Fluorescent probes of the isoxazole-dihydropyridine scaffold: MDR-1 binding and homology model Dedicated to the memory of Professor Robert O. Hutchins, peerless mentor and good friend

Article abstract of DOI:10.1016/j.bmcl.2013.11.068

Isoxazole-1,4-dihydropyridines (IDHPs) were tethered to fluorescent moieties using double activation via a lanthanide assisted Weinreb amidation. IDHP-fluorophore conjugate 3c exhibits the highest binding to date for IDHPs at the multidrug-resistance transporter (MDR-1), and IDHP-fluorophore conjugates 3c and 7 distribute selectively in SH-SY5Y cells. A homology model for IDHP binding at MDR-1 is presented which represents our current working hypothesis.

Full text of DOI:10.1016/j.bmcl.2013.11.068

Bioorganic & Medicinal Chemistry Letters 24 (2014) 117–121
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorescent probes of the isoxazole–dihydropyridine scaffold: MDR-1
binding and homology model
Monika I. Szabon-Watola b, Sarah V. Ulatowski a, Kathleen M. George a, Christina D. Hayes a,
Scott A. Steiger a, Nicholas R. Natale a,b,
⇑
a NIH COBRE Center for Structural and Functional Neuroscience, The University of Montana, Missoula, MT 59812, United States
b Department of Chemistry, University of Idaho, Moscow, ID 83843, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Isoxazole-1,4-dihydropyridines (IDHPs) were tethered to fluorescent moieties using double activation via
a lanthanide assisted Weinreb amidation. IDHP–fluorophore conjugate 3c exhibits the highest binding to
date for IDHPs at the multidrug-resistance transporter (MDR-1), and IDHP–fluorophore conjugates 3c and
7 distribute selectively in SH-SY5Y cells. A homology model for IDHP binding at MDR-1 is presented
which represents our current working hypothesis.
Received 18 October 2013
Revised 21 November 2013
Accepted 25 November 2013
Available online 4 December 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Dedicated to the memory of Professor
Robert O. Hutchins, peerless mentor and
good friend
Keywords:
Multidrug resistance transporter
Isoxazole
Dihydropyridine
Homology model
For three decades 4-aryl-1,4-dihydropyridines (DHPs) have
been used as antihypertensive agents which function as antago-
nists of the L-type voltage gated calcium channel (VGCC).1 Fluores-
cent probes of DHPs have been applied to the visualization of the
distribution of L-type calcium channels.2
The DHP scaffold has also been recognized as a privileged struc-
ture and used as for identifying lead hits for several molecular
targets.3,4 In this regard the well established structure activity rela-
tionship (SAR) of DHP antagonists of the VGCC5 can be potentially
utilized to design against antihypertensive activity, and hence,
selectivity for non-calcium channel targets.6
Of interest to us are the recent observations by the groups of An-
drus, and Chmielewski and Hrycyna that bivalent molecules can
have significant activity as inhibitors, and that consideration of
the tether connection is critical.13
Our continuing interest in the bioisosteric 4-isoxazolyl-
DHPs5b,14 (IDHPs) led us to examine a common pharmacophore
for DHP MDR-1 inhibitors (Fig. 1), in light of the similarity of the
The observation that DHPs nicardipine7 and niguldipine8 exhi-
bit activity as inhibitors of the multidrug-resistance transporter
(MDR-1, also known as p-glycoprotein or P-gp) efflux pump,9 has
stimulated renewed interest in DHPs.10 Pharmacophore modelling
for MDR-1 indicates several common features for inhibitors,11
notably at least two aryl pockets, and a tether of approximately
3–5 methylenes, leading to another functional group which
possesses H-bond donor and lipophilic groups.12 In contrast,
substrates for MDR-1 usually contain a group located near the lat-
ter distal end of the tether which is protonated at physiological pH.
Figure 1. SYBYL common pharmacophore model of nicardipine (cyan) niguldipine
(pink), and 3-[4,N-dansyl-10,40-butanediamino-]carboxamido-4-Isoxazolyl-DHP 3a
(green).
⇑
Corresponding author.
E-mail address: nicholas.natale@umontana.edu (N.R. Natale).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.068

118
M. I. Szabon-Watola et al. / Bioorg. Med. Chem. Lett. 24 (2014) 117–121
X-ray structure of Isoxazolyl-DHP 115 with the general MDR-1
inhibitor model proposed by Ecker,12 the most striking similarity
being the anchor-like conformation of the DHP relative to the
C(3) aryl moiety. Recently, we found that IDHPs exemplified by
structure 1 are in fact robust ligands of MDR-1,24 in studies
conducted in collaboration with the Psychoactive Drug Screening
Program (PDSP), and exhibit an SAR distinct from their activity at
the VGCC. In the present study we wished to test the hypothesis
as to whether bivalent IDHPs would have enhanced activity at
MDR-1.
Double activation preparation of bivalent fluorescent-labeled
isoxazolyl-DHPs (IDHPs): The isoxazolyl-DHPs (1) can be prepared
easily on a multi-gram scale using methods previously reported
by our lab,5b,14–16 however, the direct reaction of the esters is slug-
gish given their delocalized vinylogous urethane nature. We have
found that modest yields can be obtained for 2 using our double
activation method,17 which uses a lanthanide catalyst in the pres-
ence of a Weinreb activated amine.18 The resulting tethered
amines can be transformed to fluorophore-labeled analogs quite
effectively. Thus, a series of probes with differing tether lengths
(3a–d), and diverse fluorophores (3–7) were readily available.
The method, of course, is not limited to isoxazolyl-dihydropyri-
dines, and the o-, m-, and p-bromophenyl DHPs were also
converted to dansyl analogs (Supplementary data).19
derived acid chloride proceeded to the amide 5 quite effectively.
Fluorescein isothiocyanate (FITC) gave thiourea adduct 6. Finally
sulforhodamine B, similar to the danzyls, proceeded virtually
quantitatively to the adduct 7 (Scheme 1).
From a microscopist’s perspective, an important consideration
in synthesis of a library of fluorophore-conjugated compounds is
the extinction coefficient of the fluorophore groups. The extinction
coefficient defines how strongly a substance absorbs light at a par-
ticular wavelength. Hence, a fluorophore with a high extinction
coefficient will enable a microscopist to image a fluorescently la-
beled compound at a lower, more physiologically relevant concen-
tration than a compound labeled with a fluorophore with a low
extinction coefficient. Extinction coefficients of the fluorophores
listed in Table 1, span a range from 4400 for dansyl through
85,000 for fluorescein and approximately 120,000 for the
sulforhodamines.
Conformational dynamics of the fluorescent probes: The confor-
mational dynamics of IDHPs dramatically effect the topology pre-
sented to a biomolecular target.5b,14–16,24 The danzyl probes,
most notably 3c, in addition to the expected set of signals for the
isoxazolyl-DHP, also exhibited a second set of signals with pro-
nounced magnetic anisotropy in typical organic solvents (i.e.,
CDCl3), and of equal intensity. Nuclear Overhauser spectroscopy
evidenced interactions between the DHP C(2) and C(6) methyls
with the Isoxazolyl C(3) phenyl, and the isoxazolyl C(3) phenyl
with the dimethyl amino groups of the dansyl moiety, indicative
of a folded conformation. Raising the temperature resulted in a
conformational reorganization to a single set of chemical shifts
which were consistent with an extended conformation.19 The
interconversion was not reversible in wet DMSO. Computation
indicated that the folded conformation was lower in energy, in
the gas phase, consistent with its predominant presence in less po-
lar solvent. The VT and computation are shown in the Supplemen-
tar data.
The fluorophores 4–7 were selected to study a range of reactiv-
ities in the presence of the IDHP moiety. The 9-anthryl adduct 4
was prepared by reductive amination with sodium cyanoborohy-
dride from amine 2 and anthracene-9-carboxaldehyde.25 Pyrene-
2-carboxylic acid did not give synthetically useful yields with
dicyclohexy carbodiimide (DCC), likely owing to steric hindrance,
however the corresponding Schotten–Bauman reaction with the
N
O
N
O
Multidrug-resistance inhibition (MDR-1): MDR-1 inhibition was
evaluated in the Psychoactive Drug Screening Program (PDSP) as-
say, the details of which are given in the Supplementarty data,
and described on the PDSP web site http://pdsp.med.unc.edu/
pdspw/MDR.php. Compound 3c exhibited 63% inhibition, versus
0% for untreated cells and 100% for the MDR inhibitor cyclo-
sporin.21,22 This represents a significant increase in MDR-1 binding
compared to IDHP-1, which exhibited a value of 48.9% in the PDSP
assay.23,24
H3C
OC
H
H3C
EtO2C
CH3
SmCl3
N
H2N
CO2Et
CH3
CO2Et
CH3
(CH2)n
H2N-(CH2)n-NH2
Al(CH3)3
CH3
N
N
H
H
2 a, n = 4
b, n = 6
c, n = 8
1
d, n = 12
SO2
N
O
R =
Homology modeling: In the absence of a homo sapien X-ray crys-
tal structure of MDR-1 a homology mode was constructed using
Mus musculus MDR-1, PDB:3G5U,26 as a template for threading of
the human gene sequence (P08183).27–29 The sequence alignment
was generated using Clustal W,30 the alignment of and the subse-
quent threading indicated high sequence homology to the human
sequence with a sequence identify of 82.34%. To visualize the
hypothetical interactions between 3c and MDR-1 molecular mod-
eling was conducted. Ligand structures were drawn and energy
minimized (Powell method, 0.01 kcal molꢀ1 Åꢀ1 gradient termina-
tion, MMFF94s force field, MMFF94 charges, 1000 maximum itera-
tions) using the SYBYL software package (Tripos, St. Louis, MO).
Virtual dockings of energy-minimized ligand to the MDR-1 homol-
ogy model were performed using the GOLD software package
(Cambridge Crystallographic Data Center, Cambridge, UK)31 and
scored using Chem PLP with default settings. Docking algorithms
H3C
OC
H
N
HN
CO2Et
CH3
R
(CH3)2N
(CH2)n
CH3
N
3 a, n = 4
b, n = 6
c, n = 8
H
3 - 7
d, n = 12
O
R =
R =
5, n = 6
4, n = 6
N
HO
-
SO3
H
N
O
S
O
were performed with the constraint of limiting the allowed
0
O
O+
N
R =
R =
C
binding area to a 6 ÅA radius around the ARG905 residue. Ligand–
receptor ensemble structures were obtained by merging the
highest-ranked output ligand orientation structures with the input
MDR-1 homology model structure using PyMOL. An MDR-1 human
homology model protein active site analysis was performed using
Q-site finder.33 This program binds hydrophobic probes to the
S
CO2H
O
7, n = 6
6, n = 6
Scheme 1. Synthesis of isoxazole-DHP flourophone conjugates 3–7.

M. I. Szabon-Watola et al. / Bioorg. Med. Chem. Lett. 24 (2014) 117–121
119
Table 1
Bivalent fluorescent IDHPs prepared for the current study 3–7
Compds
Fluorophore
Tether length
Empirical formula
Mass spectra (m/Z)a,b
3a
3b
3c
3d
4
5
6
7
Dansyl
Dansyl
Dansyl
Dansyl
9-Anthryl
2-Pyryl-carboxy
Fluorescein
SulforhodamineB
4
6
8
12
6
6
C37H43O6N5S
C39H47O6N5S
C41H51O6N5S
C45H59O6N5S
C42H46N4O4
C44H44N4O5
C48H47N5O9S
685[M]+
713[M]+
741[M]+
797[M]+
669[M-1]+
708[M]+
869[M+1]+
1021[M]+
6
6
C54H64N6O10S
a
3a to 3d were obtained by FAB MS on a JEOL-JMS-AX505HA.
Compounds 4–7 were obtained by HR-ESMS, Micromass MS/Waters HPLC.
b
MDR-1 homology model and ranks sites with the most favorable
binding energy. These clusters are then placed in rank order of
the likelihood of being a binding site according to the sum total
binding energies for each cluster. The first binding cluster was
bound in the rhodamine binding site or R-site, the second and third
sites were located proximal to the nucleotide binding domains
(NBDs). The overlap of the dexniguldipine binding site in MDR-1
via photoaffinity labeling (shown in red in Fig. 2),32 combined with
the output of the Q-site finder gave a consensus binding site for
IDHP.33
Interestingly, the predicted binding location of the IDHP resides
near the conserved V907 of Coupling Helix 2, and an essential Mg2+
binding residue Q475 of the Walker domain required for ATP
hydrolysis. A ligand binding in this location would be expected
to limit conformational reorganization necessary for xenobiotic ef-
flux. This presents a testable working hypothesis for future studies,
and target design (see Fig. 3).
Figure 3. Close up of IDHP 3c docked into MDR-1. The fluorophore interacts at the
edge of the red dexniguldipine photoaffinity site, the DHP moiety of 3c (gold) is
mainly located near the NBD, as predicted by Q-site.
Fluorescent microscopy: SH-SY5Y human neuroblastoma cells
(American Type Cell Culture Cat no. CRL-2266) lines were cultured
in the recommended media including 10% fetal bovine serum and
antibiotics. Cells were plated on coverslips in 12-well plates at
1 ꢁ 105/well and allowed to adhere for 48 h. Cells were incubated
with DHP–sulfonorhodamine diluted in dimethyl sulfoxide
(DMSO) for a prescribed period of time (30 min to 21 h) at 37 °C/
5% CO2. After incubation, media was removed, cells were washed
twice with phosphate-buffered saline (PBS), and fixed in 4%
R
PHE 163
R
HN
R
O
O
O
PHE 904
GLN 475
R
NH
SER 474
R
O
R
VAL 478
R
ARG 543
R
O
N
HO
R
N
N
H
HN
R
O
O
N
H
H
H
N
N
H2N
H
O
O
ARG 905
O
O
ALA 544
S
O
N
H
N
H
VAL 908
N
O
O
H
N
H2N
GLN 438
R
HN
R
TYR 490
PHE 480
Figure 2. Homology model of IDHP 3c (gold) docked into MDR-1 based on pdb
Scheme 2. Summary of close contacts between 3c and MDR-1 from docking the
accession number 3G5U. The red area is the dexniguldipine photoaffinity site.
homology model.

120
M. I. Szabon-Watola et al. / Bioorg. Med. Chem. Lett. 24 (2014) 117–121
Figure 4. Confocal fluorescence Microscopy of SH-SY5Y cells with: (left), 10
(orange), imaged at 60ꢁ, with a 90 min incubation time. Scale bar indicates a distance of 10
l
M dansyl isoxazolyl-DHP 3c (green); and (right) 1
m.
lM sulforhodamine B-isoxazole DHP 7
l
paraformaldehyde in PBS (20 min, RT). Fluorescent images were ta-
ken with a Nikon Eclipse E800 fluorescent microscope and images
were processed with Nuance 1.6.2.368 alpha software (Cambridge
Research Institute) (see Scheme 2).
C.D.H. thanks the NSF REU Award Number 0649306. M.I.S. thanks
the Malcolm and Carol Renfrew Scholarship. We thank Dr. Alex
Blumenfeld for VT NMR and Dr. Gary Knerr for able assistance in
FAB MS while at the University of Idaho. We thank Dr. Mike Braden
and Dave Holley of the Core Facility for Molecular Computation
(UM) for helpful discussions during pharmacophore 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, Ph.D. at the University
of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at
NIMH, Bethesda MD, USA.
Representative results are shown in Figure 4. Dansyl isoxazole
3c and Sulforhodamine isoxazole 7 possess fluorophores with
radically different physical characteristics, yet they localize in
SH-SY5Y cells in virtually identical locations, whereas controls
for the fluorophores not conjugated to the IDHP showed non-selec-
tive binding throughout the cell. This suggests that it is the IDHP
moiety which is the driving force for the localization. The
characteristics of the fluorophores were effected by the tether.
For example, the sulforhodamine control alone had higher inten-
sity (albeit no selectivity in cellular distribution) compared to the
conjugate 7, with a tether of six methylene units. In contrast the
fluorophore conjugate 3c, with eight methylenes had a higher
intensity than the corresponding control (dansylcadaverine),
which is consistent with the former being inside of the Forster ra-
dius and exhibits quenching relative to fluorophore alone, whereas
the latter with the longer tether is beyond this radius.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
11.068.
References and notes
4-Aryl-DHPs tethered via an ester linkage to the fluorophore
BODIPY localized in the L-type calcium channel in a functional,
extracellular heparin-binding site.2 It has previously been re-
ported that expression of MDR-1 is reported to be localized at
several subcellular sites, including the nucleus, mitochondria,
and Golgi apparatus of rat brain cells and suggests a role in mul-
tidrug resistance at subcellular sites.20 The current study suggests
an analogous distribution in SH-SY5Y cells, and will be the
subject of future studies.
1. Zamponi, G. Voltage-Gated Calcium Channels; Landes Bioscience: New York,
2005.
2. Knaus, H. G.; Moshammer, T.; Friedrich, K.; Kang, H. C.; Haugland, R. P.;
Glossman, H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3586.
3. Kubinyi, H.; Mueller, G. Chemogenomics in Drug Discovery; Wiley-VCH: New
York, 2004. pp 18–19.
4. Natale, N. R. Learning from the Hantzsch Synthesis; Chemical Innovation
(American Chemical Society), 2000. pp 22–28.
5. (a) Triggle, D. J.; Langs, D. A.; Janis, R. A. Med. Res. Rev. 1989, 9, 123; (b)
Zamponi, G.; Stotz, S. C.; Staples, R. J.; Rogers, T. A.; Nelson, J. K.; Hulubei, V.;
Blumenfeld, A.; Natale, N. R. J. Med. Chem. 2003, 46, 87.
Summary: Isoxazolyl-DHPs can be readily conjugated to fluoro-
phores, once tethering moieties are connected using a further
application of our double activation methodology. A wide variety
of fluorophores can be conjugated to the IDHP using this approach.
The localization of the conjugates in SH-SY5Y cells appears to be
driven by the IDHP moiety. The distribution of the present conju-
gates are not consistent with previous reports of L-type calcium
channels,2 and appear to concentrated in the nuclear envelope
and the Golgi.20 We have developed a homology model of MDR-
1, which combined with our experimental observations, represents
our current working hypothesis. Current studies are focused on
whether the IDHPs may represent useful tools for the study of
MDR-1, and ultimately lead to clinically useful inhibitors. Our
progress will be reported in due course.
6. (a) Nagarathnam, D.; Wetzel, J. M.; Miao, S. W.; Marzabadi, M. R.; Chiu, G.;
Wong, W. C.; Hong, X.; Fang, J.; Forray, C.; Branchek, T. A.; Heydorn, W. E.;
Chang, R. S.; Broten, T.; Schorn, T. W.; Gluchowski, C. J. Med. Chem. 1998, 41,
5320; (b) Miri, R.; Mehdipour, A. Bioorg. Med. Chem. 2008, 16, 8329.
7. Nobili, S.; Landini, I.; Giglioni, B.; Mini, E. Curr. Drug Targets 2006, 7, 861.
8. (a) Zhou, X. F.; Shao, O.; Coburn, R. A.; Morris, M. E. Pharm. Res. 2005, 22, 1989;
(b) Zhou, X. F.; Zhang, L.; Tseng, E.; Scott-Ramsay, E.; Schentag, J. J.; Coburn, R.
A.; Morris, M. E. Drug Metab. Dispos. 2005, 33, 321.
9. Linton, K. J. Physiology 2007, 22, 122.
10. (a) Shah, A.; Bariwal, J.; Molnár, J.; Kawase, M.; Motohashi, N. Top. Heterocycl.
Chem. 2008, 15, 201; (b) Mehdipour, A. R.; Javindnia, K.; Hemmateenejad, B.;
Amirghorfran, Z.; Miri, R. Chem. Biol. Drug Des. 2007, 70, 337; (c) Voigt, B.;
Coburger, C.; Monar, J.; Hilgeroth, A. Bioorg. Med. Chem. 2007, 15, 5110.
11. (a) Ekins, S.; Ecker, G. F.; Chiba, P.; Swaan, P. W. Xenobiotica 2007, 37, 1152; (b)
Crivori, P.; Reinach, B.; Pezzetta, D.; Poggesi, I. Mol. Pharm. 2006, 3, 33.
12. (a) Ecker, G.; Huber, M.; Schmid, D.; Chiba, P. Mol. Pharmacol. 1999, 56, 791; (b)
Ecker, G. F.; Schwaha, R.; Demel, M.; Chiba, P. Pharmacoinformatic approaches
to target P-glycoprotein: from inhibitor design to substrate prediction. ACS
235th National Meeting, New Orleans, LA, April 6–10, 2008, Abstract MEDI 161.
13. (a) Sauna, Z. E.; Andrus, M. B.; Turner, T. M.; Ambudkar, S. V. Biochemistry 2004,
43, 2262; (b) Namanja, H. A.; Emmert, D.; Davis, D. A.; Campos, C.; Miller, D. A.;
Hrycyna, C. A.; Chmielewski, J. J. Am. Chem. Soc. 2011, 134, 2976; (c) Pires, M.
M.; Emmert, D.; Hrycyna, C. A.; Chmielewski, J. Mol. Pharmacol. 2009, 75, 92;
(d) Pires, M. M.; Hrycyna, C. A.; Chmielewski, J. Biochemistry 2006, 45, 11695.
Acknowledgements
The authors thank NIH for grants NS038444 (N.N., M.I.S.),
5U01ES016102-02 (K.M.G., S.V.U.), and P20RR015583 (N.N., S.S.).

M. I. Szabon-Watola et al. / Bioorg. Med. Chem. Lett. 24 (2014) 117–121
121
14. (a) Natale, N. R.; Rogers, M. E.; Staples, R.; Triggle, D. J.; Rutledge, A. J. Med.
Chem. 1999, 42, 3087; (b) Mirzaei, Y. R.; Simpson, B. M.; Triggle, D. J.; Natale, N.
R. J. Org. Chem. 1992, 57, 6271; (c) Natale, N. R.; Triggle, D. J.; Palmer, R. B.;
Lefler, B. J.; Edwards, W. D. J. Med. Chem. 1990, 33, 2255; (d) McKenna, J. I.;
Schlicksupp, L.; Natale, N. R.; Maryanoff, B. E.; Flaim, S. F.; Willett, R. D. J. Med.
Chem. 1988, 31, 473.
30. Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.;
McWilliam, H.; Valentin, F.; Wallace, I. M.; Wilm, A.; Lopez, R.; Thompson, J. D.;
Gibson, T. J.; Higgins, D. G. Bioinformatics 2007, 23, 2947.
31. Verdonk, M. L. et al Proteins 2003, 52, 609.
32. Borchers, C.; Boer, R.; Klemm, K.; Volker, F.; Denzinger, T.; Ulrich, W.; Haas, S.;
Ise, W.; Gekeler, V.; Przybylski, M. Mol. Pharm. 2002, 61, 1366.
15. Schauer, C. K.; Anderson, O. P.; Quincy, D. A.; Natale, N. R. Acta Crystallogr., Sect.
C, Cryst. Struct. Commun. 1986, C42, 884.
33. http://www.modelling.leeds.ac.uk/qsitefinder/help.html,
November 7, 2013.
last
accessed
16. Natale, N. R.; Quincy, D. A. Synth. Commun. 1983, 13, 817.
17. (a) Zhou, P.; Mosher, M. D.; Taylor, W. D.; Crawford, G. A.; Natale, N. R. Bioorg.
Med. Chem. Lett. 1997, 7, 2455; (b) Han, X.; Twamley, B.; Natale, N. R. J.
Heterocycl. Chem. 2003, 40, 539; (c) Han, X.; Li, C.; Mosher, M. D.; Rider, K. C.;
Zhou, P.; Crawford, R. L.; Fusco, W.; Paszczynski, A.; Natale, N. R. Bioorg. Med.
Chem. 2009, 17, 1671.
18. (a) Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett. 1977, 4171; (b)
Lipton, M. F.; Basha, A.; Weinreb, S. M. Org. Synth. 1979, 59, 49.
19. Szabon-Watola, M. I. Neurotransporters, receptors and calcium signaling. M.S.
Thesis, University of Idaho, 2006.
20. Babakhanian, K.; Bendayan, M.; Bendayan, R. Biochem. Biophys. Res. Commun.
2007, 361, 301.
21. Tiberghien, F.; Loor, F. Anticancer Drugs 1996, 7, 568.
22. Mizuno, N.; Niwa, T.; Yotsumoto, Y.; Sugiyama, Y. Pharmacol. Rev. 2003, 55,
425.
23. Hamilton, G.; Cosentini, E. P.; Teleky, B.; Koperna, T.; Zacheri, J.; Riegler, M.;
Feil, W.; Schiessel, R.; Wenzi, E. Anticancer Res. 1993, 13, 2059.
24. Hulubei, V.; Meikrantz, S. B.; Quincy, D. A.; Houle, T.; McKenna, J. I.; Rogers, M.
E.; Steiger, S.; Natale, N. R. Bioorg. Med. Chem. 2012, 20, 6613.
25. Hutchins, R. O.; Natale, N. R. Org. Prep. Proced. Int. 1979, 11, 201.
26. Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.; Harrell, P. M.;
Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G. Science 2009, 323, 1718.
27. (a) Globisch, C.; Pajeva, I. K.; Wiese, M. ChemMedChem 2008, 3, 280; (b) Sirisha,
K.; Shekhar, M. C.; Umasankar, K.; Mahendar, P.; Sadanandam, A.; Achaiah, G.;
Reddy, V. M. Bioorg. Med. Chem. 2011, 19, 3249.
Notes: Experimental for the synthesis of 5-[12-(5-dimethylamino-naphthalen-
1-ylsulfamoyl)-dodecylcarbamoyl]-2,6-dimethyl-4-(5-methyl-3-phenyl-
isoxazol-4-yl)-1,4-dihydro-pyridine-3-carboxylic acid ethyl ester, 3d.
Yield from two steps 4%, greenish oil, ratio of two conformers: 80% of folded
versus 20% of extended, 1H NMR for folded conformation: d 1.12 (t, J = 6.8 Hz,
3H, ethyl CH3), 1.18–1.4 (m, 20H, amine 10*CH2’s), 1.35 (m, 4H, amine 2*CH2),
1.85 (s, 3H, C-2 Me), 2.07 (s, 3H, C-6 Me), 2.41 (s, 3H, isoxazole C-50 Me), 2.83
(m, 2H, amine CH2), 2.86 (s, 6H, N (CH3)2), 2.98 (m, 1H, amine CH), 3.16 (m, 1H,
amine CH), 4.00 (m, 2H, CH3CH2CO2), 4.55 (m, 1H, NH), 4.81 (br s, 1H, pyridine
NH), 4.94 (s, 1H, C-4 CH), 5.19 (br s, 1H, NH), 7.18 (d, J = 7.5 Hz, 1H,
naphthalene-H), 7.38 (m, 3H, Ph-H’s), 7.51 (m, 2H, Ph-H’s), 7.53 (m, 2H,
naphthalene-H’s), 8.23 (m, 1H, naphthalene-H), 8.30 (m, 1H, naphthalene-H),
8.51 (d, 1H, naphthalene-H). 13C NMR d 11.34, 14.40, 17.87, 19.76, 26.37, 26.63,
28.88, 29.14, 29.24, 29.29, 29.34 (2C0s), 29.53, 30.73, 39.51 (2C’s), 43.34, 45.43
(2C’s), 59.63, 98.80, 106.54, 115.20, 118.94, 118.95, 123.23, 127.98 (2C’s),
128.31, 128.75, 129.26 (2C0s), 129.64, 129.68, 129.92, 130.34, 130.57, 134.58,
134.59, 145.00, 152.01, 163.11, 166.23, 167.46.
Characteristic peaks for extended conformation, 1H NMR: d 1.00 (t, J = 6.8 Hz,
3H, ethyl CH3), 2.26 (s, 3H, isoxazole C-50 Me), 2.52 (s, 3H, C-2 Me), 2.65 (s, 3H,
C-6 Me), 4.15 (m, 2H, CH3CH2CO2), 7.18 (m, 1H, naphthalene-H), 7.35 (m, 1H,
Ph-H), 7.40 (m, 2H, Ph-H’s), 7.51 (m, 2H, Ph-H’s), 7.55 (m, 2H, naphthalene-
H’s), 8.21 (m, 1H, naphthalene-H), 8.30 (d, 1H, naphthalene-H), 8.55 (m, 1H,
naphthalene-H). 13C NMR d 11.40, 13.67, 39.61, 127.30(2C’s), 129.07, 129.88,
167.02, 168.02. FAB+ (m-NBA) m/z 797.24 [M+], 798.24 [M+1]+, also observed
796.24 [M-1]+. Anal. Calcd for C45H59O6N5S.H2O: C, 66.23; H, 7.53; N, 8.58.
Found: C, 66.02; H ,7.16; N, 8.19.
28. Pearson, W. R.; Lipman, D. J. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 2444.
29. Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779.