328 (M, 62), 1329 (M+1, 97), 1330
([M+1](81Br),60) 1331 ([M+1]+(81Br),60), 1332 ([M+1]+2(81Br),40)
IR: cm-1 3014.92, 2962.68, 1746.26, 1492.10, 1369.40, 1216.41, 753.73
TLC: SiO2; EtOAc:MeOH:CH2Cl2 (4:1:2) Rf = 0.56
Then run a column in 4:1:3:2 (EtOAc:MeOH:Hexanes:CH2Cl2) Rf = 0.23
3-(1-{17-[4-({[5-(ethoxycarbonyl)-2,6-dimethyl-4-(5-methyl-3-phenyl-1,2-oxazol-4-yl)-1,4-
dihydropyridin-3-yl]carbonyloxy}methyl)-1H-1,2,3-triazol-1-yl]-3,6,9,12,15-
pentaoxaheptadecan-1-yl}-1H-1,2,3-triazol-4-yl)methyl 5-ethyl 2,6-dimethyl-4-(5-methyl-3-
phenyl-1,2-oxazol-4-yl)-1,4-dihydropyridine-3,5-dicarboxylate (4a)
1H NMR: (CDCl3) δ 7.64 (s, 2H), 7.52 (d, J = 8.28Hz, 4H), 7.25 (d, J = 8.28Hz, 4H), 4.98 (s,
2H), 4.54 (q, J = 4.77Hz, 4H), 4.11 (q, J = 7.03Hz, 2H), 4.04 (q, J = 7.03Hz, 2H), 3.87 (t, J =
5.27Hz, 3H), 3.75 (t, J = 5.77Hz, 4H), 3.65 (m, 16H), 2.36 (s, 6H), 2.03 (d, J = 5.27Hz, 9H), 1.19
(t, J = 7.03Hz, 6H)
13C NMR: (CDCl3) δ 171.15, 168.55, 167.21, 169.96, 151.03, 144.05, 130.90, 129.78, 129.44,
129.30, 128.68, 128.40, 127.51, 106.88, 101.17, 88.97, 71.28, 70.60, 70.51, 69.93, 69.37, 68.94,
66.48, 63.51, 61.56, 59.56, 55.57, 51.58, 50.65, 42.73, 37.64, 35.87, 29.57, 29.20, 23.50, 22.58,
21.50, 18.84, 15.10, 14.40, 14.02, 12.41, 11.06
HRMS: calculated for C60H71N10O15 1171.5100 found 1171.5134
MS: m/z = 1169 ([M-2] 100 % rel. intensity), 1170 ([M-1] 70), 1171([M] 40), 1172 ([M+] 10)
IR: cm-1 2972.26, 2831.20, 1734.06, 1571.92, 1357.96, 1225.00, 727.32
TLC: SiO2; EtOAc:MeOH:CH2Cl2 (4:1:2) Rf = 0.52
3-[1-(17-{4-[({4-[3-(o-bromophenyl)-5-methyl-1,2-oxazol-4-yl]-5-(ethoxycarbonyl)-2,6-
dimethyl-1,4-dihydropyridin-3-yl}carbonyloxy)methyl]-1H-1,2,3-triazol-1-yl}-3,6,9,12,15-
pentaoxaheptadecan-1-yl)-1H-1,2,3-triazol-4-yl]methyl 5-ethyl 4-[3-(o-bromophenyl)-5-
methyl-1,2-oxazol-4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate. (4b)
1H NMR: (CDCl3) δ 7.59 (d, J = 6.78Hz, 2H), 7.58 (d, J = 6.78Hz, 2H), 7.31 (d, J = 7.03Hz,
2H), 7.23 (d, 2H J = 7.53Hz), 7.15 (d, J = 7.03Hz, 2H), 4.92 (s, 2H), 4.54 (q, J = 5.52Hz, 4H),
4.10 (q, J = 8.78Hz, 2H), 3.97 (t, J = 5.77Hz, 3H), 3.81 (t, J = 6.27Hz, 4H), 3.66 (m, 16H), 2.36
(s, 6H), 1.96 (s, 9H), 1.23 (t, J = 7.03Hz, 6H)
13C NMR: (CDCl3) δ 167.24, 132.21, 131.98, 130.28, 126.40, ,100.36, 71.28, 70.56, 69.96,
69.47, 68.96, 65.56, 61.91, 59.69, 50.64, 47.62, 42.81, 37.70, 32.16, 30.48, 29.63, 23.41, 19.16,
15.14, 14.56, 11.28
HRMS: calculated for C60H71Br2N10O15 1329.3467 found 1329.3467
MS: m/z = 1327 (M (79Br) 35% rel. intensity), 1328 (M, 30), 1329 (M+1, 97), 1330
([M+1](81Br),50) 1331 ([M+1]+(81Br),100), 1332 ([M+1]+2(81Br),40)
IR: cm-1 2917.91, 2850.74, 1731.34, 1496.26, 1261.19, 1164.17, 733.12
23

Steiger, et al
IDHP dimers @ VGCC, MDR-1 & 7TM
TLC: SiO2; EtOAc:MeOH:CH2Cl2 (4:1:2) Rf = 0.48
Isoxaquidar. 3-[1-(17-{4-[({4-[3-(m-bromophenyl)-5-methyl-1,2-oxazol-4-yl]-5-
(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridin-3-yl}carbonyloxy)methyl]-1H-1,2,3-
triazol-1-yl}-3,6,9,12,15-pentaoxaheptadecan-1-yl)-1H-1,2,3-triazol-4-yl]methyl 5-ethyl 4-
[3-(m-bromophenyl)-5-methyl-1,2-oxazol-4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate. (4c).
1H NMR: (CDCl3) δ 7.73 (s, 2H), 7.64 (d, J = 5.52Hz, 2H), 7.51 (s, 2H ), 7.25 (s, 2H), 7.18 (d, J
= 10.54Hz, 2H), 4.98 (s, 2H), 4.53 (q, J = 4.02Hz, 4H), 4.38 (q, J = 4.27Hz, 2H), 4.09 (q, J =
10.79Hz, 2H), 3.86 ( t, J = 5.02Hz, 3H), 3.76 (t, J = 5.02Hz, 4H), 3.62 (m, 16H), 2.35 (s, 6H),
2.01 (s, 9H), 1.19 (t, J = 7.28Hz, 6H)
13C NMR: (CDCl3) δ 167.18, 166.31, 162.49, 145.12, 144.12, 133.04, 132.73, 132.40, 131.54,
130.44, 13.026, 129.28, 128.39, 126.18, 124.96, 121.62, 199.30, 101.11, 100.33, 71.35, 70.62,
69.39, 69.01, 66.64, 59.85, 58.44, 56.92, 50.69, 43.38, 42.79, 41.44, 37.75, 29.41, 23.17, 23.09,
19.46, 19.29, 14.48, 13.62, 11.30
HRMS: calculated for C60H71Br2N10O15 1329.3467 found 1329.3450
MS: m/z = 1327 (M (79Br) 35% rel. intensity), 1328 (M, 20), 1329 (M+1, 100), 1330
([M+1](81Br),60) 1331 ([M+1]+(81Br),100), 1332 ([M+1]+2(81Br),50)
IR: cm-1 3007.46, 2970.14, 1738.80, 1432.83, 1227.61, 1216.41, 761.51
TLC: SiO2; EtOAc:MeOH:CH2Cl2 (4:1:2) Rf = 0.52
3-[1-(2-{2-[2-(2-{4-[({4-[3-(p-bromophenyl)-5-methyl-1,2-oxazol-4-yl]-5-(ethoxycarbonyl)-
2,6-dimethyl-1,4-dihydropyridin-3-yl}carbonyloxy)methyl]-1H-1,2,3-triazol-1-
yl}ethoxy)ethoxy]ethoxy}ethyl)-1H-1,2,3-triazol-4-yl]methyl 5-ethyl 4-[3-(p-bromophenyl)-
5-methyl-1,2-oxazol-4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (4e)
1H NMR: (CDCl3) δ 7.60 (d, J = 5.52, 2H), 7.50 (t, J = 8.28Hz, 4H), 7.26 (t, J = 8.28Hz, 4H),
5.17 (m, 4H), 4.99 (s, 2H), 4.51 (q, J = 5.52Hz, 4H), 4.07 (q, J = 6.78Hz, 2H), 3.94 (q, J =
6.78Hz, 2H), 3.85 (t, J = 5.27Hz, 4H), 3.62 (m, 8H), 2.34 (s, 6H), 2.01 (d, J = 5.02Hz, 9H), 1.15
(t, J = 7.28Hz, 6H)
13C NMR: (CDCl3) δ 167.21, 162.62, 145.12, 143.89, 131.90, 131.12, 130.78, 129.92, 129.02,
122.88, 119.54, 101.29, 100.39, 72.46, 70.67, 70.06, 69.34, 61.72, 59.81, 50.67, 42.84, 29.69,
19.03, 14.45, 11.30
HRMS: calculated for C56H63 N10O13Br2 1241.2943 found 1241.2950
MS: m/z = 1239 ([M(79Br)] 60% rel. intensity), 1240 ([M] 40), 1241 ([M+1] 100), 1242
([M+1](81Br) 60), 1243([M+2] 70), 1244 ([M+3] 50)
IR: cm-1 2917.91, 2850.74, 1731.31, 1496.26, 1261.19, 1164.17, 697.76
TLC: SiO2; EtOAc:MeOH:CH2Cl2 (4:1:2) Rf = 0.52
3-(1-{2-[2-(2-{4-[({4-[3-(4-bromophenyl)-5-methyl-1,2-oxazol-4-yl]-5-(ethoxycarbonyl)-2,6-
dimethyl-1,4-dihydropyridin-3-yl}carbonyloxy)methyl]-1H-1,2,3-triazol-1-
yl}ethoxy)ethoxy]ethyl}-1H-1,2,3-triazol-4-yl)methyl 5-ethyl 4-[3-(4-bromophenyl)-5-
methyl-1,2-oxazol-4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (4.f)
1H NMR: (CDCl3) δ 7.61 (d, J = 7.53Hz 2H), 7.49 (d, J = 8.28Hz 4H), 7.25 (d 4H J = 8.53Hz),
5.17 (m 4H), 4.98 (s 2H), 4.51 (q 4H J = 5.27Hz), 4.06 (q 2H J = 6.83Hz), 3.88 (q 2H J =
5.02Hz), 3.72 (t 4H J = 6.02Hz), 3.64 (d 4H J = 6.27Hz), 2.33 (s 6H), 2.01 (s 9H), 1.15 (t 6H J =
7.28Hz)
24

Steiger, et al
IDHP dimers @ VGCC, MDR-1 & 7TM
13C NMR: (CDCl3) δ 167.25, 143.96, 131.97, 131.18, 130.81, 129.89, 129.03, 122.98, 101.28,
71.30, 70.74, 70.47, 70.32, 69.15, 61.75, 59.83, 53.51, 50.67, 42.92, 29.70, 29.36, 19.04, 15.16,
14.46, 13.64, 11.36
HRMS: calculated for C54H59 N10O12Br2 1197.2633 found 1197.2681
MS: m/z = 1195 ([M(79Br)] 80% rel. intensity), 1196 ([M] 60), 1197 ([M+1] 100), 1198
([M+1](81Br) 80), 1199 ([M+2] 100), 1200 ([M+3] 60)
IR: cm-1 3014.92, 2962.68, 1746.26, 1429.10, 1369.40, 1216.41, 753.73
TLC: SiO2; EtOAc:MeOH:CH2Cl2 (4:1:2) Rf = 0.5
General Synthesis of 4-[3-(p-Bromo-phenyl)-5-methyl-isoxazol-4-yl]-2,6-dimethyl-1,4-
dihydro-pyridine 3,5-dicarboxylic acid 3-ethyl ester 5-(1-phenethyl-1H-[1,2,3]triazol-4-
ylmethyl) ester, 5. An oven dried 100mL round bottom was charged with 0.038g (0.259mmol)
of (2-Azido-ethyl)-benzene and a magnetic stir bar. The sample was then taken up in freshly
distilled THF and the solution was allowed to stir at room temperature. Once the (2-Azido-
ethyl)-benzene was completely dissolved 0.129g (0.259mmol) of 4-[3-(p-Bromo-phenyl)-5-
methyl-isoxazol-4-yl]-2,6-dimethyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid 3-ethyl ester 5-
prop-2-ynyl ester 2d was added in portions. The round bottom was then capped with a rubber
septum and the solution was allowed to stir. The solution was allowed to stir until all reagents
where dissolved, 0.011g of Copper(I)-bromide was then added and the solution was allowed to
stir at room temperature for 48 Hr. Reaction progress was monitored via TLC, once the compete
excess solvent was removed via rotovap. The solution was then purified via silica column
yielding 5 as a yellow oil (0.0527g, 0.0816mmol, 32%). TLC: SiO2; EtOAc:MeOH:CH2Cl2
(4:1:2) Rf =0.55.
1H NMR: (CDCl3) δ 7.51(d, J = 8.53Hz, 2H), 7.30 (d, J = 7.53Hz, 2H), 7.26(s, 1H), 7.24(d, J =
9.29Hz, 2H), 7.12(d, J = 8.28Hz, 2H), 5.15(q, J = 12.30Hz, 2H), 5.03(s, 1H), 4.99(s, 1H), 4.56 (t,
J = 7.53Hz, 2H), 4.14 (q, J = 7.03Hz, 2H), 4.07 (t, J = 7.03Hz, 2H), 3.20(t, J = 7.28Hz, 2H),
2.35(s, 3H), 2.02(d, J = 5.27Hz, 6H), 1.27( t, J = 7.28Hz, 3H)
13C NMR: (CDCl3) δ 167.14, 167.04, 166.55, 144.59, 143.54, 136.89, 131.20, 130.79, 129.93,
128.85, 128.69, 127.13, 122.89, 101.52, 100.64, 60.43, 59.89, 56.88, 51.68, 45.91, 36.63, 26.39,
21.08, 19.46, 19.26, 14.45, 19.26, 14.45, 14.22, 11.33, 8.67
HRMS: calculated for C23H32BrN5O5 646.1685 found 646.1665
MS: m/z = 645 (M (79Br), 20% rel. intensity), 646 (M+, 80), 647([M+1]+, 35), 648
([M+1]+(81Br),78)
IR: cm-1 2992.36, 1734.06, 1365.56, 1255.39, 1278.18, 1221.20, 761.51, 746.32
Synthesis of 2,6-Dimethyl-4-(5-methyl-3-phenyl-isoxazol-4-yl)-pyridine-3,5-dicarboxylic
acid diethyl ester, 6. The 2,6-Dimethyl-4-(5-methyl-3-phenyl-isoxazol-4-yl)-1,4-dihydro-
pyridine-3,5-dicarboxylic acid diethyl ester (1a) was placed in a 50mL oven dried round bottom
along with a magnetic stir bar and 0.085g of deionized water. The solution was then allowed to
stir at room temperature while, 0.227g of HNO3 and 0.0246g of H2SO4 was added. The solution
was then heated to 110ºC and white foam was observed, after 10 min. at 110ºC the solution
turned a light yellow. The solution was then allowed to cool to room temperature. The solution
was then taken up in ice water and LiOH was added slowly until the solution was neutral. The
solution was then added to a separatory funnel and extracted with CHCl3 and Brine (x 3). The
solution was then dried over Na2SO4 and filtered; the excess solvent was then removed via
rotovap yielding 6 as a pale yellow oil that solidified on standing, and was then crystallized with
25

Steiger, et al
IDHP dimers @ VGCC, MDR-1 & 7TM
CH2Cl2 and Hexanes. 6, (0.100g, 0.245 mmol, 98%). TLC: SiO2; Hexanes:EtOAc (4:1) Rf =
0.31. 1H NMR: (CDCl3) 7.46 (d, J = 7.28Hz, 2H), 7.34 (d, J = 7.11Hz, 2H), 7.29 (s, 1H), 4.03
(q, J = 9.56Hz, 2H), 2.59 (s, 3H), 2.25 (s, 3H), 0.97 (t, J = 7.01Hz, 3H).
13C NMR: (CDCl3) δ 167.18, 165.91, 159.70, 155.29, 134.52, 128.66, 127.56, 126.56, 109.18,
60.63, 22.09, 12.57, 10.38
HRMS: calculated for C23H24N2O5 408.1685 found 408.1692
MS: m/z = 409 (M+1, 100), 410 (M+2, 40)
IR: cm-1 3007.46, 1737.86, 1441.54, 1365.56, 1259.19, 1217.60, 746.32
General synthesis of 2-Acetyl-3-[3-(p-bromo-phenyl)-5-methyl-isoxazol-4-yl]-acrylic acid
ethyl ester, 7d. An oven dried 50mL round bottom was charged with 0.95g of 5-methyl-3-p-
bromophenyl-1,2-oxazole-4-carbaldehyde, 0.799g of Ethyl Acetoacetate, 4 mL of Benzene
where added at room temperature. The solution was allowed to stir, and 0.1325g of glacial acidic
acid and 0.054g of piperidine was added. The round bottom was then fitted with a dean stark trap
and a condenser. The solution was then allowed to reflux for three hours; with the reaction
progress monitored my TLC. The solution was then cooled to room temperature and the excess
solvent was removed via rotovap. The solution was then purified via a silica column. The
purification yielded yellow oil. (0.761g, 2.53 mmol, 52%)
1H NMR: (CDCl3) δ 7.54 (d, J = 9.78Hz, 2H), 7.40 (d, J = 8.56Hz, 2H), 7.33 (s, 1H), 4.13 (q, J =
7.09Hz, 2H), 2.35 (s, 3H), 2.19 (s, 3H), 1.20 (t, J = 7.09Hz, 3H)
13C NMR: (CDCl3) δ 194.12, 169.17, 165.80, 160.44, 137.70, 132.11, 129.50, 131.45, 127.45,
124.46,109.55, 61.63, 13.75, 12.19
MS: m/z =377.03 (M+, 50% rel. intensity) 418.45 ([M+]+CH3CN, 100)
TLC: SiO2; Hexane:EtOAc 1:1 Rf = 0.39
2-Acetyl-3-(5-methyl-3-phenyl-isoxazol-4-yl)-acrylic acid ethyl ester, 7a.
1H NMR: (CDCl3) δ 7.57 (d, J = 12.72Hz, 2H), 7.44 (d, J = 12.72Hz, 2H), 7.25 (s, 1H), 3.99 (q,
J = 7.52Hz, 2H), 2.40 (s, 3H), 2.37 (s, 3H), 1.14 (t, J = 6.05Hz, 3H)
13C NMR: (CDCl3) δ 194.36, 168.96, 166.03, 161.50, 137.26, 132.67, 128.93, 128.05, 109.77,
61.60, 27.82, 13.79, 12.31
TLC: SiO2; Hexane:EtOAc 1:1 Rf = 0.39
2-Acetyl-3-[3-(o-bromo-phenyl)-5-methyl-isoxazol-4-yl]-acrylic acid ethyl ester, 7b.
1H NMR: (CDCl3) δ 7.65 (s, 1H), 7.54 (s, 1H), 7.36 (s, 1H) 7.24 (s, 1H), 7.10 (s, 1H), 4.17 (q, J
= 6.57Hz, 2H), 2.37 (s, 3H), 2.15 (s, 3H), 1.17 ( t, J = 6.98Hz, 3H)
13C NMR: (CDCl3) δ 194.36, 168.96, 166.03, 161.50, 138.26, 132.30, 130.71, 128.93, 128.05,
109.77, 61.60, 27.82, 13.79, 12.13
TLC: SiO2; Hexane:EtOAc 1:1 Rf = 0.41
2-Acetyl-3-[3-(3-bromo-phenyl)-5-methyl-isoxazol-4-yl]-acrylic acid ethyl ester, 7c.
1H NMR: (CDCl3) δ 7.65 (s, 1H), 7.43 (s, 1H), 7.35 (s, 1H), 7.30 (s, 1H), 7.68 (s, 1H), 4.15 (q, J
= 6.84Hz, 2H), 2.34 (s, 3H), 2.17 (s, 3H), 1.16 (t, J = 5.42Hz, 3H)
13C NMR: (CDCl3) δ 194.24, 169.12, 165.83, 162.78, 148.65, 137.68, 132.11, 130.41, 131.96,
124.93, 127.45, 110.15, 59.67, 24.65, 14.04, 12.18
TLC: SiO2; Hexane:EtOAc 1:1 Rf = 0.30
26

Steiger, et al
IDHP dimers @ VGCC, MDR-1 & 7TM
Synthesis of 3-Oxo-butyric acid prop-2-ynyl ester, 9. An oven dried round bottom (250mL)
was charged with a magnetic stir bar, 4.64g of Ethyl Acetoacetate, 2.00g of propargyl alcohol,
0.22g of boric acid, and 40mL of toluene added at room temperature. The round bottom was then
fitted with a Dean Stark trap, a condenser and a drying tube. The solution was then heated to
reflux and left for 144 hours. The reaction yielded a light red solution. The solution was then
purified via a silica column. The purification compound yielded yellow oil. (2.868 g, 20.46
mmol, 57%)
1H NMR: (CDCl3) δ 4.68 (d, J = 2.45Hz, 2H), 3.46 (s 2H), 2.49 (t, J = 2.45Hz, 1H), 2.22 (s, 3H),
13C NMR: (CDCl3) δ 200.03, 166.27, 104.98, 89.06, 52.60, 49.56, 30.08
MS: m/z = 140.047 (M+, 100% rel. intensity) 142.07(M+2, 90), 183.78([M+2]+CH3CN, 60)
TLC: SiO2; Hexane:EtOAc 4:1 Rf = 0.18
Acknowledgement
The authors thank NIH for grants NS038444 (NN), P20RR015583 (NN, SS), and CBSD
CoBRE grant P20GM103546 (NN). These studies were also funded by the ALSAM foundation
Skaggs Scholars Program Grant (DSB, NN). Computational modeling studies were
conducted at the University of Colorado Computational Chemistry and Biology Core
Facility, which is funded in part by NIH/NCATS UL1 TR001082. We thank Dr. Mike Braden
of the Core Facility for Molecular Computation (UM) for helpful discussions during
computer modeling.
MDR1 data was generously provided by the National Institute of Mental Health's Psychoactive
Drug Screening Program (NIMH PDSP), Contract # HHSN-271-2008-00025-C (NIMH PDSP).
The NIMH PDSP is Directed by Bryan L. Roth MD, PhD at the University of North Carolina at
Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. We also thank Dr.
X.P. Huang of PDSP for helpful suggestions with the manuscript.
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