Design and Synthesis of New 1,3,5-Trisubstituted Triazines for the Treatment of Cancer and Inflammation

Article abstract of DOI:10.1002/open.201800136

Low-molecular-weight synthetic molecules 1 with the general 2-(fluorophenylamino)-4,6-disubstituted 1,3,5-triazine structure and showing anti-inflammatory and anticancer activities were explored. Structure–activity relationship studies demonstrated the importance of the aminopentyl chain, the 3- or 4-fluorophenylaniline component, and the presence of at least one substituent, such as a tyramine moiety, attached directly to the triazine ring as essential for good activity. These compounds, represented by leads 4-{2-[4-(5-Aminopentylamino)-6-(3-fluorophenylamino)-1,3,5-triazin-2-ylamino]ethyl}phenol (6) and 4-{2-[4-(5-Aminopentylamino)-6-(4-fluorophenylamino)-1,3,5-triazin-2-ylamino]ethyl}phenol (10), displayed moderate and significant in vitro and in vivo dual activities, respectively, and address the molecular link between inflammation and cancer. Compound 10 demonstrated significant antitumor efficacy upon administration by the oral and intravenous routes in several animal models. This class of triazine compounds is new, safe, and nontoxic and offers a novel approach to the treatment of inflammation and cancer.

Full text of DOI:10.1002/open.201800136

DOI: 10.1002/open.201800136
Design and Synthesis of New 1,3,5-Trisubstituted Triazines
for the Treatment of Cancer and Inflammation
Boulos Zacharie,* Shaun D. Abbott, Jean-Simon Duceppe, Lyne Gagnon, Brigitte Grouix,
Lilianne Geerts, Liette Gervais, FranÅois Sarra-Bournet, ValØrie Perron, Nicole Wilb,
Christopher L. Penney, and Pierre Laurin[a]
Low-molecular-weight synthetic molecules 1 with the general
2-(fluorophenylamino)-4,6-disubstituted 1,3,5-triazine structure
and showing anti-inflammatory and anticancer activities were
explored. Structure–activity relationship studies demonstrated
the importance of the aminopentyl chain, the 3- or 4-fluoro-
phenylaniline component, and the presence of at least one
substituent, such as a tyramine moiety, attached directly to the
triazine ring as essential for good activity. These compounds,
represented by leads 4-{2-[4-(5-Aminopentylamino)-6-(3-fluoro-
phenylamino)-1,3,5-triazin-2-ylamino]ethyl}phenol (6) and 4-{2-
[4-(5-Aminopentylamino)-6-(4-fluorophenylamino)-1,3,5-triazin-
2-ylamino]ethyl}phenol (10), displayed moderate and signifi-
cant in vitro and in vivo dual activities, respectively, and ad-
dress the molecular link between inflammation and cancer.
Compound 10 demonstrated significant antitumor efficacy
upon administration by the oral and intravenous routes in sev-
eral animal models. This class of triazine compounds is new,
safe, and nontoxic and offers a novel approach to the treat-
ment of inflammation and cancer.
1. Introduction
Cancer is the second leading cause of death in the USA and
many European countries.[1] Whereas breast cancer is among
the five most common types of cancers, the five-year survival
rate is one of the lowest of all cancers.[2–4] Also, pancreatic
cancer is currently one of the deadliest of the solid malignan-
cies,[5] and surgery remains the only option for cure.[6] Another
example of major cancer is melanoma.[7] A need, therefore,
exists for the discovery of novel compounds that are more ef-
fective but less toxic for the treatment of these cancers.
inflammation. These compounds, which simultaneously exhibit
significant anticancer and anti-inflammatory properties, offer a
potential two-pronged approach that targets both genetically
unstable tumor cells (high mutation rate and subsequent re-
sistance to chemotherapy) and genetically normal cells present
in inflamed tissue. This two-pronged approach to the treat-
ment of cancer is made more compelling by the increasing
awareness that a link exists between chronic inflammation and
the subsequent development of cancer.[9] It has been shown
that chronic inflammation is associated with the development
of numerous human cancers.[10] For example, markers of a sys-
temic inflammatory response have prognostic significance in
advanced, inoperable pancreatic cancer,[11] and inflammatory
processes have emerged as key mediators of breast cancer de-
velopment and progression.[12] Inflammatory cytokines such as
tumor necrosis factor (TNF-a),[13] IL-1, IL-6,[14] and MCP-1[15] have
been shown to be involved in all stages of the malignant pro-
cess. In a breast,[16] prostate,[17] and melanoma[18] cancer study,
both serum TNF-a and IL-6 correlated with the extent of the
disease. Other inflammatory mediators, such as prostaglandins
and leukotrienes, have also been reported to play a role in the
development and progression of several cancers, including
breast,[19] melanoma,[20] and pancreatic[21] cancers.
It has long been recognized that inflammation is related to
cancer, and strong correlation between the presence of inflam-
mation and the development of precancerous lesions has
been established.[8] One novel approach to the treatment of
cancer lies in the discovery of new compounds with a new
mechanism of action that are efficacious in reducing tumor
size and/or the spread of metastasis and that can also reduce
[a] Dr. B. Zacharie, Dr. S. D. Abbott, J.-S. Duceppe, Dr. L. Gagnon, Dr. B. Grouix,
L. Geerts, L. Gervais, F. Sarra-Bournet, V. Perron, Dr. N. Wilb, Dr. C. L. Penney,
P. Laurin
Prometic Biosciences Inc.
500 boul. Cartier Ouest, Bureau 150
Laval, QuØbec, H7V 5B7 (Canada)
E-mail: b.zacharie@prometic.com
1,3,5-Triazine compounds have been studied extensively and
are the subject of many reviews.[22] The triazine scaffold has
been exploited for the design of biologically relevant mole-
cules with broad biomedical value as therapeutics.[23] For ex-
ample, these compounds possess potent antiprotozoa,[24] anti-
viral,[25] fungicidal,[22] insecticidal, bactericidal,[26] herbicidal,[27]
antimicrobial,[28] and antimalarial[29] activities. In addition, tri-
substituted triazines have been shown to display protein A
The ORCID identification number(s) for the author(s) of this article can
be found under:
https://doi.org/10.1002/open.201800136.
ꢀ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
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mimetic properties for the treatment of autoimmune diseas-
es.[30] As part of our autoimmune diseases program, small mol-
ecules were examined as potential anticancer agents. A recent
review describes the versatile bioactivities of 1,3,5-triazine de-
rivatives as potential antitumor compounds.[31] The reasons for
selecting the 1,3,5-triazine scaffold were threefold: 1) 1,3,5-tria-
zines are monocyclic, symmetrical molecules and have been
utilized as promising scaffolds for their versatile biological po-
tential;[32] 2) the presence of three nitrogen atoms in the 1,3,5-
triazine core can inherently impart polarity to the whole mole-
cule, and the partition coefficient (clog P) values of the de-
signed compounds can be about 2 or lower; 3) trifunctional-
ized 1,3,5-triazines are ideal modular scaffolds for generating li-
braries. These molecules may demonstrate remarkable pharma-
cokinetics and oral bioavailability with additional modifications.
In this paper, the synthesis and anticancer and anti-inflamma-
tory activities of trisubstituted triazines, as exemplified by the
general structure 1, are reported.
Scheme 1. For intermediates 3–5, NHR1 =4-aminobutylamino or 5-amino-
pentylamino; NHR2 =substituted anilino or phenethylamino. Reagents and
conditions: a) fluoroaniline, acetone/water, À108C to RT; b) 4-(tert-butoxycar-
bonylamino)butylamine or 5-(tert-butoxycarbonylamino)pentylamine,
NaHCO3/H2O/THF/acetone, RT; c) R2NH2 (substituted anilino or phenethylami-
no); NaHCO3, acetone/H2O; d) 4-aminobutylamine or 5-aminopentylamine,
THF/MeOH, 1308C, 10 min, microwave; e) 4-R-phenylene-(CH2)nNH2, Et3N,
THF, 658C; f) removal of the protecting group (if applicable).
2. Results and Discussion
tion of dichlorotriazine intermediate 2 as in route 1, followed
first by the reaction with aryl- and aralkylamines to give 4 and
then by the addition of aminoalkylamines at 1308C for 10 min
by using microwave irradiation to afford compound 5. The last
step was the removal of the protecting groups.
As part of an effort towards the discovery of novel therapeu-
tics for the treatment of autoimmune diseases, a search was in-
itiated to discover small molecules that possess anti-inflamma-
tory activity. It was reported that fluorophenylaminotriazine de-
rivatives possess anti-inflammatory activity (kinase inhibitors,[33]
EGFR inhibitors,[34] and glycosidase modulators[35]). Considering
that we synthesized and tested in-house many fluorophenyla-
mino-4,6-disubstituted 1,3,5-triazines[28] as protein A mimetics
and as antimicrobial agents, a number of these compounds
were selected and screened on the basis of general structure
1.
These compounds were evaluated for their anti-inflammato-
ry activity by using inhibition of TNF-a production by lipopoly-
saccharide (LPS)-stimulated J774A.1 cells (murine macrophag-
es). A total of 15 compounds were chosen upon which to
define a structure–activity relationship for a 2-(3- or 4-fluoro-
phenylamino)-4,6-disubstituted 1,3,5-triazine scaffold. In gener-
al, the analogues varied in their aminoalkyl chains [(CH2)mNH2,
m=4 or 5] and tyramine units [4-HOC6H4(CH2)nNH,
The starting material for these compounds was cyanuric
chloride. This is an inexpensive commercially available reagent,
which makes its use attractive. The ease of displacement of
the chlorine atoms in cyanuric chloride by various nucleophiles
enhances the utility of this reagent for the preparation of
mono-, di-, and trisubstituted 1,3,5-triazines at controlled tem-
peratures.[36] The general synthetic sequence for the prepara-
tion of triazine compounds is outlined in Scheme 1. It illus-
trates the route employed for the preparation of trisubstituted
triazine 5, for which the key intermediate was dichlorotriazine
2. Compound 5 was prepared by two different routes. The first
route involved the reaction of cyanuric chloride with fluoroani-
line at À108C in the presence of sodium bicarbonate to give
dichlorotriazine intermediate 2. This reaction proceeded in ex-
cellent yield (>95%) and was general for different aniline de-
rivatives.[30,37] Aminoalkylamines were then added in the pres-
ence of a base to afford triazine 3 in high yield (90%). This
compound was then treated with different aryl- and aralkyla-
mines to give products 5. Route 2 demonstrates the prepara-
4-H2NC6H4(CH2)nNH,
4-H2NCOC6H4(CH2)nNH,
4-
H2NO2SC6H4(CH2)nNH, 4-(CH3)2HNO2SC6H4(CH2)nNH, n=0–2],
and the third group was held constant as a 3- or 4-fluorophe-
nylamine group. Table 1 illustrates the variation of these sub-
stituents and demonstrates in vitro the effect of analogues of
1 on tumor necrosis factor (TNF-a) production, as measured by
ELISA (enzyme-linked immunosorbent assay) by using J774A.1
cells stimulated by LPS. J774A.1 cells were cultured in the pres-
ence or absence of LPS and the compound. The cells were pre-
treated with the compounds 1 h prior to LPS stimulation. The
supernatants were collected to determine the concentration of
TNF-a by ELISA. The data were analyzed, and the concentra-
tion of compound that inhibited 50% of TNF-a production
(IC50) was calculated. Prior work indicated the importance of
the length of the carbon chain (n-pentyl) between the amine
groups. In fact, a decrease in the length of the chain by three
carbon atoms (as in compound 12) or by one carbon atom (as
in compounds 17 and 23) reduced the activity. However, ho-
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Table 1. Effect of compounds on the inhibition of TNF-a released by LPS
Table 1. (Continued)
induction from J774A.1 cells.
Compound Structure
TNF-a inhibi-
tion
Compound Structure
TNF-a inhibi-
tion
IC50 [mm]
IC50 [mm]
20
21
>90
>90
6
7
29
50
8
>90
>90
13
22
>90
9
23
24
>90
10
11
32[a]
33
25
26
26[a]
12
13
14
>90
>90
37
>90
27
29[a]
15
16
17
>90
>90
>90
[a] Compound showed cytotoxicity at this concentration.
mologation of the alkyl chain with a one-carbon spacer chain
length (as in compound 24) resulted in some activity but also
some toxicity. Similarly, rigidifying the six-carbon chain and
keeping the terminal amine function (as in compound 25)
gave some toxicity. However, introducing a heteroatom in the
chain (as in compound 21) or increasing the rigidity of the
chain by forming a ring with the primary amine function (as in
compound 22) led to a drop in activity. It was observed that
inhibition of TNF-a was obtained with triazine compounds
containing a tyramine moiety, for example, compounds 6, 10,
and 11. However, substitution of the alkyl chain of the tyra-
mine unit with a hydroxy group, as in compound 18, or a car-
boxylate group, as in compound 19, resulted in a loss of activi-
ty. Changing the tyramine moiety to an arylsulfonamide unit,
18
19
>90
40
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as in 16, or a benzylamine, as in 20, also resulted in a loss of
activity. Similarly, compounds 13, 9, and 8, in which the hy-
droxy group of the tyramine unit was replaced with an amide
group, an amine, and a N,N-dimethylsulfonamide, displayed
lower activity. In contrast, corresponding carboxylic ester 14
and sulfonamides 7 and 15 showed weaker activity than com-
pounds 6 and 10. Also, the correct acidity of the phenol group
in the tyramine unit was important for the activity of the tria-
zine compounds. For example, adding an ortho electron-donat-
ing substituent such as a methoxy group to give compound
26 made the phenol less acidic and not active. In contrast,
adding an ortho electron-withdrawing substituent such as a
chlorine atom to give compound 27 resulted in some activity
but also some toxicity. In general, compound 10 in which the
triazine ring is substituted with a 4-fluoroaniline, tyramine, and
a pentylamine chain resulted in good activity relative to that
observed for corresponding 3-fluoroanilines 6 and 11.
On the basis of the above results, three triazine compounds
demonstrated inhibition of TNF-a production. Compounds 6
and 7 showed moderate inhibition, whereas triazine 10 gave
good TNF-a inhibition. To confirm the anti-inflammatory activi-
ty of triazine derivative 10, we studied the in vitro effect of this
compound on the production of prostaglandin E2 (PGE2) in
LPS-stimulated J774A.1 cells. The results are shown in Figure 1.
Figure 2. Effect of the intravenous administration of compound 7 or 10 on
TNF-a production induced by LPS (2 h) in an air-pouch rat model.
LPS. As expected and on the basis of the above results, com-
pound 7 had some effect on the concentration of TNF-a in the
exudates 2 h post-LPS induction. Also, similar effects were ob-
tained by intravenous administration of compounds 7 and 10
on TNF-a production induced by LPS (12 h) in the air-pouch
rat model (Figure 3).
Figure 3. Effect of the intravenous administration of compound 7 or 10 on
TNF-a production induced by LPS (12 h) in an air-pouch rat model. **:
p<0.01, ***: p<0.001: ns: not significant.
Figure 1. Effect of compound 10 on the inhibition of PGE2 released by LPS
Another experiment was performed to confirm the anti-in-
flammatory activity of compound 10 compared to that of com-
pound 7. Figure 4 represents the effect of the intravenous ad-
ministration of compound 7 or 10 on PGE2 production in-
duced by LPS (12 h after induction) in an air-pouch rat model.
Compound 10 and indomethacin significantly inhibited PGE2
induction from J774A.1 cells.
LPS-stimulated J774A.1 cells produce PGE2, a bioactive lipid as-
sociated with inflammation and cancer. Compound 10 was
demonstrated to inhibit PGE2 production in stimulated
J774A.1 cells with an IC50 of 1.82 mm. It was next demonstrated
that the in vitro activity of 7 and 10 would translate into mod-
erate and significant in vivo activity, respectively, in a relevant
model of inflammation. Therefore, compounds 7 and 10 were
assessed in an LPS-induced inflammation air-pouch model in
rats. This animal model has widely been used to study the
anti-inflammatory activity of test compounds in efficacy stud-
ies. Upon injection with LPS, the air-pouch produces an inflam-
matory response characterized by the infiltration of inflamma-
tory cells and the production of inflammation factors, such as
TNF-a and PGE2.
Figure 2 represents the effect of the intravenous administra-
tion of the two compounds on TNF-a production induced by
LPS (2 h after induction) in the air-pouch rat model. Com-
pound 10 significantly inhibited TNF-a production induced by
Figure 4. Effect of the intravenous administration of compound 7 and 10 on
PGE2 production induced by LPS (12 h) in an air-pouch rat model. *:
p<0.05, **: p<0.01: ****: p<0.0001.
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production induced by LPS. However, compound 7 demon-
strated a weak and nonsignificant effect on PGE2 production.
The next step was to examine the anticancer activity of the
two best compounds, 6 and the lead 10, in in vitro and in vivo
models. Cell proliferation, enhanced cell motility, cell adhesion,
proteolytic degradation of the extracellular matrix, and cell mi-
gration are inter-related processes that are responsible for the
invasion and metastasis of cancer. In prostate cancer, androgen
independence and bone metastasis are lethal complications in
patients. The first in vitro experiment was to evaluate the
effect of compound 10 on PC-3 prostate cancer cell line prolif-
3
eration by using DNA synthesis by H-thymidine incorporation
and the cell cycle. This compound reduced PC-3 cell prolifera-
tion with an IC50 of 43.3 mm at 24 h (Figure 5) and 72 h but ex-
Figure 6. Effect of compounds 6 and 10 on EGF-induced PC-3 cell migration
or invasion (typical experiment of three replicates).
step was to determine if this activity translated into anticanc-
er/anti-inflammatory mouse models. The first experiment was
to study the antitumor effects of compound 10 on primary
tumor P815 cells. This cell line has been used extensively as a
cancer model to establish a relationship between the tumor-in-
duced local, regional, and systemic increase of proinflammato-
ry mediators and progression of tumors in vivo.[38] Figure 7
Figure 5. Cytotoxicity effect of compound 10 on the PC-3 cell line (24 h).
EC50 =median effective concentration.
hibited no effect on the cell cycle below 20 mm. We demon-
strated, from cell-cycle experiments (Figure 5), that there was
cell-cycle arrest in the G0/G1 phase. No increase in the sub G0/
G1 phase was observed, and hence, no apoptosis occurred at
compound concentrations of 40 and 80 mm. Therefore, com-
pound 10 at a concentration of 10 mm was used for the re-
maining in vitro experiments. In a second experiment, it was
desired to demonstrate the effect of the compounds on epi-
dermal growth factor (EGF)-induced migration of the PC-3
prostate cancer cell line in an in vitro scratch wound healing
assay. A migration assay was used to assess cell mobility in
two dimensions. Confluent cells were quiesced by starvation
and mitomycin C treatment to prevent the confounding issue
of cell proliferation. Cells were incubated in the presence or
absence of EGF and 6 or 10 for 24 h. Photographs were taken
at 24 h. Figure 6 represents the effects of EGF and compounds
6 and 10 on PC-3 cell migration or invasion. EGF promotes the
migration or invasion of PC-3 cells treated with mitomycin
compared to a control (without growth factor). The addition of
different concentrations of 10 to the cell culture resulted in in-
hibition of the EGF-induced PC-3 migration or invasion
(Figure 6). However, as expected, compound 6 performed less
well than compound 10 (Figure 6). The addition of different
concentrations of compound 6 to the cell culture produced
partial inhibition of the EGF-induced PC-3 migration or inva-
sion after culture for 24 h.
Figure 7. Antitumor efficacy of compound 10 and acetylsalicylic acid on
P815 primary tumor.
shows the effect of the oral administration (PO) of triazine de-
rivative 10 (50 mgkgÀ1) and acetylsalicylic acid (positive con-
trol, 50 mgkgÀ1) on primary tumor P815 cells. Compound 10
induced a significant reduction T/C (mean relative tumor
volume of treated group/mean relative tumor volume of con-
trol group) between 40 and 50% of tumor growth. Further-
more, the efficacy of this compound was comparable to that
of the gold standard, soluble acetylsalicylic acid (lysine salt).
To validate this observation, another in vivo experiment was
performed to demonstrate the effect of the oral administration
of compound 10 (25 mgkgÀ1) on primary melanoma tumor
B16F10 cells. Triazine 10 induced a significant reduction (T/C<
40%, p=0.001) in the tumor volume compared to the control,
Cytoxan (cyclophosphamide, 100 mgkgÀ1) (Figure 8).
Compound 10 showed in vitro activity on PC-3 prostate
cancer, which is a model that involves inflammation. The next
Also, additional experiments were undertaken with other
types of cancer. Figure 9 shows the antitumor efficacy of com-
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Figure 8. Antitumor efficacy of compound 10 (25 mgkgÀ1) and Cytoxan (cy-
clophosphamide, 100 mgkgÀ1) on B16F10 primary tumor.
Figure 10. Antitumor effects of compound 10 (IV) on a primary DA-3 breast
cancer.
Figure 9. Antitumor activity of compound 10 on pancreatic cancer.
Figure 11. Antitumor effects of compound 10 (PO) on a primary DA-3 breast
pound 10 on pancreatic cancer (PAN02 primary tumor), which
is one of the most malignant tumors of the gastrointestinal
tract. To evaluate long-term tumor growth, the study lasted
35 days. The syngeneic tumor PAN02 cells were grown in
RPMI-1640 with 10% fetal bovine serum (FBS). At day 0, 50 mL
of 7.5”105 viable PAN02 cells were intradermally injected to
produce localized tumors in 6-to-8-week-old C57BL/6 mice.
Mice were treated daily with oral administration of vehicle
(negative control) or compound 10 (50 mgkgÀ1) and with in-
traperitoneal injection of gemcitabine (50 mgkgÀ1) at day 6
and day 12. Mice were sacrificed at day 35. Figure 9 shows the
effect of oral administration of compound 10 and gemcitabine
(positive control) on primary tumor PAN02 cells. The effect of
10 was comparable to that of the gold standard, gemcitabine
(T/C between 52 and 77%), which is used for pancreatic
cancer therapy.
cancer.
(100 mgkgÀ1) was undertaken at days 11 and 18 and with in-
travenous administration (5 mgkgÀ1
)
or oral treatment
(50 mgkgÀ1) of compound 10 at days 11, 12, 13, 15, 18, and
20. Tumors were palpable 7 to 10 days postinoculation. In
both types of administration, compound 10 induced significant
(p<0.05) inhibition of tumor volume with T/C between 35 and
57%, which is comparable to cyclophosphamide, which indu-
ces significant (p<0.04) inhibition of tumor volume with T/C
between 33 and 59%.
We then sought to demonstrate if compound 10 could in-
hibit other types of cancer, especially those that spread (meta-
stasize) to other parts of the body. Figure 12 shows the antitu-
mor efficacy of the oral administration of compound 10
(50 mgkgÀ1) or cyclophosphamide [100 mgkgÀ1, intravenous
(IV) administration] on xenograft human prostate PC-3 tumor.
Compound 10 significantly inhibited (p<0.05) tumor volume
with T/C between 14 and 40%. Cyclophosphamide induced
significant inhibition (p<0.05) of tumor volume with T/C be-
tween 1 and 39%. Injection of Cytoxan was undertaken at
days 29 and 36.
The fact that compound 10 showed similar anticancer and
anti-inflammatory activities suggests that there is a direct rela-
tionship between the two diseases. Indeed, it was reported
that a few small molecules[39] and several natural products[40]
exhibited a strong correlation between anti-inflammatory and
anticancer activities.
An additional in vivo experiment was performed by testing
the activity of compound 10 (good anti-inflammatory activity)
on a metastatic tumor model. Mice were treated with intrave-
nous administration (5 mgkgÀ1, Figure 10) or by oral adminis-
tration (50 mgkgÀ1, Figure 11) of compound 10 with intraperi-
toneal injection (IP) of Cytoxan (cyclophosphamide) on primary
DA-3 breast tumor. IP injection of cyclophosphamide
The mechanism by which most-active compound 10 demon-
strates anti-inflammatory and anticancer properties in vivo is
under investigation. To address this issue, different experi-
ments were undertaken to identify the target. Compound 10
inhibits TNF-a synthesis and activity without affecting TNF-a
binding to its receptors (TNF-RI and TNF-RII). It inhibits PGE2
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significant levels, respectively, in anticancer/anti-inflammatory
models. This addresses the molecular link between inflamma-
tion and cancer. Compound 10 demonstrated significant anti-
tumor efficacy in several animal models, such as the breast
DA-3 adenocarcinoma and human xenogeneic prostate PC-3
cancer models. This was confirmed by the ability of compound
10 to inhibit PGE2 in vitro in stimulated J774 cell (murine mac-
rophage) and in vivo (LPS-induced air-pouch assay). Also, com-
pound 10 reduced PC-3 cell proliferation with an IC50 of 20 mm
by inducing cell-cycle arrest at the G0/G1 phase. In an in vitro
model of wound healing, compound 10 inhibited PC-3 cell mi-
gration/invasion. This class of triazine compounds is new,
safe,[42] and nontoxic[43] and offers a novel approach to the
treatment of inflammation and cancer.
Figure 12. Antitumor efficacy of compound 10 and Cytoxan (cyclophospha-
mide) on xenograft human prostate PC-3 tumor.
Experimental Section
production[41] with no effect on Cox-1 and Cox-2 activity. It has
weak in vitro cytotoxicity (IC50 >10 mm) on NHDF (normal
human dermal fibroblasts), HUVEC (human umbilical vein en-
dothelial cells), PC-3, K562, P815, and resting PBML (peripheral
blood mononuclear leukocytes) (Table 2). Even though com-
pound 10 showed some toxicity in vitro on normal cells, the
in vivo air-pouch rat model showed that this compound inhib-
ited both TNF-a and PGE2. These results indicate the effect of
compound 10 on proinflammatory cytokines that can affect
tumor growth. In fact, in all types of cancers studied in vivo,
we observed no regression of tumors but a slow down of
growth, which is an indication that compounds 6, 7, and 10
affect cancer cell proliferation but do not kill the cells directly.
It is most likely scaffold linked/dependent. Furthermore, com-
pound 10 had no effect on PLA2, 5-Lox, and 15-Lox enzymatic
activities or modulation of iNOS expression. On PC-3 prostate
cancer cells, compound 10 (20 mm) induced cell-cycle block in
G1. Compound 10 had no effect on microtubule
polymerization.
General Methods
All HPLC chromatograms and mass spectra were recorded with an
HP 1100 LC-MS Agilent instrument by using a diode array detector.
An analytical C18 column (75”4.6 mm, 5 mm) with a gradient of 1–
40% acetonitrile/water containing 0.01% trifluoroacetic acid (TFA)
in 6 min and a flow of 2 mLminÀ1 (method 1), an analytical C18
column (75”4.6 mm, 5 mm) with a gradient of 15–99% acetoni-
trile/water containing 0.01% TFA in 6 min and a flow of 2 mLminÀ1
(method 2), an analytical C18 column (75”4.6 mm, 5 mm) with a
gradient of 0.1–20% acetonitrile/water containing 0.01% TFA in
5 min and a flow of 1 mLminÀ1 (method 3), or an analytical C18
column (75”4.6 mm, 5 mm) with a gradient of 1–50% acetonitrile/
water containing 0.01% TFA in 5 min and a flow of 1 mLminÀ1
(method 4) was used.
Syntheses
4-{2-[4-(5-Aminopentylamino)-6-(3-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}phenol Hydrochloride Salt (6)
Cyanuric chloride (10.0 g, 54.2 mmol) was added in small portions
to a cooled (À108C) mixture of water (50 mL) and acetone (50 mL).
A
solution of 3-fluoroaniline (5.2 mL, 54.2 mmol) in acetone
Table 2. In vitro cytoxicity of compound 10.
(50 mL) was added slowly over 50 min, maintaining the tempera-
ture of the reaction below À58C. The mixture was then stirred at
ambient temperature for 1 h. The pH of the mixture was adjusted
from 2 to 8 with saturated aqueous sodium bicarbonate (200 mL),
and stirring was continued for another 30 min. The precipitated
solid was collected by filtration, washed with water, and dried in
vacuo. This gave 2,4-dichloro-3-fluorophenylamino-1,3,5-triazine as
Time [h]
IC50 [mm]
NHDF
HUVEC
PC-3
K562
P815
Resting PBML
24
72
78.6
45.8
31.8
51
43.3
26.8
36.0
15.5
23.5
7.5
>10
nd[a]
[a] nd: not determined.
1
a white solid (13.3 g, 94%): H NMR (400 MHz, [D6]DMSO): d=6.97–
7.01 (1H, m), 7.38–7.43 (2H, m), 7.52–7.55 (1H, m), 11.25 ppm (1H,
br); LRMS (ESI): m/z: 259 [M+H]+; HPLC (method 2): tR =4.1 min.
The product was used in the next step without further purification.
This dichlorotriazine derivative (6.4 g, 24.7 mmol) was dissolved in
THF (70 mL) at RT and was treated with a solution of 5-(tert-butoxy-
carbonylamino)pentylamine (7.5 g, 37.0 mmol) in a mixture of ace-
tone (50 mL) and water (50 mL). The resulting solution was then
treated with saturated aqueous sodium bicarbonate (70 mL). The
mixture was stirred at RT for 2.5–3 h. The mixture was then con-
centrated in vacuo, and the residue was diluted with water and ex-
tracted with ethyl acetate. The combined organic extract was
washed with saturated aqueous sodium chloride, 2m aqueous HCl,
saturated sodium chloride, saturated sodium bicarbonate, and sa-
3. Conclusions
Low-molecular-weight synthetic molecules 1 with a general 2-
(fluorophenylamino)-4,6-disubstituted 1,3,5-triazine structure
and showing anti-inflammatory and anticancer activities were
described. Basic structure–activity relationship studies demon-
strated the importance of the 3- or 4-fluorophenylaniline
group attached to the triazine ring, which played a role in the
activity of these compounds. Two lead triazines, 6 and 10, dis-
played both in vitro and in vivo dual activity at moderate and
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turated sodium chloride. The solution was then dried (magnesium
sulfate/charcoal), filtered through Celite diatomaceous earth, and
concentrated in vacuo to 200 mL. This solution was poured, with
stirring, into hexane (1.2 L), and the precipitate was collected by fil-
tration, washed with hexane, and dried in vacuo to yield the mon-
ochloro-1,3,5-triazine derivative as a white solid (6.6 g, 63%):
1H NMR (400 MHz, [D6]DMSO): d=1.23–1.30 (2H, m), 1.31–1.56 (2H,
m), 1.34 (9H, s), 1.44–1.56 (2H, m), 2.85–2.91 (2H, m), 3.20–3.30
(2H, m), 6.70–6.77 (1H, m), 6.79–6.85 (1H, m), 7.25–7.33 (1H, m),
7.38–7.43 (1H, m), 7.67–7.75 & 7.76–7.85 (1H, br), 8.14–8.21 &
8.22–8.30 (1H, br), 10.05–10.11 & 10.15–10.26 ppm (1H, br); LRMS
(ESI): m/z: 425 [M+H]+, 447 [M+H+Na]+; HPLC (method 2): tR =
4.5 min. A solution of the monochlorotriazine (6.6 g, 15.6 mmol) in
THF (300 mL) was treated with tyramine (6.4 g, 46.7 mmol) and
triethylamine (77.7 mmol, 10.9 mL). The mixture was heated at 65–
708C for 16–60 h, cooled to ambient temperature, and concentrat-
ed in vacuo. The residue was extracted with ethyl acetate and fil-
tered. The filtrate was washed with 1m aqueous HCl, saturated
sodium chloride, saturated aqueous sodium bicarbonate, and satu-
rated sodium chloride; dried (magnesium sulfate/charcoal); filtered
through Celite diatomaceous earth; and concentrated in vacuo.
The residue was then dissolved in ether (150 mL), and this solution
was added dropwise to hexane (1.4 L) with vigorous stirring. The
precipitated solid was collected by filtration and dried in vacuo to
yield the tri(amino-substituted) 1,3,5-triazine derivative as an off-
white solid (6.5 g, 80%): 1H NMR (400 MHz, [D6]DMSO): d=1.21–
1.29 (2H, m), 1.32–1.41 (2H, m), 1.34 (9H, s), 1.44–1.54 (2H, m),
2.65–2.71 (2H, m), 2.88 (2H, dt, J=6.5, 6.5 Hz), 3.15–3.27 (2H, m),
3.33–3.42 (2H, m), 6.61–6.70 (1H, m), 6.67 (2H, d, J=8.5 Hz), 6.71–
6.76 (1H, m), 6.84–7.02 (1H, m), 7.01 (2H, d, J=8.5 Hz), 7.16–7.23
(1H, m), 7.39–7.47 (1H, m), 7.87–7.91 (1H, m), 8.92–8.94 & 9.00–
9.06 (1H, 2”br), 9.13 ppm (1H, s); LRMS (ESI): m/z: 526 [M+H]+,
548 [M+H+Na]+; HPLC (method 2): tR =2.9 min. A solution of the
tert-butoxycarbonyl (Boc)-protected compound (6.5 g, 12.4 mmol)
in 4m HCl/1,4-dioxane (100 mL) and water (10 mL) was stirred at
RT for 2 h. The solvents and the excess amount of acid were
evaporated in vacuo, and the trace amounts of water were re-
moved by co-evaporation (2”) with 2-propanol (25 mL). The dried
residue was dissolved in 2-propanol (25 mL), and the solution was
added dropwise to ether (450 mL) with vigorous stirring. The pre-
cipitated solid was collected by filtration, dried in vacuo, and then
dissolved in pyrogen-free water (800 mL), filtered (0.22 mm), and
lyophilized to give deprotected compound 6 as an off-white solid
À114.5 to À113.8 ppm (1F, m); LRMS (ESI): m/z: 489 [M+H]+; HPLC
(method 2): tR =1.6 min.
4-{2-[4-(5-Aminopentylamino)-6-(4-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}phenol Hydrochloride Salt (10)
2,4-Dichloro-4-fluorophenylamino-1,3,5-triazine was prepared by
using the same method as that outlined for compound 6 by using
4-fluoroaniline (18 mL, 190 mmol) instead of 3-fluoroaniline to
yield a white solid (44.3 g, 90): LRMS (ESI): m/z: 259 [M+H]+ HPLC
(method 2): tR =4.0 min. The dichlorotriazine (44.2 g, 0.2 mole) was
coupled with tyramine (35.1 g, 0.3 mole) according to compound 6
by using tyramine instead of 5-(tert-butoxycarbonylamino)pentyla-
mine to yield a white solid (56.1 g, 91%): LRMS (ESI): m/z: 360
[M+H]+, 382 [M+H+Na]+; HPLC (method 2): tR =3.7 min. A solu-
tion of the monochlorotriazine (15.0 g, 41.8 mmol) and 1,5-diami-
nopentane (24.5 mL, 209 mmol) in tetrahydrofuran (125 mL) and
methanol (60 mL) was divided into nine portions. Each portion was
heated in a chemistry microwave apparatus at 1308C for 10 min.
The portions were then recombined and concentrated in vacuo,
and the residue was dissolved in ethyl acetate. The ethyl acetate
solution was washed with water and saturated sodium chloride
and then extracted with 2m aqueous HCl. The aqueous extract
was treated with ethyl acetate and then saturated aqueous sodium
bicarbonate. The precipitate was extracted with ethyl acetate, and
the combined extract was washed with saturated sodium chloride,
dried (magnesium sulfate/charcoal), filtered through Celite dia-
tomaceous earth, and concentrated in vacuo. The residue was dis-
solved in methanol (300 mL), and the solution was treated with
1m HCl in ether (60 mL). The solution was then concentrated in va-
cuo. The residue was dissolved in hot 2-propanol (150 mL), and
this solution was added dropwise to ether (1.5 L) with vigorous
stirring. The precipitated solid was collected by filtration, dried in
vacuo, and then dissolved in pyrogen-free water (1.6 L), filtered
(0.22 mm), and lyophilized to give compound 10 as the hydrochlo-
ride salt (14.9 g, 72%): m.p. 130–1338C; 1H NMR (400 MHz, D2O):
d=1.16–1.27 (2H, m), 1.37–1.54 (4H, m), 2.53–2.64 (2H, m), 2.76–
2.83 (2H, m), 3.09–3.17 (2H, m), 3.21–3.48 (2H, m), 6.56–6.64 (2H,
m), 6.85–7.02 (4H, m), 7.16–7.27 ppm (2H, m); 19F NMR (376.5 MHz,
CD3OD): d=À118.1 to À116.0 ppm (1F, m); LRMS (ESI): m/z: 426
[M+H]+; HPLC (method 2): tR =1.6 min.
1
(5.5 g, 89%): H NMR (400 MHz, [D6]DMSO): d=1.26–1.35 (2H, m),
1.47–1.57 (4H, m), 2.64–2.73 (4H, m), 3.24–3.31 (2H, m), 3.32–3.55
(5H, m, CH2 +NH3+), 6.63 (2H, d, J=8.5 Hz), 6.82–6.89 (1H, m),
6.93–7.06 (2H, m), 7.24–7.39 (2H, m), 7.61–7.73 (1H, m), 7.81–7.93
(3H, m), 8.15–8.25, 8.40–8.60, 9.10–9.30, 10.25–10.40 & 10.55–
10.65 ppm (2H, br); 19F NMR (376.5 MHz, CD3OD): d=À114.5 to
À113.8 ppm (1F, m); LRMS (ESI): m/z: 426 [M+H]+, 448
[M+H+Na]+; HPLC (method 2): tR =1.6 min.
4-{2-[4-(5-Aminopentylamino)-6-(3-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}-N,N-dimethylbenzenesulfonamide
Dihydrochloride Salt (8)
This compound was prepared according to the procedure outlined
for compound 10 by using N,N-dimethyl-4-[2-aminoethyl]benzene-
sulfonamide instead of tyramine. N,N-Dimethyl-4-(2-aminoethyl)-
benzenesulfonamide was synthesized as follows: A solution of 4-
(2-aminoethyl)benzenesulfonamide (26.5 g, 0.1 mole) in anhydrous
DMF (120 mL) was treated with phthalic anhydride (23.5 g,
0.2 mole), and the mixture was heated at 708C for 4 h. The mixture
was cooled to ambient temperature and 1,1’-carbonyldiimidazole
(21.5 g, 0.1 mole) was added in small portions; the mixture was
stirred at ambient temperature overnight. The solvent was evapo-
rated in vacuo, and the residue was washed with water, dried, and
4-{2-[4-(5-Aminopentylamino)-6-(3-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}benzenesulfonamide Dihydrochloride
Salt (7)
This compound was prepared by using the same method as that
outlined for compound 6 by using 4-(2-aminoethyl)benzenesulfo-
namide instead of tyramine. White solid; 77% yield; m.p. 145– triturated with ethyl acetate to give the phthaloyl-protected com-
1
1478C; 1H NMR (400 MHz, D2O): d=1.14–1.26 (2H, m), 1.33–1.44
(2H, m), 1.46–1.55 (2H, m), 2.64–2.84 (4H, m), 3.04–3.15 (2H, m),
3.33–3.56 (2H, m), 6.68–6.84 (1H, m), 6.88–6.99 (1H, m), 7.09–7.32
(4H, m), 7.44–7.63 ppm (2H, m); 19F NMR (376.5 MHz, CD3OD): d=
pound as a white solid (38.1 g, 89): H NMR (400 MHz, [D6]DMSO):
d=2.98 (2H, t, J=7.0 Hz), 3.82 (2H, t, J=7.0 Hz), 7.29 (2H, s), 7.38
(2H, d, J=8.0 Hz), 7.69 (2H, t, J=8.0 Hz), 7.76–7.84 ppm (4H, m);
LRMS (ESI): m/z: 331 [M+H]+, 348 [M+H+Na]+; HPLC (method 2):
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tR =2.9 min. A solution of the phthaloyl-protected compound
(12.7 g, 38.6 mmol) in anhydrous DMF (120 mL) was treated with
NaH (60% dispersion in oil; 3.5 g, 88.8 mmol) in small portions
under a N2 atmosphere at 08C over 15 min, and the mixture was
stirred under a N2 atmosphere at 08C for 1 h. Iodomethane
(4.8 mL, 77.2 mmol) was then added dropwise over 15 min, and
the mixture was stirred under a N2 atmosphere at 08C to RT over-
night. The resultant yellow suspension was poured onto ice/water
(1.4 L) and was stirred for 30 min. The precipitate was collected by
filtration; washed sequentially with water, hexane, and ether; and
then dried in vacuo to give the N,N-dimethylbenzenesulfonamide
derivative as a white solid (11.3 g, 81%): LRMS (ESI): m/z: 359
[M+H]+, 381 [M+H+Na]+; HPLC (method 2): tR =3.7 min. A solu-
tion of the phthaloyl-protected, N,N-dimethyl compound (11.3 g,
31.5 mmol) and hydrazine hydrate (4.6 mL, 44.6 mmol) in 95% eth-
anol (125 mL) was heated at reflux for 2 h. The white solid that
formed was removed by filtration and was washed with ethanol.
The filtrate and washings were combined, and the solution was
concentrated in vacuo. The solid that formed was removed by fil-
tration and was washed with ethanol. This procedure was repeated
(3”), and the final filtrate was concentrated to dryness in vacuo.
The solid was extracted with ethyl acetate. The extracts were con-
centrated in vacuo to give the free amine as a yellow oil (4.8 g,
67%): 1H NMR (400 MHz, CD3OD): d=2.65 (6H, s), 2.84–2.92 (4H,
m), 7.47 (2H, d, J=8.5 Hz), 7.71 ppm (2H, d, J=8.5 Hz); LRMS (ESI):
m/z: 229 [M+H]+, 251 [M+H+Na]+; HPLC (method 2): tR =2.3 min.
This compound was treated with dichlorotriazine followed by the
alkylamine and was then deprotected to give the final product as
a white solid (2.2 g, 92%): m.p. 143–1468C; 1H NMR (400 MHz,
CD3OD): d=1.42–1.53 (2H, m), 1.64–1.78 (4H, m), 2.60 & 2.64 (6H,
2 x s), 2.92–2.99 (2H, m), 3.01–3.07 (2H, m), 3.39–3.48 (2H, m),
3.68–3.78 (2H, m), 6.83–6.92 (1H, m), 7.24–7.37 (2H, m), 7.42–
7.71 ppm (5H, m); LRMS (ESI): m/z: 517 [M+H]+, 539 [M+H+Na]+;
HPLC (method 1): tR =4.3 min.
4-{2-[4-(2-Aminoethylamino)-6-(4-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}phenol Dihydrochloride Salt (12)
This compound was prepared according to the procedure outlined
for compound 10 by using ethylenediamine instead of 1,5-diami-
nopentane. White solid (566 mg): 1H NMR (400 MHz, CD3OD): d=
2.77–2.82 (2H, m), 3.14–3.20 (2H, m), 3.54–3.70 (4H, m), 6.70 (2H,
d, J=7.8 Hz), 6.98–7.13 (4H, m), 7.52–7.62 ppm (2H, m); LRMS
(ESI): m/z: 384 [M+H]+; HPLC (method 1): tR =1.6 min.
4-{2-[4-(5-Aminopentylamino)-6-(3-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}benzamide Dihydrochloride Salt (13)
This compound was prepared according to the procedure outlined
for compound 10 by using 4-(2-aminoethyl)benzamide and 3-fluo-
roaniline instead of tyramine and 4-fluoroaniline, respectively. 4-(2-
Aminoethyl)benzamide was prepared as follows: A suspension of
4-(2-aminoethyl)benzoic acid hydrochloride (5.0 g, 24.8 mmol) in
methanol (200 mL) was treated with a 4m solution of HCl in 1,4-di-
oxane (10 mL, 40 mmol), and the mixture was heated at reflux
overnight. The solvents and the excess amount of acid were re-
moved in vacuo. The residue was triturated with ether and dried in
vacuo to give the ester as a white solid (5.5 g, quant.): 1H NMR
(400 MHz, CD3OD): d=3.04 (2H, t, J=7.0 Hz), 3.21 (2H, td, J=7.0,
0.5 Hz), 3.89 (3H, s), 7.41 (2H, dd, J=8.0, 0.5 Hz), 8.00 ppm (2H, d,
J=8.0 Hz).
A suspension of this hydrochloride salt (5.4 g,
24.8 mmol) in tetrahydrofuran (60 mL) and methanol (30 mL) was
treated with diisopropylethylamine (4.8 mL, 27.3 mmol) and di-tert-
butyl dicarbonate (8.1 g, 37.2 mmol). The mixture was stirred at
ambient temperature under a N2 atmosphere for 5 h. The solvents
were evaporated in vacuo, and the residue was dissolved in ethyl
acetate. The solution was washed with water and saturated
sodium chloride and was then dried (magnesium sulfate), filtered,
and concentrated in vacuo. The residue was triturated with cold
ether and dried in vacuo to give the protected compound as a
white solid (5.6 g, 81%): LRMS (ESI): m/z: 192 [M+H]+, 302
[M+H+Na]+; HPLC (method 2): tR =3.9 min. A solution of the ester
(5.6 g, 20.0 mmol) in 1,4-dioxane (36 mL) was treated with saturat-
ed aqueous ammonia (36 mL). The mixture was heated in a sealed
tube at 1008C overnight. After cooling, the precipitated solid was
collected by filtration, washed with water, and dried in vacuo to
give the amide as a white solid (4.4 g, 82%): LRMS (ESI): m/z: 287
[M+H+Na]+; HPLC (method 2): tR =2.6 min. Deprotection of the
tert-butoxycarbonyl compound (4.4 g, 16.5 mmol) was undertaken
by modifying the procedure outlined for compound 6, in that the
water co-solvent was omitted and the solid was dried in vacuo
rather than lyophilized to yield a white solid (3.3 g, quant.): LRMS
(ESI): m/z: 165 [M+H]+, 187 [M+H+Na]+; HPLC (method 2): tR =
0.3 min. This compound was treated with dichlorotriazine and the
alkylamine and was then deprotected to give the final product as
a white solid (1.0 g, 20%): m.p. 190–1928C; 1H NMR (400 MHz,
D2O): d=1.13–1.26 (2H, m), 1.31–1.55 (4H, m), 2.54–2.84 (4H, m),
2.99–3.12 (2H, m), 3.23–3.49 (2H, m), 6.66–6.82 (1H, m), 6.86–7.14
(3H, m), 7.16–7.25 (2H, m), 7.36–7.57 ppm (2H, m); LRMS (ESI): m/
z: 453 [M+H]+; HPLC (method 2): tR =1.5 min.
N-(5-Aminopentyl)-N’-[2-(4-aminophenyl)ethyl]-N’’-(4-fluoro-
phenyl)-1,3,5-triazine-2,4,6-triamine Dihydrochloride Salt (9)
This compound was prepared according to the procedure outlined
for compound 6 by using 2-(4-aminophenyl)ethylamine instead of
tyramine. Yellow solid (97%): m.p. 155–1588C; 1H NMR (400 MHz,
D2O): d=1.42–1.53 (2H, m), 1.63–1.76 (4H, m), 2.87–3.02 (4H, m),
3.40–3.48 (2H, m), 3.62–3.77 (2H, m), 7.07–7.15 (2H, m), 7.28–7.38
(3H, m), 7.40–7.49 (1H, m), 7.52–7.63 ppm (2H, m); LRMS (ESI): m/
z: 425 [M+H]+, 447 [M+H+Na]+; HPLC (method 3): tR =1.9 min.
N-{5-[4-(3-Fluorophenylamino)-6-(4-hydroxyphenethylamino)-
1,3,5-triazin-2-ylamino]pentyl}acetamide Hydrochloride Salt
(11)
This compound was prepared according to the procedure outlined
for compound 6 by using N-(5-aminopentyl)acetamide instead of
5-(tert-butoxycarbonylamino)pentylamine. Orange solid (37 mg):
1H NMR (400 MHz, CD3OD): d=1.34–1.41 (2H, m), 1.46–1.62 (4H,
m), 1.90 (3H, s), 2.76 (2H, t, J=7.1 Hz), 3.14 (2H, t, J=7.1 Hz), 3.26–
3.40 (2H, m), 3.48–3.56 (2H, m), 6.62–6.71 (1H, m), 6.70 (2H, d, J=
8.4 Hz), 7.05 (2H, d, J=8.2 Hz), 7.18–7.30 (1H, m), 7.75–7.83 ppm
(1H, m); LRMS (ESI): m/z: 468 [M+H]+; HPLC (method 2): tR =
2.3 min.
Methyl 4-{2-[4-(5-aminopentylamino)-6-(3-fluorophenylami-
no)-1,3,5-triazin-2-ylamino]ethyl}benzoate Dihydrochloride
Salt (14)
This compound was prepared according to the procedure outlined
for compound 6 by using methyl 4-(2-aminoethyl)benzoate instead
of tyramine. Off-white solid (88 mg): 1H NMR (400 MHz, CD3OD):
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d=1.44–1.53 (2H, m), 1.65–1.76 (4H, m), 2.93–3.04 (4H, m), 3.43– 7.00 (2H, m), 7.13 (2H, d, J=8.5 Hz), 7.56–7.67 ppm (2H, m); LRMS
3.50 (2H, m), 3.69–3.76 (2H, m), 3.84 (3H, s), 6.87–6.96 (1H, m),
7.24–7.43 (4H, m), 7.57–7.68 (1H, m), 7.88–7.96 ppm (2H, m); LRMS
(ESI): m/z: 468 [M+H]+; HPLC (method 2): tR =1.9 min.
(ESI): m/z: 442 [M+H]+; HPLC (method 1): tR =3.3 min.
Methyl (S)-2-[4-(5-aminopentylamino)-6-(3-fluorophenylami-
no)-1,3,5-triazin-2-ylamino]-3-(4-hydroxyphenyl)propanoate
Dihydrochloride Salt (19)
4-{2-[4-(5-Aminopentylamino)-6-(4-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}benzenesulfonamide Dihydrochloride
Salt (15)
This compound was prepared according to the procedure outlined
for compound 6 by using methyl (S)-tyrosine instead of tyramine.
Colorless residue (1 mg): LRMS (ESI): m/z: 484 [M+H]+; HPLC
(method 2): tR =1.8 min.
This compound was prepared according to the procedure outlined
for compound 7 by using 4-fluoroaniline in place of 3-fluoroaniline.
The final Boc-protected intermediate (4.8 mmol) was deprotected
by using a variation of the procedure outlined for compound 6. In
this case, 4m HCl/1,4-dioxane (36 mL) in CH2Cl2 (30 mL) was used
at 08C to ambient temperature to yield a low-density, white solid
(87%): m.p. 165–1688C; 1H NMR (400 MHz, CD3OD): d=1.40–1.51
(2H, m), 1.62–1.74 (4H, m), 2.87–3.04 (4H, m), 2.93 (2H, t, J=
7.5 Hz), 3.01 (2H, t, J=6.5 Hz), 3.41 (2H, t, J=7.5 Hz), 3.64–3.77
(2H, m), 7.07–7.16 (2H, m), 7.29–7.48 (2H, m), 7.50–7.62 (2H, m),
7.77–7.86 ppm (2H, m); 19F NMR (376.5 MHz, CD3OD): d=À120.2
to À119.8 ppm (1F, m); LRMS (ESI): m/z: 245 [M+H]+, 489
[M+H+Na]+; HPLC (method 1): tR =3.6 min.
N-(4-Aminobenzyl)-N’-(5-aminopentyl)-N’’-(3-fluorophenyl)-
1,3,5-triazine-2,4,6-triamine Trihydrochloride Salt (20)
This compound was prepared according to the procedure outlined
for compound 6 by using 4-aminobenzylamine instead of tyra-
mine/triethylamine in step 3. Orange solid (quant.): m.p. 197–
1998C; 1H NMR (400 MHz, CD3OD): d=7.59–7.73 (m, 1H), 7.53 (d,
J=7.9 Hz, 2H), 7.37 (d, J=7.9 Hz, 2H), 7.09–7.33 (m, 2H), 6.82–6.96
(m, 1H), 4.69 (s, 2H), 3.44–3.53 (m, 2H), 2.90–2.97 (m, 2H), 1.62–
1.76 (m, 4H), 1.42–1.55 ppm (m, 2H); 13C NMR (101 MHz, CD3OD):
d=complex rotamers; 19F NMR (377 MHz, CD3OD): d=À114.5 to
À113.9 (1F, m); LRMS (ESI): m/z (%): 411.2 (100) [M+H]+; HPLC (10–
99% 5 min): tR =0.89 min.
4-[4-(5-Aminopentylamino)-6-(3-fluorophenylamino)-1,3,5-tri-
azin-2-ylamino]benzenesulfonamide Dihydrochloride Salt (16)
This compound was prepared according to the procedure outlined
for compound 10 by using 4-aminobenzenesulfonamide and 3-flu-
oroaniline instead of tyramine and 4-fluoroaniline, respectively.
Pale-beige solid (95% yield): m.p. 162–1638C; 1H NMR (400 MHz,
CD3OD): d=1.47–1.56 (2H, m), 1.67–1.78 (4H, m), 2.95 (2H, t, J=
7.5 Hz), 3.52 (2H, t, J=7.0 Hz), 6.92–7.00 (1H, m), 7.29–7.42 (2H,
m), 7.60–7.78 (1H, m), 7.82–7.95 ppm (4H, m); 19F NMR (376.5 MHz,
CD3OD): d=À114.2 to À113.7 ppm (1F, m); LRMS (ESI): m/z: 461
[M+H]+, 483 [M+H+Na]+; HPLC (method 4): tR =3.7 min.
4-(2-{4-[2-(2-Aminoethoxy)ethylamino]-6-(4-fluorophenylami-
no)-1,3,5-triazin-2-ylamino}ethyl)phenol bis(Trifluoroacetate)
Salt (21)
This compound was prepared according to the procedure outlined
for compound 10 by using 2,2’-oxydiethylamine dihydrochloride
instead of 1,5-diaminopentane. White solid (33 mg): 1H NMR
(400 MHz, CD3OD): d=2.75–2.83 (2H, m), 3.12–3.17 (2H, m), 3.54–
3.73 (8H, m), 6.67–6.72 (2H, m), 6.96–7.14 (4H, m), 7.55–7.60 ppm
(2H, m); LRMS (ESI): m/z: 428 [M+H]+; HPLC (method 4): tR =
3.2 min.
4-{2-[4-(4-Aminobutylamino)-6-(3-fluorophenylamino)-1,3,5-tri-
azin-2-ylamino]ethyl}benzenesulfonamide Dihydrochloride
Salt (17)
4-(2-{4-(4-Fluorophenylamino)-6-[2-(piperazin-1-yl)ethylami-
no]-1,3,5-triazin-2-ylamino}ethyl)phenol Trifluoroacetate Salt
(22)
This compound was prepared according to the procedure outlined
for compound 10 by using 4-(2-aminoethyl)benzenesulfonamide
and 4-aminobutylamine instead of tyramine and 5-aminopentyla-
mine, respectively. White solid (74%): m.p. 181–1848C; 1H NMR
(400 MHz, CD3OD): d=1.65–1.77 (4H, m), 2.93–3.04 (4H, m), 3.42–
3.54 (2H, m), 3.68–3.78 (2H, m), 6.86–6.95 (1H, m), 7.24–7.50 (4H,
m), 7.57–7.66 (1H, m), 7.78–7.86 ppm (2H, m); 19F NMR (376.5 MHz,
CD3OD): d=À116.10 to À115.43 ppm (1F, m); LRMS (ESI): m/z: 475
[M+H]+, 497 [M+H+Na]+; HPLC (method 1): tR =3.6 min.
This compound was prepared according to the procedure outlined
for compound 10 by using 2-[4-(tert-butoxycarbonyl)piperazin-1-
yl]ethylamine instead of 1,5-diaminopentane with a final deprotec-
tion step as for compound 6. White solid (15 mg): 1H NMR
(400 MHz, CD3OD): d=2.60–2.82 (6H, m), 2.81 (2H, t, J=7.0 Hz),
3.08–3.17 (2H, m), 3.64 (2H, t, J=7.0 Hz), 3.82–3.98 (4H, m), 6.71
(2H, d, J=8.4 Hz), 7.05 (2H, d, J=8.4 Hz), 7.10–7.17 (2H, m), 7.46–
7.57 ppm (2H, m); LRMS (ESI): m/z: 453 [M+H]+; HPLC (method 4):
tR =3.1 min.
(RS)-4-{2-[4-(5-Aminopentylamino)-6-(4-fluorophenylamino)-
1,3,5-triazin-2-ylamino]-1-hydroxyethyl}phenol Dihydrochlor-
ide Salt (18)
4-{2-[4-(4-Aminobutylamino)-6-(4-fluorophenylamino)-1,3,5-tri-
azin-2-ylamino]ethyl}phenol Trifluoroacetate Salt (23)
This compound was prepared according to the procedure outlined
for compound 10 by using [Æ]-octopamine instead of tyramine.
1
White solid (36%): m.p. 122–1258C; H NMR (400 MHz, CD3OD): d=
This compound was prepared according to the procedure outlined
for compound 10 by using 1,4-diaminobutane instead of 1,5-diami-
nopentane. White solid (1.9 mg): LRMS (ESI): m/z: 412 [M+H]+;
1.36–1.43 (2H, m), 1.50 (2H, tt, J=7.0, 7.0 Hz), 1.57–1.63 (2H, m),
2.62 (2H, t, J=7.0 Hz), 3.28–3.40 (2H, m), 3.45 (1H, J=13.5, 8.0 Hz),
3.54–3.63 (1H, m), 4.67–4.75 (1H, m), 6.69 (2H, d, J=8.5 Hz), 6.96– HPLC (method 2): tR =4.1 min.
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and the concentration of compound that inhibited 50% of PGE2
production (IC50) was calculated by using Prism software.
4-{2-[4-(6-Aminohexylamino)-6-(4-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}phenol Dihydrochloride Salt (24)
This compound was prepared according to the procedure outlined
for compound 10 by using 1,6-diaminohexane instead of 1,5-dia-
minopentane. White solid (30 mg): H NMR (400 MHz, CD3OD): d=
In Vitro Effect of Compounds on PC-3 Cell Migration or
Invasion
1
1.39–1.47 (4H, m), 1.61–1.70 (4H, m), 2.76–2.84 (2H, m), 2.88–2.94
(2H, m), 3.37–3.48 (2H, m), 3.56–3.67 (2H, m), 6.71 (2H, d, J=
7.8 Hz), 6.97–7.13 (4H, m), 7.52–7.62 ppm (2H, m); LRMS (ESI): m/z:
440 [M+H]+; HPLC (method 4): tR =4.0 min.
An in vitro migration assay was used to assess cell mobility in two
dimensions. PC-3 cells were plated on a 12-well plate and were
grown to confluence in RPMI+10% FBS. A rubber policeman was
used to create a denuded area. Confluent cells were quiesced by
mitomycin C treatment (0.5 mm) at the concentration used to pre-
vent the confounding issue of cell proliferation and protein synthe-
sis. These cells were also incubated in the presence or absence of
endothelial growth factor (EGF) and the compound for 24 h, and
they were then photographed.
4-{2-[4-{[4-(Aminomethyl)cyclohexyl]methylamino}-6-(4-fluoro-
phenylamino)-1,3,5-triazin-2-ylamino]ethyl}phenol (25)
This compound was prepared according to the procedure outlined
for compound 10 by using 1,4-bis(aminomethyl)cyclohexane in-
stead of 1,5-diaminopentane. Pale-pink solid (13 mg): 1H NMR
(400 MHz, CD3OD): d=0.86–1.02 (2H, m), 1.33–1.58 (6H, m), 1.68–
1.90 (2H, m), 2.47–2.60 (2H, m), 2.69–2.79 (2H, m), 3.16–3.24 (2H,
m), 3.45–3.53 (2H, m), 6.70 (2H, d, J=8.2 Hz), 6.95–7.03 (4H, m),
7.53–7.68 ppm (2H, m); 19F (377 MHz, CD3OD): d=À124.1 to
À123.7 ppm (1F, m); LRMS (ESI): m/z: 466 [M+H]+; HPLC
(method 4): tR =3.5 min.
In Vitro Effect of Compounds on the Production of PGE2 in
LPS-Stimulated J774A.1
J774A.1 cells were cultured in the presence or absence of LPS and
the compounds. Cells were cultured at 378C for 24 h, and there-
after, the supernatants were collected for determination of the
concentration of PGE2 by ELISA, as recommended by the manufac-
turer (GE Healthcare). Data were analyzed in Microsoft Excel soft-
ware, and the concentration of compound that inhibited 50% of
PGE2 production (IC50) was calculated by using Prism software.
4-{2-[4-(5-Aminopentylamino)-6-(3-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}-2,6-dimethoxyphenol Dihydrochloride
Salt (26)
In Vivo Experiments
This compound was prepared according to the procedure outlined
for compound 6 by using 4-(2-aminoethyl)-2,6-dimethoxyphenol
instead of tyramine. Pink solid (22 mg): H NMR (400 MHz, CD3OD):
All animal studies were reviewed and approved by the animal care
and use committee of the National Institute of Scientific Research,
INRS-Institut-Armand-Frappier Center (Laval, QC, Canada).
1
d=1.45–1.53 (2H, m), 1.64–1.77 (4H, m), 2.82–2.88 (2H, m), 2.90–
2.98 (2H, m), 3.39–3.50 (2H, m), 3.66–3.74 (2H, m), 3.76–3.82 (2H,
m), 3.78 (3H, s), 3.81 (3H, s), 6.47–6.60 (2H, m), 6.84–6.93 (1H, m),
7.26–7.37 (2H, m), 7.60–7.67 ppm (1H, m); 19F (377 MHz, CD3OD):
d=À114.5 to À113.8 ppm (1F, m); LRMS (ESI): m/z: 486 [M+H]+;
HPLC (method 4): tR =3.1 min.
Antitumor Effects of Compounds on a Primary P815
Mastocytoma Tumor
The syngeneic tumor P815 is a DBA/2 (H-2d)-derived mastocytoma
obtained from ATCC (TIB64). P815 cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine
serum. At day 0, 5”105 viable P815 cells (50 mL) were intradermally
injected to produce localized tumors in 6-to-8-week-old DBA/2
mice. The animals were then serially monitored by manual palpa-
tion for evidence of tumor. Mice were then treated every day with
oral administration of vehicle (negative control), acetylsalicylic acid
(positive control, 50 mgkgÀ1), or compound (50 mgkgÀ1). Mice
were sacrificed at day 23. Serial tumor volume was obtained by bi-
dimensional diameter measurements with calipers by using Equa-
tion (1):
4-{2-[4-(5-Aminopentylamino)-6-(3-fluorophenylamino)-1,3,5-
triazin-2-ylamino]ethyl}-2-chlorophenol, tris(trifluoroacetate)
Salt (27)
This compound was prepared according to the procedure outlined
for compound 6 by using 4-(2-aminoethyl)-2-chlorophenol instead
of tyramine. White solid (18 mg): 1H NMR (400 MHz, CD3OD): d=
1.43–1.52 (2H, m), 1.65–1.74 (4H, m), 2.77–2.83 (2H, m), 2.90–2.96
(2H, m), 3.43–3.49 (2H, m), 3.58–3.65 (2H, m), 6.78–6.91 (2H, m),
6.94–7.03 (1H, m), 7.13–7.21 (1H, m), 7.30–7.36 (2H, m), 7.54–
7.69 ppm (1H, m); 19F (377 MHz, CD3OD): d=À114.6 to
À113.9 ppm (1F, m); LRMS (ESI): m/z: 460 [M+H]+; HPLC
(method 4): tR =3.7 min.
Volume 0:4 ab2
1
in which a is the major tumor diameter and b is the minor perpen-
dicular diameter. Tumors were palpable, in general, 3 days to
5 days postinoculation.
Anticancer Activity
In Vitro Effect of Compounds on the Inhibition of TNF-a
Released by LPS Induction from J774A.1 Cells
Antitumor Effects of Compounds on Pancreatic PAN02
J774A.1 cells were cultured in the presence or absence of LPS and
compounds. Cells were cultured at 378C for 24 h, and thereafter,
the supernatants were collected for determination of the concen-
tration of PGE2 by ELISA, as recommended by the manufacturer
(GE Healthcare). Data were analyzed in Microsoft Excel software,
The syngeneic tumor PAN02 is a pancreatic tumor cell line ob-
tained from NCI (0507232). PAN02 cells were grown in RPMI-1640
containing 10% fetal bovine serum. At day 0, 7.5”105 viable
PAN02 cells (50 mL) were intradermally injected to produce local-
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Conflict of Interest
Authors are employees of Prometic
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Keywords: autoimmune diseases · cancer · inflammation ·
structure–activity relationships · triazines
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Received: July 5, 2018
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