Reaction between harmaline and vanillin to produce dimeric scaffolds that exhibit anti-proliferative activity

Article abstract of DOI:10.1016/j.tetlet.2021.153139

Herbal medicine is used as a complement to modern medicine for the treatment of human diseases such as cancer, inflammation, and diabetes. Nutraceutical components in foods such as vanillin produce antioxidant and anticancer activities and many of these p

Full text of DOI:10.1016/j.tetlet.2021.153139

Tetrahedron Letters 73 (2021) 153139
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Reaction between harmaline and vanillin to produce dimeric scaffolds
that exhibit anti-proliferative activity
Vishal C. Birar a, Gene Zaid b, Brian S.J. Blagg a,
⇑
a Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA
b Genzada Pharmaceuticals, LLC, 205 S. Broadway, Sterling, KS 67579, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herbal medicine is used as a complement to modern medicine for the treatment of human diseases such
as cancer, inflammation, and diabetes. Nutraceutical components in foods such as vanillin produce
antioxidant and anticancer activities and many of these produce minimal adverse effects in humans.
Therefore, strategies that combine both herbal medicine and nutraceutical components could produce
compounds that exhibit reduced toxicity. Recently, we developed GZ16.007, which is a combination of
harmaline and curcumin that is currently undergoing clinical evaluation for the treatment of cancer. In
contrast to the utilization of curcumin, we report herein the synthesis of a novel scaffold that utilizes har-
maline and vanillin as nutraceutical components to form a newly identified anti-cancer scaffold. It was
determined that the inclusion of two molecules of harmaline and a single equivalent of vanillin produced
a dimeric product that was active against various human cancer cell lines. The synthesis, evaluation and
preliminary SAR studies for the dimeric scaffold is discussed herein.
Received 28 January 2021
Revised 22 April 2021
Accepted 27 April 2021
Available online 1 May 2021
This article is dedicated to Professor Dale
Boger for his contributions to the fields of
organic and medicinal chemistry along with
his encouragement for the next generation
of scientists.
Keywords:
Harmaline
Vanillin
Ó 2021 Published by Elsevier Ltd.
b-Carboline dimer
Cytotoxic
GZ16.007
Traditional medicine is widely accepted as a complementary
approach to modern medicine for the treatment of human disease
[1]. According to the World Health Organization (WHO), a large
portion of the population (80%) in developing countries is depen-
dent upon herbal plants as a source of medicine [2]. Various case
studies have examined the use of herbal therapies to treat cancer
and suggest that ~40% of the population could benefit from herbal
therapy as a complement to modern anticancer drugs [1]. It has
also been reported that the use of herbal plant extracts containing
a nutraceutical mixture can produce potent anti-cancer activity [3].
For example, Arum palaestinum (Black Calla lily) and Peganum har-
mala are plants that are well known for their anti-cancer proper-
ties. Arum palaestinum and Peganum harmala are particularly
interesting because the ethanolic extract from these plants is
widely used for the treatment of cancer [4]. In fact, suspensions
that contain the plant extract from Arum palaestinum or Peganum
harmala and a nutraceutical component (such as curcumin or iso-
vanillin) were evaluated for their activity against cancer cell lines
[5]. The suspension, GZ17S, contained the combined plant extracts
from Arum palaestinum, Peganum harmala and Curcuma longa;
whereas GZ17-05.00 contains synthetic components such as cur-
cumin, harmine and isovanillin. Evaluation of these suspensions
against cancer cell lines as well as in animal studies revealed the
selective inhibition of cancer growth without observable toxicity.
GZ17-6.02, the most effective suspension, was investigated for
prostate cancer in mice at doses of 0.015 to 0.060 g/kg and showed
promising results, which eventually led to Phase-I clinical
investigation.
A study was pursued to identify the active components from the
Black lily plant extract suspension, which identified harmaline (1),
vanillin (2), and linolenic acid as major contributors [5]. Subse-
quent studies with the purified components revealed an unex-
pected reaction that resulted in formation of the dimeric scaffold
(4) as shown in Scheme 1. Consequently, the aim of the present
study was to develop analogs based on this chemistry and to estab-
lish preliminary structure-activity relationships (SAR) for this
dimeric scaffold.
A condensation reaction between harmaline and vanillin in
methanol resulted in formation of dimeric compounds that feature
two harmaline moieties and one vanillin. Presumably, the methyl
imine of harmaline (tricyclicpyrido[3,4-b] in-dole ring) reacts
favorably via ene-amine chemistry with the carbonyl of vanillin
to form these novel harmaline analogs (Scheme 1). Vanillin (2)
⇑
Corresponding author.
https://doi.org/10.1016/j.tetlet.2021.153139
0040-4039/Ó 2021 Published by Elsevier Ltd.

V.C. Birar, G. Zaid and Brian S.J. Blagg
Tetrahedron Letters 73 (2021) 153139
Scheme 1. Synthesis of dimeric harmaline analogs and proposed modifications.
and benzaldehyde (3) were selected to react with harmaline in an
effort to determine the role played by substituents on the aldehyde
linker. Vanillin was chosen because it exhibits well known antiox-
idant and anti-proliferative activities [6]. Initially, our hypothesis
was that the methoxy and hydroxyl substituents on vanillin might
be critical for anti-proliferative activity, and therefore we prepared
the unsubstituted harmaline dimer containing benzaldehyde as a
linker. We also synthesized dimeric analogs to determine the role
played by the linker and to optimize the tether length between the
two harmaline moieties. The harmaline ring system (1) was also
investigated to determine the requisite substituents for the quino-
line and iso-quinoline ring systems and to evaluate the role played
by each. In total, eleven new analogs were prepared, evaluated, and
initial SAR determined for this scaffold. Formation of these prod-
ucts required a condensation reaction between two harmaline
molecules and an aldehyde in methanol to form the desired prod-
ucts. Nucleophilic addition of the harmaline enamine to the alde-
hyde resulted in formation of hydroxyl intermediate (I), which
underwent subsequent nucleophilic attack by the enamine of a
second harmaline to give the dimeric compounds (Scheme 2).
Although other solvents were explored, methanol was found to
provide the best yield for this reaction.
The same procedure was also utilized to synthesize dimeric
analogs of harmaline with vanillin and benzaldehyde (Scheme 3,
analogs 4 and 5). The next objective was to determine the effect
of an aliphatic linker and to determine the optimal linker length
based on the resulting biological activity. Previously identified con-
ditions were not suitable for the reaction between dialdehydes
(gloxal, malondialdehyde bis(dimethyl acetal) or succinaldehyde
bis(dimethyl acetal)) and harmaline. Therefore, the synthetic route
was modified for the utilization of carboxylic acid linkers wherein
two, three or four methylene units were included (Scheme 3).
Tryptamine 18 was coupled with the corresponding diacid using
an amide coupling reaction to give the diamide products 19a-e,
which were subjected to a Bischler–Napieralski reaction to give
dimeric analogs 6, 7 and 8, which contain three and four methylene
units, respectively. Unfortunately, the cyclization process was not
successful for linkers bearing none, one or two methylene units,
which is consistent with earlier reports and reflects the strain
introduced during cyclization [7]. The in vitro cytotoxicity of com-
pounds 4–10, 12, and 15–17 against various human cancer cells
was evaluated using an MTT assay. Three pancreatic cancer cell
lines (MIA PaCa-2, ASPC-1, BxPC-3), two endometrial cancer cell
lines (AN3CA, HEC-1a), three triple negative breast cancer cell lines
(MDA-MB-231, MDA-MB-468, HCC70), two lung cancer cell lines
(H1975, H1650 both EGFR mut), two ovarian cancer cell lines
(A2780, A2780CP), a renal cancer cell line (A498), two cervical,
H&N cancer cell lines (SiHA, FaDu), two B-cell lymphoma cancer
cell lines (DoHH-2, OCI-LY3), and six myeloma cancer cell lines
(JIM1, KMM-1, KMS-34, RPMI-8226, L363 and MOLP-8) were used.
The IC50 values (effective concentration inhibiting 50% of tumor
cell proliferation) are listed in Table 1.
We evaluated the activity of 4–10, 12, and 15–17 against various
human cancer cells and all of them demonstrated growth inhibi-
tion. Compounds 4 and 5 displayed cancer cell growth inhibition
at 4–7 mM, which is more potent than harmaline and vanillin (25
and 40 mM for harmaline and vanillin, respectively). The data also
revealed the hydroxyl and methoxy groups present in 4 and 9 are
not required for activity, which was confirmed via compounds 5
and 10, which lack these moieties but continue to inhibit cancer cell
growth at similar concentrations (IC50 2–4 mM). In addition, reduc-
tion of the imine in analogs 4 and 5 to the corresponding secondary
Scheme 2. Formation of dimeric harmaline analogs and the ORTEP diagram of 4.
2

V.C. Birar, G. Zaid and Brian S.J. Blagg
Tetrahedron Letters 73 (2021) 153139
Scheme 3. Synthesis of dimeric harmaline analogs. Reagent and conditions: a) 2-vanillin R1 = OH, R2 = OMe, R3 = H, MeOH, reflux, 65%; b) 4-benzaldehyde, R1-3 = H, MeOH,
reflux, 70%; c) NaBH4, MeOH/CH2Cl2, RT; d) toluene, p-TSA, reflux; e) MeOH, reflux; f) heating 190 °C; g) POCl3 (4–6 eq.) toluene reflux.
Table 1
The IC50 values (in
lM) of harmaline analogs against 23 human cancer cell lines. Growth inhibition effect on cancer cells were determined by MTT assay and represented by
average IC50 value. nd = not determined. IC50 values for analogs 12, 15–17 were more 30 mM and not shown here.
Cancer
Cell line
4
5
6
7
8
9
10
Pancreatic
MIA Paca-2
ASPC-1
BxPC-3
AN3CA
HEC-1a
MDA-MB-231
MDA-MB-468
HCC70
H1975 (EGFR mut)
H1650 (EGFR mut)
A2780
A2780CP
A498
SiHA
2.8
6.7
2.1
2.5
5
2.5
2.7
2.3
6.1
2.7
5
3.7
3.5
4.8
3.8
1.8
4.5
3.1
3.4
4.7
3.1
2.6
3.3
1.8
2.0
nd
nd
nd
nd
nd
nd
nd
nd
2.9
nd
nd
nd
1.5
1.9
nd
2.2
1.9
nd
nd
1.4
nd
4.5
7.9
3.2
3.0
7.0
3.1
2.6
5.1
5.7
4.9
2.5
4.1
5.3
12.6
2.3
2.4
8.8
6.1
5.1
4.4
1.9
2.4
1.8
1.2
2.0
0.9
1.9
2.2
1.5
1.9
1.3
2.0
1.7
1.3
2.2
1.7
2.8
0.9
2.2
2.5
2.5
1.4
1.7
0.8
1.2
0.6
6.5
2.5
4.6
nd
nd
nd
nd
nd
nd
nd
nd
6.1
nd
nd
nd
2.6
2.2
nd
2.2
2.5
nd
nd
2.1
nd
2.2
2.4
nd
nd
nd
nd
nd
nd
nd
nd
3.4
nd
nd
nd
2.2
2.3
nd
2.4
2.0
nd
nd
1.9
nd
>20
>10
>20
>20
>10
7.0
>10
>10
>10
>10
>10
>10
>10
6.8
6.4
6.9
>20
>10
>10
5.5
Uterus/endometrium
Breast
Lung
Ovarian
Kidney
Throat
FaDu
Lymphoma
Myeloma
DoHH-2
OCI-LY3
JIM1
KMM-1
KMS-34
RPMI-8226
L363
MOLP-8
8.3
3.6
amines (9 and 10) did not significantly affect the IC50 values. Four
quinoline and isoquinoline-based dimeric analogs were prepared
to investigate the tricyclicpyrido[3,4-b] indole ring system present
in these analogs (12 and 15–17). Interestingly, analogs 12 and 15–
17 were not efficacious when compared to 4–5 and 9–10 as evi-
denced by increased IC50 values, indicating the tricyclicpyrido
[3,4-b]indole ring system is necessary for anti-proliferative activity
(12, 15–17 IC50 values not shown in Table 1 were >30 mM).
A prior report suggested that the b-carboline subunit is more
potent as a dimeric analog than its monomeric counterpart [8].
3

V.C. Birar, G. Zaid and Brian S.J. Blagg
Tetrahedron Letters 73 (2021) 153139
Therefore, dimeric analogs that linked two harmaline molecules
with an aliphatic linker containing three or four carbon units at
the 9-position of harmaline were pursued. Our attempt to synthe-
size the corresponding one and two carbon linkers failed, which is
consistent with earlier synthetic reports [5]. However, replacement
of the phenyl group of 4 and 5 with an aliphatic linker (n = 3) was
successful and the resulting compounds manifested IC50 values of
0.6–2 mM, which reflects a 3–6-fold increase in anti-proliferative
activity. When a carbon linker of n = 4 (8) was evaluated, IC50 values
between 10 and 15 mM were obtained, indicating a loss of activity.
Interestingly, when the linker contained three methylene units
and the gem-dimethyl group (7), it was among the most potent com-
pounds identified and manifested IC50 values between 0.6 and 2 mM.
In conclusion, we report the synthesis and evaluation of dimeric
harmaline analogs as a potent inhibitory scaffold against various
cancer cell lines. Most of the new compounds exhibit improved
anti-cancer activity compared to harmaline or vanillin. The dimeric
harmaline analogs 6 and 7, were found to be the most potent com-
pounds and exhibited IC50 values between 0.6 and 2 mM against
various human cancer cell lines. SAR analysis revealed the dimeric
harmaline analogs containing three methylene units were most
potent, providing evidence for spatial limitations within the cog-
nate binding partner. Studies are now underway to optimize this
class of compounds and to determine their mechanism of action.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2021.153139.
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