Synthesis and in vitro anticancer evaluation of some 4,6-diamino-1,3,5-triazine-2-carbohydrazides as Rad6 ubiquitin conjugating enzyme inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.02.085

Series of 4-amino-6-(arylamino)-1,3,5-triazine-2-carbohydrazides (3a-e) and N′-phenyl-4,6-bis(arylamino)-1,3,5-triazine-2-carbohydrazides (6a-e), for ease of readership, we will abbreviate our compound names as 'new triazines', have been synthesized, base

Full text of DOI:10.1016/j.bmcl.2016.02.085

Accepted Manuscript
Synthesis and in vitro anticancer evaluation of some 4,6-diamino-1,3,5-tria-
zine-2-carbohydrazides as Rad6 ubiquitin conjugating enzyme inhibitors
Hend Kothayer, Sebastian M. Spencer, Kaushlendra Tripathi, Andrew D.
Westwell, Komaraiah Palle
PII:
S0960-894X(16)30206-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.085
BMCL 23639
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 December 2015
24 February 2016
27 February 2016
Please cite this article as: Kothayer, H., Spencer, S.M., Tripathi, K., Westwell, A.D., Palle, K., Synthesis and in
vitro anticancer evaluation of some 4,6-diamino-1,3,5-triazine-2-carbohydrazides as Rad6 ubiquitin conjugating
enzyme inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.02.085
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Synthesis and in vitro anticancer evaluation of some
4,6-diamino-1,3,5-triazine-2-carbohydrazides as Rad6
ubiquitin conjugating enzyme inhibitors
Hend Kothayer a, Sebastian M. Spencer b, Kaushlendra Tripathi b,
Andrew D. Westwellc and Komaraiah Palle.b
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Egypt; b Department
of Oncologic Sciences, USA Mitchell Cancer Institute,1660 Springhill Avenue, Mobile, Alabama
36604, USA; cSchool of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood
Building, King Edward VII Avenue, Cardiff, CF10 3NB, Wales, U.K.
Abstract— Series of 4-amino-6-(arylamino)-1,3,5-triazine-2-
carbohydrazides (3a-e) and N'-phenyl-4,6-bis(arylamino)-1,3,5-triazine-
2-carbohydrazides (6a-e), for ease of readership, we will abbreviate our
compound names as “ new triazines”, have been synthesized, based on
the previously reported Rad6B-inhibitory diamino-triazinylmethyl
benzoate anticancer agents TZ9 and 4-amino-N'-phenyl-6-(arylamino)-
1,3,5-triazine-2-carbohydrazides. Synthesis of the target compounds was
readily accomplished in two steps from either bis-aryl/aryl biguanides via
reaction of phenylhydrazine or hydrazinehydrate with key 4-amino-6-
bis(arylamino)/(arylamino)-1,3,5-triazine-2-carboxylate
intermediates.
These new triazine derivatives were evaluated for their abilities to inhibit
Rad6B ubiquitin conjugation and in vitro anticancer activity against
several human cancer cell lines: ovarian (OV90 and A2780), lung
(H1299and A549), breast (MCF-7 and MDA-MB231) and colon (HT29)
cancer cells by MTS assays. All the 10 new triazines exhibited superior
Rad6B inhibitory activities in comparison to selective Rad6 inhibitor TZ9
that was reported previously. Similarly, new triazines also showed better
 Corresponding author. Tel. 00201224578259; E-mail: hendo1311@hotmail.com

IC50 values in survival assays of various tumor cell lines. Particularly,
new triazines 6a-c, exhibited lower IC50 (3.3 to 22M) values compared
to TZ9.
Keywords: Ubiquitination; E2 ubiquitin conjugating enzyme;
Rad6B; anticancer; triazines.
The ubiquitin-proteasome system controls the turnover of
regulatory proteins involved in critical cellular processes including cell
cycle progression, cell development and differentiation, apoptosis,
angiogenesis and cell signaling pathways 1-2. This system requires the
action of three enzymes: E1 ubiquitin activating enzyme, E2 ubiquitin
3
conjugating enzyme and E3 ubiquitin ligase . Firstly, the ubiquitin is
activated by the E1 activating enzyme and once it is activated, it is then
transferred to E2 conjugating enzyme. The final step is the formation of
an iso-peptide bond between a lysine of the target protein and the C-
terminal glycine of ubiquitin (carried by E2). This step usually requires
the action of an E3 ubiquitin ligase 4.
Interference with the proteasome activity was proven to be
effective in cancer therapeutics since the clinical approval of bortezomib
(Velcade®) as a proteasome inhibitor for treatment of relapsed multiple
5
myeloma and mantle cell lymphoma . However, the requirement for
more specific inhibiting targets like the design of potential E2 or E3
inhibitors, has appeared in order to reduce the side effects resulting from
6
bortezomib . Recently, many E1 and E3 ligase inhibitors such as PYR-
41, Nutlin-3a, P013222 and SCF-I2 have been successful and progressed
to preclinical/clinical development. Also, the approved myeloma drug
thalidomide has been recently identified as an E3 ligase inhibitor 7.

Among the E2 ubiquitin conjugating enzyme family, Rad6B is of
special interest since it is found to be over-expressed in many human
8-9
cancer cell lines and tumors . Constitutive over-expression of Rad6B in
the non-transformed human breast epithelial cell line MCF 10A induces a
number of adverse effects associated with cancer progression such as
formation of multinucleated cells, centrosome amplification, abnormal
10
mitosis, aneuploidy, and transformation . Most importantly, Rad6B has
been shown to positively regulate β-catenin stabilization and activity that
drives the malignant progression of breast cancer cells 11-13. Since β-
catenin- mediated signaling has been implicated in many human
malignancies, including lung, colon, breast, and ovarian, it has been an
important therapeutic target. Furthermore, Rad6B plays a central role in
regulation of multiple DNA repair pathways through its interactions with
different E3 ubiquitin ligases. For example, Rad6 partners with the E3
ubiquitin ligase Rad18 and monoubiquitinates PCNA in response to
replication fork-stalling lesions to promote trans-lesion synthesis (TLS)
or the DNA damage tolerance pathway 14-17. Rad18/Rad6 ubiquitin ligase
complex is also important in the activation of the Fanconi anemia tumor
suppressor pathway, which plays critical roles in genome integrity and
tumor resistance to a variety of chemotherapeutic agents, including those
that induce DNA crosslinks and DNA double strand breaks 17-18. Rad6 has
also been shown to associate with RNF168 to monoubiquitinate histone
H1.2 thereby enabling chromatin relaxation and allowing DNA damage
response factors access to damage sites 19. Moreover, increased
expression or activation of these DNA damage response (DDR) signaling
9, 20-22
and repair genes accounts for tumor resistance to chemotherapy
.
Therefore, development of DNA damage response and repair signal
inhibitors are important to effectively treat these tumors.

We have recently reported [4-amino-6-(arylamino)-1,3,5-triazin-2-
yl]methyl 4-nitrobenzoates TZ8-TZ9 (Fig. 1) as novel and selective
23
Rad6B-inhibitory lead compounds . These inhibitors were identified
by virtual screening of a pharmacophore model generated from the
conserved key residues stabilizing the E2-ubiquitin thioester intermediate
against a pre-prepared database using drug-like filters which determined
the substituted diamino-triazine core structure as a starting point for
analogue synthesis. Triazine analogue synthesis coupled to in vitro
anticancer evaluation led to the identification of lead compounds TZ8-
TZ9 23.
3
4 NO2
2
1
O
5
6
5
2
2
O
R
6
1 N
N 3
4
3
4
1
6
N
H
N
NH2
5
TZ8 R=CH3
TZ9 R=H
Fig.1: Chemical structures of Rad6B-inhibitory lead compounds TZ8 and TZ9
Using a molecular modeling approach to guide the design of new
derivatives of TZ8 and TZ9, we reported 4-amino-N'-phenyl-6-
(arylamino)-1,3,5-triazine-2-carbohydrazides (Fig. 2) with IC50 values
(2.48–4.79 µM) superior to those of TZ8 and TZ9 when tested on the
24
Rad6B-expressing MDA-MB-231 cell line . In docking studies, such
triazinecarbohydrazide derivatives were found to be incorporated deep
inside the Rad6B binding pocket, making key interactions between the
hydrazine nitrogen atoms and the Rad6B active site residues Cys88 and
Asp90. Additional interactions between the aniline nitrogens and
Asn119/Gln93 and between the phenyl (hydrazide) ring and Leu89 were

also apparent from our docking analysis. The importance of these active
site residues to the allosteric effect on Rad6B induced by E3 ligases, and
the observation that no other E2 family members (with the exception of
Rad6A) have residues corresponding to Gln93 or Asn119, suggest that
these triazinecarbohydrazides could be selective Rad6B inhibitors 24.
H
O
N
N
N
N'
H
R
N
N
NH2
H
R=H, p-CH3,p-OCH3
o-OCH3 or p-Cl
Fig.2: 4-amino-N'-phenyl-6-(arylamino)-1,3,5-triazine-2-carbohydrazides
In the current work, we studied the effect of removal of the phenyl
(hydrazide) ring (3a-e) or the addition of aryl substituent to the free
amino group (6a-e) of our previously reported 4-amino-N'-phenyl-6-
(arylamino)-1,3,5-triazine-2-carbohydrazides (Fig. 2) on the Rad6B
inhibitory activity and cellular anticancer activity. (Fig. 3)
Removal
H
O
N
N
N
NH2
NH2
3
6
4
5
2
H
N
(3a-e)
O
N
R
N'
H
1
N
N
H
3'
N
2'
4'
5'
R
H
1'
N
N
NH2 addition
O
N
H
N'
H
6'
3
3
6
R=H, -CH
OCH or -Cl
o-
OCH
3
p
3,p-
p
4
5
2
2
1
4
5
N
N
R
3
R
R
1
N
N
N
H
6
H
(6a-e)
Fig. 3: SAR modifications of previous lead compounds

The synthesis of the 4-amino-6-(arylamino)-1,3,5-triazine-2
carbohydrazide (3a-e) was accomplished in two steps from arylbiguanide
hydrochloride salts (1a-e), which were prepared from commercially
available substituted aniline and dicyandiamide according to previously
reported procedures 25-26. Neutralization of the arylbiguanide
hydrochloride salt using sodium methoxide/methanol was followed by
reaction with dimethyloxalate in refluxing methanol. This gave the
intermediate methyl 4-amino-6-(arylamino)-1,3,5-triazine-2- carboxylates
(2a-e) in 83–92% isolated yield following recrystallization from
methanol. Reaction of intermediates (2a-e) with hydrazinehydrate in
refluxing ethanol produced the target new triazines (3a-e) in high yield
(81–92%) following recrystallization from ethanol (Scheme 1).
H
O
N
N
O
N
OCH3
N
NH2
NH2
NH NH
N
(i), (ii)
R
.HCl
NH2
(iii)
R
R
N
N
H
N
N
NH2
N
N
H
H
H
3a-e
1a-e
2a-e
R
a
H
b 4-OMe
4-Me
Reagents and conditions: (i) NaOCH3, CH3OH, room temp. 3 h; (ii) dimethyloxalate,
CH3OH, reflux, 4 h; (iii) hydrazine hydrate, EtOH, reflux 3 h.
c
d
2-OMe
e 4-Cl
Scheme.1: Synthetic pathway for compounds 3a-e
A similar strategy was used to synthesize the N'-phenyl-4,6-
bis(arylamino)-1,3,5-triazine-2-carbohydrazides (6a-e) from bis-
arylbiguanide hydrochloride salts (4a-e), which were prepared from
commercially available substituted aniline and sodium dicyanamide
according to previously reported procedures 25-26. Neutralization of the
bis-arylbiguanide hydrochloride salt using sodium methoxide/methanol
was followed by reaction with dimethyloxalate in refluxing methanol.
This produced the intermediate methyl 4,6-bis(arylamino)-1,3,5-triazine-
2-carboxylates (5a-e) in 72-87% isolated yield following recrystallization
from methanol. Reaction of intermediates (5a-e) with phenylhydrazine in

refluxing ethanol, catalyzed by glacial acetic acid produced the target
new triazines (6a-e) in good yield (76-86%) following recrystallization
from ethanol: water (3:1) (Scheme 2).
H
O
N
N
N
O
N
OCH3
N
N
H
.HCl
NH NH
R
R
R
R
R
R
(i), (ii)
(iii)
N
N
N
H
N
N
H
N
N
H
N
N
H
H
H
H
6a-e
5a-e
4a-e
R
a
H
Reagents and conditions: (i) NaOCH3, CH3OH, room temp. 3 h; (ii) dimethyloxalate,
CH3OH, NaOCH3 reflux, 12 h; (iii) phenylhydrazine, EtOH,AcOH, reflux 18 h.
b
4-OMe
c
4-Me
d
2-OMe
e
4-Cl
Scheme.2: Synthetic pathways for compounds 6a-e
Evaluation of newly synthesized 4-amino-6-(arylamino)-1,3,5-
triazine-2-carbohydrazide (3a-e) and N'-phenyl-4,6-bis(arylamino)-1,3,5-
triazine-2-carbohydrazide (6a-e) was carried out in the human cancer cell
lines OV90 and A2780 (ovarian), H1299 and A549 (lung), MCF-7 and
MDA-MB231 (breast), and HT29 (colon) using MTS assay reagent
(Promega) to assess the cell viability (Table 1). TZ9 was used as a
positive control and aqueous DMSO as a negative control.
To assess the efficacy of these compounds in cell cultures, MTS
assays were performed using multiple tumor cell lines from different
tissue origins. We have randomly chosen these cell lines as many of these
tumors were previously shown to either over express Rad6 or -catenin
mediated signaling. The IC50 values for each of the compounds against
the individual cell line were calculated from their respective dose
response curves. As shown in Table 1, almost all the compounds
inhibited survival of cancer cells, although their IC50 concentrations and
specificities to the cell lines varied. For example, compounds 3a-e, 6a-e
and TZ9 showed IC50 values in the low micromolar range (3.3–16 µM)
when tested on HT-29 and MCF-7 cell lines, whereas only compounds

6a-d show low micromolar IC50 values (3.6–12 µM) when tested on OV
90 and A2780 cell lines. These values compare favorably with the
studies on TZ9. The compound 6b shows the lowest IC50 value (5 µM)
when tested on the H1299 cell line. However, the reasons for these
biological selectivities to particular cell lines and differences in IC50
values of the compounds need further evaluation. Moreover, these
variations could be attributed to differences in functional groups of these
compounds and inherent molecular signatures of the tumor cells, which
may include their Rad6B status and their inherent dependence on its
mediated signaling networks.
Name of
Formula
Weight
366.3
OV 90
A2780
MCF-7
MDA-
MB231
4.6
A549
H1299
HT-29
Compound
TZ9
3a
3b
3c
60
75
80
60
90
85
8
7.8
7.3
14.7
5.5
5.6
36.6
7.1
6.3
3.6
5
5
5.9
6.2
16
7.2
7.8
45
65
65
40
50
30
11
5
8.3
3.3
5.4
4.5
8.6
4.6
9.5
5.8
5.2
8.9
4.1
245.24
275.27
259.27
275.27
279.69
397.43
457.48
425.49
457.48
466.32
2.6
7.6
5.2
12.7
15.7
24.6
2.5
9.3
3d
3e
6a
6b
6c
11.8
8.8
6
7.5
7.5
14.6
10.8
11.6
6.9
12
5
7.2
4.2
7.2
4.2
4.2
3.5
22
26
15
6d
6e
10
60
3.9
8.2
3.5
7.8
Table 1: IC50 Values for New Triazine Analogues.
As described in the methods, the MTS assay was used to evaluate cell survival in the
presence of each new triazine analogue in the range of 0 to 125 µM concentrations.
For each cell line, the MTS assays were performed at least three independent
experiments and each time in triplicates. The results presented in the table are average
values from multiple experiments. The IC50 values were calculated from the data of %
survival vs. drug concentration using Microsoft Excel 2010.

To evaluate the efficacy of newly synthesized 4-amino-6-
(arylamino)-1,3,5-triazine-2-carbohydrazide (3a-e) and N'-phenyl-4,6-
bis(arylamino)-1,3,5-triazine-2-carbohydrazide (6a-e) analogues for their
Rad6 inhibitory activities, in vitro ubiquitin conjugation assays were
performed in comparison with our previously reported Rad6 inhibitor
TZ9. In these assays (Fig. 4A and 4B), ubiquitin conjugation to Rad6
enzyme readily occurs in the positive control (lane 3 in Fig. 4A and lane
2 in Fig. 5A), but not in the negative controls, which lacks either Rad6
(lane 1 in Fig. 4A) or ubiquitin (lane 2 in Fig. 4A and lane 1 in Fig. 5A)
in these assays. Consistent with the previous data, pretreatment of Rad6
with TZ9 inhibited its ability to conjugate with ubiquitin 23. Interestingly,
in the initial screening experiments, all the new triazines exhibited Rad6
inhibitory activity (Fig 4A and 4B). As shown in the figures (4B and 5B)
most of the new triazines are better Rad6 inhibitors than TZ9 at the
equimolar concentration (25 nM), for both inhibition of Rad6 conjugation
to ubiquitin and its substrate H2A ubiquitination in these assays.

Figure 4: New triazine analogues inhibit conjugation of ubiquitin to Rad6B
more effectively than TZ9. A). Representative western blot of in vitro ubiquitin
conjugation experiment showing all new triazine analogues in comparison with TZ9 ,
and probed with Rad6 antibody. Rad6-; negative control with all experimental
components except Rad6B. Ub-; negative control with all experimental components
except ubiquitin. Control: positive control with all experimental components that
shows Rad6B conjugation to ubiquitin. Experiment was repeated for reproducibility at
a minimum of two times. B). Densitometry calculations of Rad6B-Ub bands were
made using ImageJ software on the western blot shown in (A).
To further assess their inhibitory effects on Rad6B ability to
transfer ubiquitin to the substrate, in vitro ubiquitination assays were
23
performed using H2A as substrate . We have selected four compounds
6a, b, c, e that exhibited more favorable IC50 values and better inhibitory
effects on Rad6 conjugation to ubiquitin in the in vitro ubiquitin assays
compared to TZ9. As shown in Figure 5A and 5B, compounds 6a, b, c, e
exhibited superior Rad6B inhibitory properties, both in its conjugation to
ubiquitin and substrate ubiquitination (H2A-Ub) when compared to TZ9.
Together, these studies report the synthesis of new triazines with better
Rad6B inhibitory activities and anticancer properties compared to
previously reported Rad6B inhibitor TZ9 in in vitro evaluations.

Figure 5: Rad6-mediated ubiquitination of H2A is inhibited by new triazine
analogues. A). Western blot showing Rad6-mediated ubiquitination of histone H2A in
the presence of new triazine analogues probed with ubiquitin, H2A and Rad6
antibodies. B). Represents densitometric values of western blot shown in (A), were
calculated using ImageJ software and presented as relative to positive control. All
experiments were repeated at least twice to verify reproducibility.
In summary, Series of novel 4-amino-6-(arylamino)-1,3,5-triazine-
2-carbohydrazides (3a–e) and N'-phenyl-4,6-bis(arylamino)-1,3,5-
triazine-2-carbohydrazides (6a-e) have been synthesized. Compared to
the previously reported Rad6B-inhibitor TZ9, new triazines showed
better IC50 values in survival assays of various tumor cell lines.

Particularly, new triazines 6a-c exhibited much lower IC50 (3.3 to 22M)
values.
Moreover, when compared to TZ9 in in vitro ubiquitin conjugation
assays, compounds 6a, b, c, e exhibited superior Rad6B inhibitory
properties, both in its conjugation to ubiquitin and to the substrate H2A.
Acknowledgements.
This work is supported by Zagazig University to HK, in part by NIH grant
R01GM098956, and Abraham Mitchell Cancer Research Scholar Endowment grant to
K.P.
Supplementary Data available:
1
Experimental procedure ; full compound characterisation data (m.p., H, 13C NMR
spectroscopy, mass spectrometry, % CHN analysis); In vitro ubiquitin conjugating
assays and MTS assay and IC50 calculations.
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Graphical abstract
H
N
H
N
R
O
N
O
N
N
H
NH2
NH2
H
a
4-OMe
4-Me
2-OMe
b
c
N
N
R
R
R
d
N
N
N
N
N
H
H
H
e
4-Cl
3a-e
6a-e
Series of 4-amino-6-(arylamino)-1,3,5-triazine-2-carbohydrazides
(3a-e) and N'-phenyl-4,6-bis(arylamino)-1,3,5-triazine-2-carbohydrazides
(6a-e) have been synthesized. New triazines showed better IC50 values in
survival assays of various tumor cell lines. Particularly, new triazines 6a-
c, exhibited much lower IC50 (3.3 to 22M) values compared to TZ9 (the
previously reported Rad6B-inhibitory diamino-triazinylmethyl benzoate
anticancer agent). Moreover, when compared to TZ9 in in vitro ubiquitin
conjugation assays, compounds 6a, b, c, e exhibited superior Rad6B
inhibitory properties, both in its conjugation to ubiquitin and to the
substrate H2A.

Highlights
 Novel
(3a-e)
4-amino-6-(arylamino)-1,3,5-triazine-2-carbohydrazides
and N'-phenyl-4,6-bis(arylamino)-1,3,5-triazine-2-
carbohydrazides (6a-e) have been synthesized.
 To evaluate the efficacy of newly synthesized triazines in Rad6
inhibitory activities, in vitro ubiquitin conjugation assays were
performed in comparison with our previously reported Rad6
inhibitor TZ9 as well as their in vitro anticancer activity against
several human cancer cell lines.
 Our new triazines showed better Rad6B inhibitory activities and
anticancer properties compared to previously reported Rad6B
inhibitor TZ9 in in vitro evaluations.