Synthesis and SAR of novel capsazepine analogs with significant anti-cancer effects in multiple cancer types

Article abstract of DOI:10.1016/j.bmc.2018.11.040

We previously demonstrated that capsazepine (CPZ), a synthetic transient receptor potential Vanilloid subtype 1 (TRPV1) antagonist, has significant anti-cancer effects in vivo. The purpose of this study was to develop more potent analogs based upon CPZ pharmacophore and structure–activity relationships (SAR) across analogs. We generated 30 novel compounds and screened for their anti-proliferative effects in cultured HeLa cervical cancer cells. Cell viability assays identified multiple compounds with IC_50s < 15 μM and one compound, 29 with an IC₅₀ < 5 μM; six fold more potent than CPZ. We validated the anti-proliferative efficacy of two lead compounds, 17 and 29, in vivo using HeLa-derived xenografts in athymic nude mice. Both analogs significantly reduced tumor volumes by day 8 compared to control treated animals (p < 0.001) with no observable adverse effects. Calcium imaging determined that compound 17 activates TRPV1 whereas 29 neither activates nor inhibits TRPV1; indicating a unique mechanism-of-action that does not involve TRPV1 signaling. Cell viability assays using a panel of additional tumor types including oral squamous cell carcinoma, non-small cell lung cancer (NSCLC), breast cancer, and prostate cancer cell lines (HSC-3, H460, MDA-231, and PC-3 respectively) demonstrated that both lead compounds were efficacious against every cancer type tested. Compounds 29 displayed IC_50s of 1–2.5 μM in HSC-3and PC-3cells. Thus, we propose that these novel CPZ analogs may serve as efficacious therapeutic agents against multiple tumor types that warrant further development for clinical application.

Full text of DOI:10.1016/j.bmc.2018.11.040

Accepted Manuscript
Synthesis and SAR of Novel Capsazepine Analogs with Significant Anti-Cancer
Effects in Multiple Cancer Types
Jorge De La Chapa, Matthew Valdez, Franscisco Ruiz III, Keith Gonzales, Wes
Mitchell, Stanton F. McHardy, Matthew Hart, Srikanth R. Polusani, Cara B.
Gonzales
PII:
S0968-0896(18)31332-4
https://doi.org/10.1016/j.bmc.2018.11.040
BMC 14644
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 August 2018
27 November 2018
28 November 2018
Please cite this article as: Chapa, J.D.L., Valdez, M., Ruiz, F. III, Gonzales, K., Mitchell, W., McHardy, S.F., Hart,
M., Polusani, S.R., Gonzales, C.B., Synthesis and SAR of Novel Capsazepine Analogs with Significant Anti-Cancer
Effects in Multiple Cancer Types, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.11.040
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Synthesis and SAR of Novel Capsazepine Analogs with Significant Anti-Cancer Effects in
Multiple Cancer Types
Jorge De La Chapaa, Matthew Valdezb, Franscisco Ruiz IIIb, Keith Gonzalesb, Wes Mitchellb,
Stanton F. McHardy* b, c, Matthew Hartb, c, Srikanth R. Polusanib, and Cara B. Gonzales* a, c
aDepartment of Comprehensive Dentistry, University of Texas Health Science Center at San
Antonio School of Dentistry, San Antonio, Texas, 78229, USA
bCenter for Innovative Drug Discovery, Department of Chemistry, University of Texas at San
Antonio, San Antonio Texas 78249, USA
cMays Cancer Center, University of Texas Health Science Center at San Antonio, San Antonio,
Texas, 78229, USA
To whom correspondence should be addressed:
Cara B. Gonzales DDS, PhD
University of Texas Health Science Center at San Antonio
Department of Comprehensive Dentistry
7703 Floyd Curl Drive, MCS 8258
San Antonio, Texas 78229-3900
Telephone: (210) 567-3332
FAX: (210) 567-3334
E-Mail: gonzalesc5@uthscsa.edu
Stanton F. McHardy, Ph.D.
University of Texas San Antonio
Center for Innovative Drug Discovery, Department of Chemistry
One UTSA Circle, San Antonio, TX, 78249. U.S.A.
Telephone: (210) 458-8676
Email: Stanton.mchardy@utsa.edu

Abstract
We previously demonstrated that capsazepine (CPZ), a synthetic transient receptor
potential Vanilloid subtype 1 (TRPV1) antagonist, has significant anti-cancer effects in vivo.
The purpose of this study was to develop more potent analogs based upon CPZ pharmacophore
and structure-activity relationships (SAR) across analogs. We generated 30 novel compounds
and screened for their anti-proliferative effects in cultured HeLa cervical cancer cells. Cell
viability assays identified multiple compounds with IC50s < 15 μM and one compound, 29 with
an IC50 < 5 μM; six fold more potent than CPZ. We validated the anti-proliferative efficacy of
two lead compounds, 17 and 29, in vivo using HeLa-derived xenografts in athymic nude mice.
Both analogs significantly reduced tumor volumes by day 8 compared to control treated animals
(p<0.001) with no observable adverse effects. Calcium imaging determined that compound 17
activates TRPV1 whereas 29 neither activates nor inhibits TRPV1; indicating a unique
mechanism-of-action that does not involve TRPV1 signaling. Cell viability assays using a panel
of additional tumor types including oral squamous cell carcinoma, non-small cell lung cancer
(NSCLC), breast cancer, and prostate cancer cell lines (HSC-3, H460, MDA-231, and PC-3
respectively) demonstrated that both lead compounds were efficacious against every cancer type
tested. Compounds 29 displayed IC50s of 1-2.5 μM in HSC-3and PC-3cells. Thus, we propose
that these novel CPZ analogs may serve as efficacious therapeutic agents against multiple tumor
types that warrant further development for clinical application.

Introduction
Our previous studies demonstrate that capsazepine (CPZ), a synthetic transient receptor
potential Vanilloid-1 (TRPV1) channel antagonist, has significant anti-tumor effects against oral
squamous cell carcinoma (OSCC) via a secondary mechanism-of-action that is independent of
TRPV1 interactions.1 The TRPV1 agonist, capsaicin, is shown to inhibit cellular respiration by
virtue of its structural similarity to coenzyme Q (CoQ) and poor redox potential resulting in
competitive inhibition of electron transport, production of reactive oxygen species (ROS), and
induction of apoptosis.2 Given that CPZ is structurally similar to capsaicin (Figure 1), we
postulated that its anti-cancer
effects may also be due to
disruption of mitochondrial
function.
Indeed,
we
Figure 1. Structure of Capsazepine and Capsaicin
confirmed that CPZ induced high ROS levels and apoptosis in vitro.1 In addition, we
demonstrated the efficacy of CPZ to treat OSCC in mouse xenograft models, which displayed
significant reductions in tumor growth and increased apoptosis compared to control treated
tumors.1
Based upon these findings, we endeavored to generate potent CPZ analogues that yield
even greater anti-proliferative effects than the parent compound. To this end, our synthesis and
SAR strategy was focused on manipulating both the electronic and steric nature of CPZ, while
maintaining good “drug-like” physiochemical properties.3, 4 There are several reports on the
synthesis of CPZ-like analogs, however, the anti-proliferative effects of CPZ analogs and
corresponding SAR has not been communicated to date.5-12 Since our preliminary studies
1
suggested that CPZ provides anti-proliferative activity independent of TRPV1 , the known

TRPV1 SAR from the literature on CPZ structural analogs would not likely be a good guide to
identify more potent anti-proliferative analogs. Here we describe the synthesis and development
of CPZ analogs with significant anti-proliferative effects in vitro and in vivo. We first screened
our compounds based upon their anti-proliferative effects against cultured HeLa cells. Anti-
tumor efficacy of lead compounds was validated in HeLa-derived mouse xenograft models.
Effects on TRPV1 activity were evaluated by calcium imaging. Lastly, we evaluated the anti-
proliferative effects, in vitro, against a panel of cancer types (HSC-3, H460, MDA-231, and PC-
3) demonstrating their potential to treat multiple types of solid tumors.
Results and Discussion
Synthesis
The synthesis shown in Scheme 1 was used to access the desired CPZ analogs. Various
analogs of the secondary amines, represented by compounds 1, 2, and 3 were prepared according
to literature precedent. Analogs of compound 1 were prepared from the corresponding
tetralones.10 Derivatives of compound 2 were prepared from the corresponding phenethyl amines
via a Pictet-Spengler process under thermal or microwave conditions.13 Analogs of the 5,6-di-
Scheme 1. General route for the synthesis of thioureas 6 and ureas 7

substited isoindoline 3, were prepared from the corresponding xylene derivatives via benzylic
bromination and ring closure.14 The structural analogs of amines 1, 2 and 3, represented by the
general structure 4 could then be taken forward to the corresponding thiourea 6 and urea 7.
Treatment of amine 5 with thiocarbonyl diimidazole, followed by treatment of amine 4 produced
the corresponding thiourea 6 in moderate to good yield.15 Alternatively, the amine 5 could be
reacted with trichloroacetyl chloride, followed by amine 4 to produce the urea 7.16
Structure-Activity Relationship Studies
Our SAR studies focused on a systematic manipulation of the phenol substituents, ring
size, thiourea and phenethyl side chain moieties. The anti-proliferative SAR on the phenol group
of CPZ itself is summarized in Table 1. Removal of one of the phenol groups at either position
(8 and 9) provided a two-
fold decrease in potency
1HeLa
IC50
relative to CPZ. Similarly,
R1
R2
Compound
MW
log P
tPSA
(μM)
replacement of the phenols
with methoxy groups (10
and 11) produced analogs
completely devoid of anti-
CPZ
8
OH
H
OH
OH
H
30
65
376.9
360.9
4.02
4.72
4.72
4.87
4.87
55.7
35.5
35.5
24.5
24.5
9
OH
50
360.9
OCH3
10
11
H
>100
>100
374.93
374.93
OCH3
H
Table 1. SAR on 7-member ring analogs. 1 MTS cell viability assay
of HeLa cells treated with 7-member ring analogs for 24h (n=4 per
group). Positive control compound CPZ IC50 = 30 μM.
proliferative
Manipulation
activity.
of the
tetrahydro-1H-benzo[c]azepine ring produced some interesting results. Complete removal of the
ring, as represented by the acyclic analogs 12-16 in Table 2 produced a significant reduction in
potency relative to CPZ. Interestingly, similar analogs developed by Walpole et. al. (1994),
demonstrated TRPV1 agonist activity; thus loss of the tetrahydro-1H-benzo[c]azepine ring

structure appears to be critical to CPZ analogs’ anti-proliferative effects, but not to their TRPV1
activity.3 Based on these results, we next turned our attention to 5- and 6-membered ring analogs
and studied the
effect on three key
1HeLa
IC50
SAR points; 1)
Compound
n
R1
R2
R3
X
MW
log P
tPSA
(μM)
substitution effects
on the R3 and R4
position of the
fused ring systems,
2) thiourea, and
12
13
14
15
16
0
1
0
1
0
OH
OH
OH
OH
Cl
Cl
Cl
Cl
Cl
S
S
>100
30
350.86
364.89
362.85
348.83
334.8
3.93
4.22
3.33
3.33
3.04
55.73
55.73
50.8
OCH3 OCH3
O
O
O
>30
>30
30
OH
OH
72.8
OH
OH
72.8
Table 2. SAR on 7-member ring analogs. 1 MTS cell viability assay of
HeLa cells treated with 7-member ring analogs for 24h (n=4 per group).
Positive control compound CPZ IC50 = 30 μM.
urea
derivatives,
and 3) length and substitution (R5 and R6) of the phenethyl side chains (Table 3). These studies
produced analogs with improvements in potency relative to CPZ. Installation of the
unsubstituted tetrahydroisoquinoline 6-membered ring (17) produced significant improvement in
anti-proliferative activity (<10 uM) and the corresponding urea analog (18) provided analogous
activity. Similar to earlier observations, replacement of the phenols with methyl ethers (19 and
20) decreased activity substantially. Replacement of the tetrahydroisoquinoline 6-membered
ring in 17 with the 5,6-isoindoline 5-membered ring (21) also resulted in a marked decrease in
cytotoxicity. The observed anti-proliferative effects of these analogs were also found to be
dependent on the nature of the phenethyl side chain and the phenyl substitution pattern (R5 and
R6). Replacement of the para-chloro substituent (R6) with hydrogen, fluorine, or methoxy
decreased the anti-proliferative activity (22, 23 and 24). Similar results were obtained with the
incorporation of the 2,4-dichlorophenethyl or substituted benzyl groups, as represented by 25 and

1HeLa
IC50 (μM)
<10
15
>100
>100
85
>100
25
25
30
>100
<5
55
<5
Compound
n
R1
R2
R3
R4 R5
R6
X
m
MW
log P tPSA
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
Cl
Cl
H
H
H
H
Cl
Cl
Cl
Cl
Cl
OCH3
F
H
Cl
H
Cl
Cl
Cl
H
H
Cl
Cl
OCH3
F
S
O
S
O
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
1
1
1
1
1
1
1
1
1
0
1
1
0
1
1
1
1
1
1
1
0
362.87
346.81
390.93
374.87
348.85
358.46
346.42
328.43
397.31
314.4
438.97
404.95
390.5
404.53
370.51
473.41
439.4
3.97
3.08
4.26
3.37
3.68
3.21
3.51
3.37
4.58
3.08
5.76
5.28
4.86
5.15
4.67
6.36
5.88
4.99
5.29
4.81
4.38
55.73
72.8
33.73
50.8
OCH3 OCH3
OCH3 OCH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
55.73
64.96
55.73
55.73
55.73
55.73
55.73
55.73
55.73
55.73
55.73
55.73
55.73
64.96
55.73
55.73
55.73
H
phenyl
isoPr
phenyl
phenyl
isoPr
phenyl
isoPr
phenyl
phenyl
isoPr
isoPr
25
67
20
46
24
17
28
35
434.55
422.52
388.5
F
Cl
356.48
Table 3. SAR on 5- and 6-membered cyclic analogs. 1 MTS cell viability assay of HeLa cells treated
with 5- and 6-membered cyclic analogs for 24h (n=4 per group). Positive control compound CPZ IC50 =
30 μM.
26. Modification of the R3 and R4 positions of the tetrahydroisoquinoline 6-membered ring
produced some interesting results. Introduction of a phenyl group at the R4 position while
maintaining the para-chlorophenethyl group produced the thiourea 27, which showed a marked
increase in anti-proliferative activity; however the corresponding isopropyl derivative (28) was
completely devoid of any appreciable activity. Maintaining the phenyl group in the R4 position,
while shorting the phenethyl chain to para-chlorobenzyl produced the thiourea 29, which
displayed potent anti-proliferative effects similar to 27. As shown in table 3, (compounds 30 to
37), further modifications of the R3, R4, R5 and R6 positions provided analogs with either
equivalent activity or decreased activity as compared to 27 and 29. Based on these SAR studies

and evaluation of physiochemical properties, compounds 17 and 29 were taken forward and
assessed in a variety of cancer cell lines, and in a mouse xenograft model.
Compounds 17 and 29 have significant anti-tumor effects in HeLa-derived mouse xenografts.
To validate the anti-cancer activities of these compounds, we generated HeLa-derived tumors in
athymic nude mice. Once tumors reached 150 mm3, mice were treated every other day with 17,
29, or vehicle control. Both compounds significantly reduced tumor growth by day 8 compared
to control treated tumors and maintained significantly reduced tumor growth throughout the
remainder of the study
(p<0.001; Figure 2A).
No
significant
difference in efficacy
was seen between 17
and 29.
At the
conclusion of the
experiment, (day 14)
the average tumor
volumes for control,
17, and 29 treated
Figure 2. Anti-tumor effects of 17 and 29 in HeLa-derived mouse
xenograft models. Panel A: Tumor volumes of HeLa-derived xenografts
(n=4 per group) treated every other day with 40 μg of indicated analog or
vehicle control for a period of 14 days. Significant reduction in tumor
volumes is seen by day 8 and continued throughout the duration of the
study (*p<0.05 and ***p<0.001). Panel B: Scatter plot of HeLa-derived
tumors’ final volumes at day 14. Median tumor volume for control tumors
was 562 mm3 whereas median tumor volume for 17-treated tumors was 351
mm3 and median tumor volume for 29-treated tumors was 283 mm3.
mice were 562 mm3, 351 mm3, and 283 mm3 respectively (Figure 2B; p<0.01). There were no
observable adverse effects on adjacent non-malignant tissues, neurological function, and
respiration in mice treated with 17 and 29 and no reduction of body weight, mobility, or motor
functions were noted.

Compounds 17 and 29 have anti-proliferative effects against multiple cancer types in
vitro that does not require TRPV1 interactions. The anti-proliferative effects of these lead
compounds were tested
against a panel of cancer
cell lines including OSCC
(HSC-3), NSCLC (H460),
breast cancer (MDA-231)
and prostate cancer (PC-
3).
Both
analogs
significantly reduced cell
viability of each cancer
type tested (Figures 3A
and 3B; Table 4). Slight
differences in potencies
were noted for each
analog depending on the
cell line tested. Calcium
Figure 3: Effects of CPZ analogs on proliferation of multiple
cancer types and TRPV1 activation in vitro. Panels A and B: MTS
cell viability assays of HSC-3 (OSCC), MDA-231 (breast cancer),
H460 (NSCLC) and PC-3 (prostate cancer) cell lines treated with CPZ
analogs 17 and 29 for 24 h; n=4 per group. Panel C: Calcium imaging
of CHO-TRPV1 cells treated with increasing concentrations of CPZ
analogs 17 and 29. Panel D: Calcium imaging of CHO-TRPV1 cells
pre-treated with CPZ or 29 followed by capsaicin; *p<0.05.
imaging revealed that 17 is a TRPV1 agonist
(Figure 3C) and elicits a concentration dependent
influx of calcium ions into CHO-TRPV1 cells. In
contrast, 29 failed to activate TRPV1 channels in
vitro. In addition, pre-treatment of CHO-TRPV1
Cell Line
17
29
HSC-3
H460
MDA-231
PC-3
20 μM
23 μM
5 μM
2 μM
42 μM
32 μM
2.5 μM
13 μM
Table 4. IC50s of Compound 17 and 29
in cancer cell lines.
cells with 29 followed by capsaicin determined that 29 does not inhibit TRPV1 activation

(Figure 3D). Thus, 29 is neither a TRPV1 agonist nor an antagonist, whose anti-cancer
mechanism is independent of TRPV1.
Conclusion
These studies provide evidence that analogs of CPZ can be identified with potent anti-
proliferative activity in vitro and substantial in vivo activity in reducing tumor growth in mouse
xenograft models. SAR studies leading to the identification of 17 and 29 were focused on a
three-point SAR strategy to assess the anti-proliferative activity of analogs, while providing
minimal changes in physiochemical properties. Given that TRPV1 mediates thermoregulation,
bronchoconstriction, and pain16-20 and that TRPV1 interactions do not play a role in CPZ anti-
cancer activity, our goal was to eliminate TRPV1 effects, while improving the anti-cancer
efficacy. Calcium imaging studies confirm that 17 is a TRPV1 agonist; however 29 neither
activates nor inhibits TRPV1 making it an attractive lead compound for further development.
Additional SAR studies are underway to determine any other potential TRP channel activity
including TRPA1, which can form heterotetromer channels with TRPV1.21 Notably, no adverse
reactions associated with TRPV1 activity were observed with either analog (erythema,
ulcerations, or respiratory distress); thus CPZ analogs may provide a novel therapeutic approach
for treating cancers.
Experimental Methods
Chemistry
Chemicals were purchased from established commercial suppliers, including Sigma
Aldrich (St. Louis, MO), Chembridge Corporation (San Diego, CA), ChemDiv (San Diego, CA),
and Specs (Hopkinton, RI), Cayman (Ann Arbor, MI) and Pfaltz & Bauer (Waterbury, CT). The

1
identity of all the tested compounds was confirmed by H NMR and HPLC-MS, and the purity
was ensured to be ≥ 95%.
General procedures. Unless otherwise indicated all reactions were conducted in standard
commercially available glassware using standard synthetic chemistry methods and setup. All air-
and moisture-sensitive reactions were performed under nitrogen atmosphere with dried solvents
and glassware under anhydrous conditions. Starting materials and reagents were commercial
compounds of the highest purity available and were used without purification. Solvents used for
reactions were indicated as of commercial dry or extra-dry or analytical grade. Analytical thin-
layer chromatograph (TLC) was carried out using silica gel 60 F254 TLC plates. TLC
visualization was achieved with a UV lamp or by staining in an iodine chamber. Flash
chromatography was done on a system using prepacked silica gel columns or using silica gel
60A (230-400 mesh) or with preparative thin-layer chromatography plates (1000 micron F254), or
using a Biotage Isolera One 2.2, using commercial columns that were pre-packed with Merck
Kieselgel 60 (230– 400 mesh) silica gel. Solvent systems employed consisted of (EtOAc/Hex or
DCM/MeOH or DCM/MeOH/Conc. NH4OH). All moisture- and air-sensitive reactions and
reagent transfers were carried out under dry nitrogen. Final compounds for biological testing are
all ≥95% purity as determined by HPLC-MS and 1H NMR.
1
NMR. H NMR experiments were recorded on Agilent DD2 400MHz spectrometers at ambient
temperature. Samples were dissolved and prepared in deuterated solvents (CDCl3, CD3OD and
DMSOd6) with residual solvents being used as the internal standard in all cases. All deuterated
solvent peaks were corrected to the standard chemical shifts (CDCl3, dH = 7.26 ppm; CD3OD, dH
= 3.31 ppm; DMSO-d6, dH = 2.50 ppm). Spectra were all manually integrated after automatic
baseline correction. Chemical shifts (d) are given in parts per million (ppm), and coupling

constants (J) are given in Hertz (Hz). The proton spectra are reported as follows: d (multiplicity,
coupling constant J, number of protons). The following abbreviations were used to explain the
multiplicities: app = apparent, b = broad, d = doublet, dd = doublet of doublets, ddd = doublet of
doublet of doublets, dddd = doublet of doublet of doublet of doublets, m = multiplet, s = singlet,
t = triplet, ABq = AB quartet.
HPLC-MS. All samples were analyzed on Agilent 1290 series HPLC system comprised of binary
pumps, degasser and UV detector, equipped with an auto-sampler that is coupled with Agilent
6150 mass spectrometer. Purity was determined via UV detection with a bandwidth of 170nm in
the range from 230-400nm. The general LC parameters were as follows: Column - Zorbax
Eclipse Plus C18, size 2.1 X 50 mm; Solvent A: 0.10 % formic acid in water, Solvent B: 0.00 %
formic acid in acetonitrile; Flow rate – 0.7 mL/min; Gradient: 5 % B to 95 % B in 5 min and
hold at 95 % B for 2 min; UV detector – channel 1 = 254 nm, channel 2 = 254 nm. Mass detector
Agilent Jet Stream – Electron Ionization (AJS-ES).
General Procedures
Compounds of general structure 1, 2 and 3 were prepared according to the referenced literature
precedent.
General procedure for the synthesis of thiourea 6
To a stirring solution of 1,1’-thiocarbonyldiimidazole (1.2 equiv) in 5 mL of DMF at 50
oC was added a solution of TEA (1 equiv) and amine 5 (1 equiv) in DMF dropwise. The
resulting solution stirred at room temperature for 1-24 hours and was monitored by TLC. The
resulting crude solution of the isothiocyanate was used in the next step without purification.
To a stirring solution of amine 4 (1 equiv) and TEA (3 equiv) in DMF (2 mL) was added
the crude isocyanate solution prepared above (1.2 equiv) and the resulting mixture was stirred at

room temperature for 2-4 hours. Once complete by TLC, the mixture was diluted with ethyl
acetate and washed with excess water and brine. The ethyl acetate layer was collected, dried
with sodium sulfate and concentrated in vacuo. The resulting crude material was subjected to
SiO2 flash chromatography (eluent: ethyl acetate/DCM gradient) to provide the desired products
of general structure 6 in 5-95 % yield.
General procedure for the synthesis of urea 7
To a stirring solution of amine 5 (1 equiv) in THF (0.2 M) was add trichloroacetyl
chloride (1 equiv) dropwise at room temperature. After 4 hours, the solution was concentrated in
vacuo to afford the corresponding trichloroacetamide derivatives as a white solids that was used
in the next step without purification.
To the amine 4 (1 equiv), in DMSO was added DBU (2 equiv) and the trichloroacetamide
(1 equiv) at room temperature. The reaction was heated to 80 oC and monitored by TLC. After
cooling to room temperature, the mixture was diluted with DCM and washed with 1M HCl,
saturated NaHCO3 and brine. The organic layer was concentrated in vacuo and the resulting
crude material was subjected to SiO2 flash chromatography (eluent: ethyl acetate/DCM gradient)
to provide the desired products of general structure 7 in 5-41 % yield.
The following compounds were prepared using the general procedures described above.
N-[2-(4-chlorophenyl)ethyl]-7-hydroxy-2,3,4,5-tetrahydro-1H-2-benzazepine-2-
1
carbothioamide (8). H NMR (400 MHz, CD3OD) δ 7.22 – 7.17 (m, 2H), 7.11 – 7.07 (m, 2H),
7.04 (d, J = 8.1 Hz, 1H), 6.60 (d, J = 2.6 Hz, 1H), 6.48 (dd, J = 8.1, 2.6 Hz, 1H), 4.69 (s, 2H),
4.06 (s, 2H), 3.74 (dd, J = 7.8, 6.8 Hz, 2H), 2.87 – 2.77 (m, 4H), 1.76 (qt, J = 11.3, 5.5 Hz, 2H).
ESI-MS m/z [M + Na]+ : 383.4

N-[2-(4-chlorophenyl)ethyl]-8-hydroxy-2,3,4,5-tetrahydro-1H-2-benzazepine-2-
1
carbothioamide (9). H NMR (400 MHz, CD3OD) δ 7.23 – 7.13 (m, 2H), 7.13 – 7.03 (m, 2H),
6.94 (d, J = 8.1 Hz, 1H), 6.80 (d, J = 2.6 Hz, 1H), 6.58 (dd, J = 8.1, 2.6 Hz, 1H), 4.80 – 4.74 (m,
J = 3.9 Hz, 2H), 4.01 (s, 2H), 3.78 – 3.69 (m, J = 8.0, 6.8 Hz, 2H), 2.88 – 2.77 (m, J = 14.1, 7.0
Hz, 4H), 1.74 (quint, J = 11.2, 5.6 Hz, 2H). ESI-MS m/z [M + H]+ : 361.3
N-[2-(4-chlorophenyl)ethyl]-7-methoxy-2,3,4,5-tetrahydro-1H-2-benzazepine-2-
1
carbothioamide (10). H NMR (400 MHz, CD3OD) δ 7.23 – 7.19 (m, 2H), 7.15 (d, J = 8.3 Hz,
1H), 7.13 – 7.09 (m, 2H), 6.74 (d, J = 2.7 Hz, 1H), 6.62 (dd, J = 8.2, 2.7 Hz, 1H), 4.75 (s, 2H),
4.09 (s, 2H), 3.79 – 3.74 (m, 5H), 2.94 – 2.83 (m, J = 20.0, 10.2, 4.9 Hz, 4H), 1.79 (quint, J =
11.4, 5.6 Hz, 2H). ESI-MS m/z [M + H]+ : 375.3
N-[2-(4-chlorophenyl)ethyl]-8-methoxy-2,3,4,5-tetrahydro-1H-2-benzazepine-2-
1
carbothioamide (11). H NMR (400 MHz, CD3OD) δ 7.22 – 7.13 (m, 2H), 7.11 – 7.03 (m, 3H),
6.93 (d, J = 2.7 Hz, 1H), 6.73 (dd, J = 8.3, 2.7 Hz, 1H), 4.83 – 4.77 (m, J = 4.2 Hz, 2H), 4.08 (s,
2H), 3.81 – 3.69 (m, 5H), 2.91 – 2.81 (m, 4H), 1.78 (quint, J = 11.3, 5.6 Hz, 2H). ESI-MS m/z
[M+ H]+: 375.3
1
1-[2-(4-chlorophenyl)ethyl]-3-[(3,4-dihydroxyphenyl)methyl]-3-methylthiourea (12). H
NMR (400 MHz, CD3OD) δ 7.29 – 7.23 (m, 2H), 7.23 – 7.18 (m, 2H), 6.75 – 6.69 (m, J = 5.2
Hz, 2H), 6.56 (dd, J = 8.1, 2.1 Hz, 1H), 4.59 (s, 3H), 3.85 – 3.79 (m, 2H), 2.97 (s, 2H), 2.93 (t,
2H). ESI-MS m/z [M+H]+: 351.2
1
1-[2-(4-chlorophenyl)ethyl]-3-[2-(3,4-dihydroxyphenyl)ethyl]-3-methylthiourea (13). H
NMR (400 MHz, CDCl3) δ 7.30 – 7.27 (m, 2H), 7.16 – 7.11 (m, 2H), 6.80 (d, J = 8.0 Hz 1H),
6.71 (d, J = 2.0 Hz, 1H), 6.60 (dd, J = 8.0, 2.0 Hz, 1H), 3.89 – 3.79 (m, J = 12.2, 4.8 Hz, 4H),
2.98 (s, 3H), 2.85 (t, J = 6.9 Hz, 2H), 2.78 (t, J = 7.2 Hz, 2H). ESI-MS m/z [M+H]: 365.3

1-[2-(4-chlorophenyl)ethyl]-3-[(3,4-dimethoxyphenyl)methyl]-3-methylurea (14).
1H
NMR (400 MHz, CDCl3) δ 7.25 – 7.20 (m, 2H), 7.09 – 7.04 (m, 2H), 6.80 (d, J = 8.1 Hz, 1H),
6.74 (d, J = 1.9 Hz, 1H), 6.70 (dd, J = 8.1, 2.0 Hz, 1H), 4.38 (s, 2H), 3.88 (s, 3H), 3.86 (s, 3H),
3.48 (dd, J = 12.1, 6.7 Hz, 2H), 2.84 (s, 3H), 2.78 (dd, J = 8.3, 5.3 Hz, 2H). ESI-MS m/z [M+H]+
: 363.3
1-[2-(4-chlorophenyl)ethyl]-3-[2-(3,4-dihydroxyphenyl)ethyl]-3-methylurea (15).
1H
NMR (400 MHz, CDCl3) δ 7.25 – 7.20 (m, 2H), 7.09 – 7.04 (m, 2H), 6.80 (d, J = 8.1 Hz, 1H),
6.74 (d, J = 1.9 Hz, 1H), 6.70 (dd, J = 8.1, 2.0 Hz, 1H), 4.38 (s, 2H), 3.88 (s, 3H), 3.86 (s, 3H),
3.48 (dd, J = 12.1, 6.7 Hz, 2H), 2.84 (s, 3H), 2.78 (dd, J = 8.3, 5.3 Hz, 2H). ESI-MS m/z
[M+H]:363.3
1-[2-(4-chlorophenyl)ethyl]-3-[(3,4-dihydroxyphenyl)methyl]-3-methylurea (16).
1H
NMR (400 MHz, CD3OD) δ 7.20 – 7.15 (m, 2H), 7.12 – 7.07 (m, 2H), 6.63 (d, J = 8.0 Hz, 1H),
6.57 (d, J = 2.1 Hz, 1H), 6.42 (dd, J = 8.0, 2.1 Hz, 1H), 4.24 (s, 2H), 3.33 – 3.27 (m, 2H), 2.71
(d, J = 7.6 Hz, 2H), 2.68 (s, 3H). ESI-MS m/z [M+H]+: 335.3
N-[2-(4-chlorophenyl)ethyl]-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (17). H NMR (400 MHz, DMSO) δ 8.73 (d, J = 6.8 Hz, 2H), 7.65 (t, J = 5.2
Hz, 1H), 7.30 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H), 6.50 (s, 1H), 6.46 (s, 1H), 3.82 (t, J =
13
5.8 Hz, 2H), 3.65 (dd, J = 14.1, 6.1 Hz, 2H), 2.60 (t, J = 5.7 Hz, 2H). C NMR (100 MHz,
DMSO) δ 180.91, 144.41, 144.16, 139.05, 131.08, 130.93, 128.66, 125.70, 124.17, 115.43,
113.41, 49.11, 46.91, 45.88, 34.58, 27.79. ESI-MS m/z [M+H]+:363.2
N-[2-(4-chlorophenyl)ethyl]-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-2-
1
carboxamide (18). H NMR (400 MHz, CD3OD) δ 7.23 – 7.20 (m, 2H), 7.17 – 7.13 (m, 2H),

6.53 (s, 1H), 6.51 (s, 1H), 4.32 (s, 2H), 3.50 (t, J = 5.9 Hz, 2H), 3.39 – 3.32 (m, 2H), 2.76 (t,
2H), 2.63 (t, J = 5.9 Hz, 2H). ESI-MS m/z [M+H]+: 347.3
N-[2-(4-chlorophenyl)ethyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (19). H NMR (400 MHz, CDCl3) δ 7.29 – 7.25 (m, 2H), 7.17 – 7.12 (m, 2H),
6.66 (s, 1H), 6.62 (s, 1H), 4.76 (s, 2H), 3.98 – 3.91 (m, J = 11.7, 5.9 Hz, 2H), 3.88 – 3.80 (m,
8H), 2.95 (t, J = 6.9 Hz, 2H), 2.82 (t, J = 5.9 Hz, 2H). ESI-MS m/z [M+H]+: 391.3
N-[2-(4-chlorophenyl)ethyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-
1
carboxamide (20). H NMR (400 MHz, CDCl3) δ 7.29 – 7.26 (m, 2H), 7.15 – 7.10 (m, 2H), 6.64
(s, 1H), 6.59 (s, 1H), 4.40 (s, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.57 – 3.53 (m, 2H), 3.53 – 3.48 (m,
2H), 2.82 (t, J = 6.9 Hz, 2H), 2.76 (t, J = 6.0 Hz, 2H). ESI-MS m/z [M+H]+: 375.3
N-[2-(4-chlorophenyl)ethyl]-5,6-dihydroxy-2,3-dihydro-1H-isoindole-2-carbothioamide
1
(21). H NMR (400 MHz, CD3OD) δ 7.32 – 7.22 (m, 4H), 6.70 (s, 2H), 3.85 – 3.78 (m, J = 8.3,
6.8 Hz, 2H), 2.95 (t, 2H). ESI-MS m/z [M+H]+: 349.2
6,7-dihydroxy-N-[2-(4-methoxyphenyl)ethyl]-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (22). H NMR (400 MHz, CD3OD) δ 7.15 – 7.10 (m, 2H), 6.84 – 6.79 (m, 2H),
6.59 (s, 1H), 6.55 (s, 1H), 4.68 (s, 2H), 3.90 (t, J = 5.9 Hz, 2H), 3.83 – 3.77 (m, J = 7.5, 5.9 Hz,
2H), 3.75 (s, 3H), 2.87 (t, J = 8.0, 2H), 2.73 (t, J = 5.9 Hz, 2H). ESI-MS m/z [M+H]+: 359.1
N-[2-(4-fluorophenyl)ethyl]-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (23). H NMR (400 MHz, CD3OD) δ 7.24 – 7.17 (m, 2H), 7.01 – 6.92 (m, 2H),
6.57 (s, 1H), 6.54 (s, 1H), 4.67 (s, 2H), 3.88 (t, J = 5.9 Hz, 2H), 3.79 (t, 2H), 2.90 (t, 2H), 2.71 (t,
J = 5.9 Hz, 2H). ESI-MS m/z [M+H]+: 347.1
6,7-dihydroxy-N-(2-phenylethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbothioamide (24).
1H NMR (400 MHz, CD3OD) δ 7.31 – 7.13 (m, 5H), 6.59 (s, 1H), 6.55 (s, 1H), 4.69 (s, 2H), 3.90

(t, J = 5.9 Hz, 2H), 3.83 (t, 2H), 2.94 (t, 2H), 2.73 (t, J = 5.9 Hz, 2H). ESI-MS m/z [M+H]+:
329.1
N-[2-(2,4-dichlorophenyl)ethyl]-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (25). H NMR (400 MHz, CD3OD) δ 7.41 (d, J = 2.1 Hz, 1H), 7.27 (d, 1H),
7.20 (dd, J = 8.0, 2.2 Hz, 1H), 6.59 (s, 1H), 6.55 (s, 1H), 4.69 (s, 2H), 3.94 – 3.83 (m, 4H), 3.10
(d, J = 4.0 Hz, 2H), 2.73 (t, J = 5.9 Hz, 2H). ESI-MS m/z [M+H]+: 397.1
1
N-benzyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-2-carbothioamide (26). H NMR
(400 MHz, CD3OD) δ 7.41 (d, J = 2.1 Hz, 1H), 7.27 (d, 1H), 7.20 (dd, J = 8.0, 2.2 Hz, 1H), 6.59
(s, 1H), 6.55 (s, 1H), 4.69 (s, 2H), 3.94 – 3.83 (m, 4H), 3.10 (d, J = 4.0 Hz, 2H), 2.73 (t, J = 5.9
Hz, 2H). ESI-MS m/z [M+H]+: 397.1
N-[2-(4-chlorophenyl)ethyl]-6,7-dihydroxy-1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (27). H NMR (400 MHz, CDCl3) δ 7.24 – 7.18 (m, 5H), 7.14 – 7.05 (m, 4H),
6.90 (s, 1H), 6.70 (s, 1H), 6.63 (s, 1H), 5.54 (s, 1H), 3.93 (q, J = 12.0, 8.0 Hz, 2H), 3.71 – 3.62
13
(m, 2H), 2.90 (td, J = 6.8, 2.4 Hz, 2H), 2.68 – 2.54 (m, 2H). C NMR (100 MHz, DMSO) δ
180.90, 144.99, 143.93, 142.62, 138.97, 131.09, 130.99, 128.64, 128.46, 127.64, 127.32, 125.87,
115.67, 115.38, 47.09, 40.59, 34.50, 26.92. ESI-MS m/z [M+H]+: 439.1
N-[2-(4-chlorophenyl)ethyl]-6,7-dihydroxy-1-(propan-2-yl)-1,2,3,4-
1
tetrahydroisoquinoline-2-carbothioamide (28). H NMR (400 MHz, CDCl3) δ 7.25 (d, J = 7.3
Hz, 2H), 7.12 (d, J = 8.3 Hz, 2H), 6.55 (s, 2H), 5.51 (s, 1H), 4.00 – 3.87 (m, 2H), 3.57 – 3.47 (m,
1H), 3.00 – 2.84 (m, 3H), 2.72 (s, 1H), 2.00 – 1.91 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H), 0.86 (d, J =
6.6 Hz, 3H). ESI-MS m/z [M+H]+: 405.2
N-benzyl-6,7-dihydroxy-1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-carbothioamide (29).
1H NMR (400 MHz, CDCl3) δ 7.36 – 7.22 (m, 10H), 6.76 (s, 1H), 6.67 (s, 1H), 5.77 (s, 1H), 4.90

(qd, J = 14.4, 4.7 Hz, 2H), 3.90 – 3.70 (m, 2H), 2.84 – 2.59 (m, 2H). 13C NMR (100 MHz,
Acetone) δ 183.35, 145.41, 144.28, 143.38, 140.69, 129.00, 128.96, 128.87, 128.43, 128.33,
128.31, 128.19, 127.75, 127.55, 127.53, 115.85, 115.82, 62.42, 50.06, 49.93, 43.18, 27.88. ESI-
MS m/z [M+H]+: 391.2
6,7-dihydroxy-1-phenyl-N-(2-phenylethyl)-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (30). H NMR (400 MHz, CDCl3) δ 7.34 – 7.12 (m, 10H), 6.97 (s, 1H), 6.73 (s,
1H), 6.67 (s, 1H), 5.53 (s, 1H), 5.33 (d, J = 6.5 Hz, 1H), 3.99 (s, 2H), 3.74 – 3.60 (m, 2H), 3.02 –
2.87 (m, 2H), 2.73 – 2.53 (m, 2H). ESI-MS m/z [M+H]+: 405.2
6,7-dihydroxy-N-(2-phenylethyl)-1-(propan-2-yl)-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (31). H NMR (400 MHz, CDCl3) δ 7.33 – 7.27 (m, 2H), 7.23 – 7.17 (m, 2H),
6.61 (s, 1H), 6.59 (s, 1H), 5.40 (t, J = 2.0 Hz, 1H), 5.32 – 5.24 (m, 1H), 4.01 – 3.86 (m, 2H), 3.58
– 3.49 (m, 1H), 2.99 – 2.86 (m, 3H), 2.79 – 2.68 (m, 1H), 2.03 – 1.92 (m, 1H), 0.95 (d, J = 6.9
Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H). ESI-MS m/z [M+H]+: 371.2
N-[2-(2,4-dichlorophenyl)ethyl]-6,7-dihydroxy-1-phenyl-1,2,3,4-tetrahydroisoquinoline-
1
2-carbothioamide (32). H NMR (400 MHz, CDCl3) δ 7.36 (d, J = 1.8 Hz, 2H), 7.29 – 7.21 (m,
3H), 7.19 – 7.11 (m, 3H), 7.03 (s, 2H), 6.74 (s, 2H), 6.67 (s, 2H), 5.63 (s, 2H), 3.97 (ddd, J =
12.0, 6.8, 2.6 Hz, 2H), 3.70 (d, 1H), 3.08 (octet, J = 13.8, 6.8 Hz, 2H), 2.76 – 2.57 (m, 2H). ESI-
MS m/z [M+H]+: 473.1
N-[2-(2,4-dichlorophenyl)ethyl]-6,7-dihydroxy-1-(propan-2-yl)-1,2,3,4-
1
tetrahydroisoquinoline-2-carbothioamide (33). H NMR (400 MHz, DMSO) δ 8.82 – 8.59 (m,
2H), 7.52 – 7.50 (m, 1H), 7.29 – 7.21 (m, 2H), 6.49 (s, 1H), 6.45 (s, 1H), 3.77 – 3.64 (m, 2H),
3.54 – 3.40 (m, 1H), 3.28 – 3.17 (m, 1H), 3.00 – 2.89 (m, 2H), 2.75 – 2.61 (m, 2H), 1.98 – 1.85
(m, 1H), 0.86 (d, J = 6.4 Hz, 7H). ESI-MS m/z [M+H]+: 423.2

6,7-dihydroxy-N-[2-(4-methoxyphenyl)ethyl]-1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (34). H NMR (400 MHz, CDCl3) δ 7.24 – 7.20 (m, 1H), 7.11 (dd, J = 18.9, 7.7
Hz, 4H), 6.93 (s, 1H), 6.83 (d, J = 8.5 Hz, 2H), 6.71 (s, 1H), 6.66 (s, 1H), 5.52 (s, J = 4.0 Hz,
1H), 5.38 (d, J = 27.8 Hz, 2H), 3.99 – 3.88 (m, 2H), 3.80 (s, 1H), 3.74 – 3.60 (m, 2H), 2.95 –
2.82 (m, 1H), 2.73 – 2.55 (m, 1H). ESI-MS m/z [M+H]+: 435.2
N-[2-(4-fluorophenyl)ethyl]-6,7-dihydroxy-1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (35). H NMR (400 MHz, CD3OD) δ 7.33 – 7.14 (m, 7H), 6.97 (t, J = 8.8 Hz,
2H), 6.63 (s, 2H), 3.92 – 3.77 (m, 3H), 3.53 – 3.36 (m, 2H), 2.95 (t, J = 7.3 Hz, 2H), 2.77 – 2.68
(m, 1H), 2.54 – 2.46 (m, 1H). ESI-MS m/z [M+H]+: 423.2
N-[2-(4-fluorophenyl)ethyl]-6,7-dihydroxy-1-(propan-2-yl)-1,2,3,4-
1
tetrahydroisoquinoline-2-carbothioamide (36). H NMR (400 MHz, CD3OD) δ 7.22 – 7.16 (m,
2H), 6.98 – 6.92 (m, 2H), 6.58 (s, 2H), 3.84 – 3.77 (m, 2H), 3.61 (quintet, J = 12.7, 6.3 Hz, 1H),
2.91 (dt, J = 8.0, 2.4 Hz, 2H), 2.82 – 2.73 (m, 1H), 2.06 – 2.00 (m, 1H), 1.01 – 0.92 (m, 6H).
ESI-MS m/z [M+H]+: 389.1
N-benzyl-6,7-dihydroxy-1-(propan-2-yl)-1,2,3,4-tetrahydroisoquinoline-2-
1
carbothioamide (37). H NMR (400 MHz, CD3OD) δ 7.33 – 7.09 (m, 5H), 6.60 (d, J = 2.5 Hz,
2H), 4.92 (d, J = 15.6 Hz, 2H), 3.72 (dt, J = 12.6, 6.3 Hz, 1H), 2.98 – 2.78 (m, 2H), 2.12 – 2.01
(m, 1H), 1.03 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.6 Hz, 3H). ESI-MS m/z [M+H]+:357.2
Physiochemical Property Calculations
Data were archived and analyzed using the CDD Vault from Collaborative Drug
Discovery (Burlingame, CA. https://www.collaborativedrug.com). Within CDD Vault,
ChemAxon's JChem Base was used for structure searching, chemical database access and

management, and chemical property calculations. JChem v17.7.0, 2017, ChemAxon
(http://www.chemaxon.com)
Reagents
Working stocks of CPZ, 17, and 29 were made fresh prior to each experiment by diluting
each compound in 100% EtOH to a final stock concentration of 100 mM. If needed, stocks were
stored at -20 ºC for periods no longer than 48 h.
Cell Lines
HeLa (cervical cancer), MDA-231 (breast cancer), H460 (NSCLC), and PC-3 (prostate
cancer) cell lines were obtained from American Type Culture Collection (Manassas, VA, USA).
HSC-3 cells were kindly provided by Dr. Brian Schmidt at New York University College of
Dentistry. Cell lines were authenticated prior to use by Genetica DNA Laboratories (Burlington,
NC). Cells were maintained in DMEM culture medium with high glucose (Gibco, Carlsbad,
CA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and
maintained at 37°C in 5% CO2. Fore calcium imaging studies we obtained CHO cells
overexpressing TRPV1 (CHO-TRPV1), kindly provided by Dr. Ardem Patapoutian at the
22
Scripps Research Institute and cultured as previously described at 37°C in 5% CO2.
MTS Cell Viability Assays
The Cell Titer 96 ® Aqueous Non-Radioactive Cell Proliferation Assay (Promega,
Madison, WI) was used to assess cell viability in response to treatments according to
manufacturer’s protocol. Cells were plated and treated for 24 h with serum free DMEM
1
containing CPZ analogs at the indicated concentrations as previously described . Final EtOH
concentrations were maintained at 0.1% or less. Absorbance values of test groups were
compared to vehicle-treated controls (n=4).

Mouse Efficacy Studies
All studies were approved by the UT Health Institutional Animal Care and Use
Committee and follow the ARRIVE guidelines. Six week-old female athymic nude mice
(Envigo Laboratories, Indianapolis, IN) were used in a laminar air-flow cabinet under pathogen-
free conditions. Mice were provided with a 12 h light/dark schedule at controlled temperature
and humidity with food and water ad libitum and acclimated for one week prior to study
initiation.
Mice were injected subcutaneously in the right flank with 6 × 106 HeLa cells in 0.1 ml of
1
sterile PBS as previously described. Two weeks post-inoculation, tumors grew to an average
volume of 170 mm3 and mice were stratified into three experimental groups (n=4 per group)
which received the following treatments via intra-tumor injection: vehicle control (100 μl of 5%
EtOH diluted in sterile saline) or 40 μg of 17 and 29 diluted in 100 μl sterile saline (final
concentration of 5% EtOH). Treatments were repeated every day for a period of 14 days. Mice
were monitored daily for tumor growth (using digital calipers), cachexia, and weight loss.
Tumor volumes were calculated by the elliptical formula: 1/2(length x width2). 1, 23
Calcium Imaging
Calcium imaging was performed using FLIPR Calcium 6 Evaluation Kit (Molecular
Devices, LLC, Sunnyvale, CA, USA) according to the manufacturer's protocol and as previously
described.1 CHO-TRPV1 cells (9x103) were plated in a 384 well plate, loaded with Calcium 6
dye for 2 h and the experiment run on Pherastar FS multimode plate reader (BMG Labtech,
Cary, NC, USA). Dose response curves were performed followed by analysis of 1 μM of
compounds 17 and 29 compared to 100 nM capsaicin (positive control) and 1μM CPZ (negative
control; n=3 per group). Effects on calcium influx were measured by changes in fluorescent

intensity (535 nm). In addition, cells were pre-treated with 1 μM CPZ, 17, or 29 followed by
100nM capsaicin to determine potential TRPV1 inhibitory effects of CPZ analogs.
Statistical Analyses
Statistical analyses were performed using GraphPad Prism4 (San Diego, CA, USA).
Calcium imaging results were analyzed by one-way ANOVA and Dunnett’s multiple comparison
post-tests. Tumor growth was analyzed by two-way ANOVA with repeated measures and
Bonferroni’s post-hoc tests. A p value less than 0.05 was considered statistically significant.
Key Words: Capsazepine, TRPV1, Structure-Activity Relationships, Solid Tumors, Cancer
Therapy

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