Optimised synthesis of diamino-triazinylmethyl benzoates as inhibitors of Rad6B ubiquitin conjugating enzyme

Article abstract of DOI:10.1016/j.tetlet.2014.10.122

Recently, we have identified the first inhibitors of Rad6B, an E2 enzyme essential for post-replication DNA repair and a potential new drug target for the treatment of breast cancer. We report two newly optimised synthetic routes to our [4-amino-6-(phenylamino)-1,3,5-triazin-2-yl]methyl 4-nitrobenzoate target compounds TZ8 and TZ9 with general applicability for further structure-activity relationship studies around this pharmacophore. The key step involved the condensation/cyclisation between phenylbiguanide and either ethyl bromoacetate or dimethyloxalate in good yield.

Full text of DOI:10.1016/j.tetlet.2014.10.122

Accepted Manuscript
Optimized synthesis of diamino-triazinylmethyl benzoates as inhibitors of
Rad6B ubiquitin conjugating enzyme
Hend Kothayer, Matteo Morelli, Ghali Brahemi, Abdalla A. Elshanawani,
Mansour E. Abu Kull, Osama I. El-Sabbagh, Malathy P.V. Shekhar, Andrew D.
Westwell
PII:
S0040-4039(14)01828-0
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.10.122
TETL 45351
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 August 2014
8 October 2014
22 October 2014
Please cite this article as: Kothayer, H., Morelli, M., Brahemi, G., Elshanawani, A.A., Abu Kull, M.E., El-Sabbagh,
O.I., Shekhar, M.P.V., Westwell, A.D., Optimized synthesis of diamino-triazinylmethyl benzoates as inhibitors of
Rad6B ubiquitin conjugating enzyme, Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/j.tetlet.
2014.10.122
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Leave this area blank for abstract info.
Optimized synthesis of diamino-
triazinylmethyl benzoates as inhibitors of
Rad6B ubiquitin conjugating enzyme
Hend Kothayer, Matteo Morelli, Ghali Brahemi, Abdalla A. Elshanawani, Mansour E. Abu Kull, Osama I. El-
Sabbagh, Malathy P. V. Shekhar, Andrew D. Westwell
NH2
R
a) (CO2Me)2, MeOH, reflux
b) LiAlH4, THF
NH
NH
R
N
N
N
N
NH2
O
c) O2NC6H4COCl, Et3N,
CH2Cl2
H
H
N
H
N
NO2
(R = Me, H, OMe)
O

1
Tetrahedron Letters
journal homepage: www.els evier.com
Optimized synthesis of diamino-triazinylmethyl benzoates as inhibitors of Rad6B
ubiquitin conjugating enzyme
Hend Kothayera,b, Matteo Morellia, Ghali Brahemia, Abdalla A. Elshanawanib, Mansour E. Abu Kullb, Osama I. El-
Sabbaghb, Malathy P. V. Shekharc, Andrew D. Westwella*
a School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, Wales, U.K.
b Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
c Karmanos Cancer Institute, Wayne State University School of Medicine, 110 E. Warren Avenue, Detroit, MI 48201, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Recently, we have identified the first inhibitors of Rad6B, an E2 enzyme essential for post-
replication DNA repair and a potential new drug target for treatment of breast cancer. We report
two newly optimized synthetic routes to our [4-amino-6-(phenylamino)-1,3,5-triazin-2-
yl]methyl 4-nitrobenzoate target compounds TZ8 and TZ9 with general applicability to further
structure-activity relationship studies around this pharmacophore. The key step involved the
Received in revised form
Accepted
Available online
condensation/cyclisation between
dimethyloxalate in good yield.
a
phenylbiguanide and either ethyl bromoacetate or
Keywords:
Triazines
2009 Elsevier Ltd. All rights reserved.
Biguanides
Rad6B inhibitors
Antitumour agents
Nutlins, currently being studied in clinical trials for cancer.4 A
further example is provided by the disulfiram-based inhibitors of
the E3 ligase enzyme BCA2 (Breast Cancer Associated protein-
2) discovered in our laboratories.5
The highly regulated ubiquitin-proteasome system responsible
for degradation of >80% of cellular proteins, has proved to be a
popular target for the development of new anticancer agents in
recent years. Research in this field has culminated in the clinical
approval of bortezomib (Velcade®) as the first-in-class small
molecule proteasome inhibitor for treatment of relapsed multiple
myeloma and mantle cell lymphoma.1 A major function of the
protein ubiquitination system serves to polyubiquitinate (tag with
small proteins) cellular proteins destined for proteasomal
degradation,2 however the role of ubiquitination in other
important cellular functions such as cell signalling and DNA
repair is now becoming more widely appreciated.3 Three
successive classes of enzymes of increasing structural and
mechanistic class diversity mediate protein ubiquitination. The
process starts with the ubiquitin-activating enzyme (E1) that
forms a thioester bond between its active site cysteine and the
carboxyl terminus of the ubiquitin protein. Transfer of ubiquitin
to the family of thioester-linked E2 ubiquitin conjugating
enzymes results in either direct transfer of ubiquitin to the protein
substrate, or interaction with the larger family of E3 ubiquitin-
protein ligases resulting in substrate mono- or poly-
ubiquitination.
Inhibitors of E2 ubiquitin conjugating enzymes are less well
studied than their E3 counterparts, despite their potential as
cancer drug targets in a number of cases. For example, the E2
enzyme Rad6 has been found to be essential for post-replication
DNA repair, and Rad6B over-expression is reported in breast
cancer cell lines and tumours. Constitutive Rad6B over-
expression in non-transformed cells is associated with induction
of cancer phenotypic changes including centrosome
amplification, abnormal mitosis and aneuploidy.6 The ability of
Rad6B to ubiquitinate β-catenin leads to conjugates insensitive to
proteasomal degradation. Consequent stabilization and activation
of oncogenic β-catenin provides further evidence of the
therapeutic potential of Rad6B as a drug target, particularly in
breast cancer.7
An important therapeutic advance in this area was the recent
report from our laboratories of the first selective inhibitors of
Rad6 ubiquitin conjugating enzyme.8 Virtual screening of a
library of drug-like structures against a pharmacophore model
generated from the conserved key residues stabilizing the E2-
ubiquitin thioester intermediate, identified a substituted diamino-
triazine core structure as a starting point for analogue synthesis.
Triazine analogue synthesis coupled to in vitro anticancer
evaluation in Rad6B-relevant models led to the identification of
Extensive efforts in recent years to target various classes of E3
ubiquitin ligases deregulated in cancer cells has led to the
development of a number of potential cancer therapeutics.
Amongst the most well developed examples are the Mdm2 (E3
———
ligase)–p53 protein-protein interaction inhibitors known as the
*
Corresponding author. Tel.: +44 (0)2920875800. E-mail address: WestwellA@cf.ac.uk (A. Westwell).

2
Tetrahedron Letters
4-
[4-amino-6-(phenylamino)-1,3,5-triazin-2-yl]methyl
NH
R
R
nitrobenzoates TZ8 and TZ9 as novel and selective Rad6B-
inhibitory anticancer lead compounds. TZ8 and TZ9 were found
to inhibit proliferation, colony formation and migration, leading
to G2-M cell cycle arrest and apoptosis in MDA-MB-231 breast
cancer cells.
The trisubstituted 1,3,5-triazine scaffold plays an important
role in medicinal chemistry, since a number of triazine-based
compounds are reported to possess useful biological properties.
Biologically relevant trisubstituted triazines include the
antigastric ulcer agent irsogladine,9 commonly used in Japan and
also shown to possess anti-angiogenic/antimetastatic activity;10
plus other agents with anticancer,11 antimalarial,12 antimicrobial13
and anti-angiogenic14 properties.
(a)
NH NH
OEt
+
+
NC
.HCl
NH2
HO
N
NH2
H
NH2
N
H
N
H
O
2
1a (R=Me)
1b (R=H)
4
3a,b
(b)
N
NH2
NH2
NO2
N
N
N
(c)
R
R
O
OH
N
H
N
N
H
N
O
TZ 8 (R=Me)
TZ 9 (R=H)
5a,b
aReagents and conditions: (a) 3 M HCl (aq), 90 oC, 5h; (b) NaOEt, EtOH, reflux, 3 h;
(c) 4-nitrobenzoyl chloride, Et3N, CH2Cl2, reflux, 12 h.
Scheme 1. Initial synthetic approach to substituted diamino-s-triazinylmethyl
benzoates TZ8 and TZ9.
The reagent, ethyl glycolate, was considered to be the main
reason for the complex mixture of products arising from the
second step of Scheme 1, since we postulated that the unprotected
nucleophilic alcohol function would be capable of participating in
side reactions (for example self-condensation) under the strongly
basic conditions of the reaction. We therefore tested a number of
related functionalized esters in the triazine ring-forming step.
These alternative synthetic approaches are outlined in Scheme 2.
There are two major conceptual approaches used for the
synthesis of trisubstituted 1,3,5-triazines. The first main method
involves the successive base-promoted nucleophilic substitution
of chlorine atoms starting from commercially available cyanuric
chloride (2,4,6-trichloro-1,3,5-triazine). Although widely used,
this method has significant drawbacks mainly relating to the
harsh conditions and low yields associated with nucleophilic
substitution of the third chlorine atom to obtain the final
trisubstituted product. The method has recently been extended to
the synthesis of substituted triamino-triazine derivatives using an
efficient microwave-promoted aryl and heteroaryl amination to
install the third triazine substituent.15 In the case of our synthetic
triazine targets TZ8 and TZ9, this approach was discounted due
to the difficulty of forming the required carbon-carbon bond to
the methyl benzoate substituent. Alkyl group substitution into
triazines is often accomplished through the use of highly reactive
Grignard reagents, which was not appropriate for our target
compounds.
The second major approach to the synthesis of trisubstituted
triazines involves construction of the triazine ring through
condensation/cyclisation chemistry. For example, a common
approach involves the reaction of substituted biguanides with
carboxylic acid derivatives such as esters,16 nitriles,17 acyl
chlorides or acid anhydrides. This method has been used for
example in the microwave-promoted synthesis of 2-
(arylmethyl)amino-4-arylamino-6-alkyl-1,3,5-triazines via the
reaction of phenylbiguanide derivatives and simple aliphatic
esters.16
NH2
R
NO2
NH NH
N
N
EtO
R
+
X
O
N
H
N
H
NH2
N
H
N
O
O
6
TZ 8 (R=Me)
TZ 9 (R=H)
3a,b
(X = OTIPS, OBn,
OCH2C6H4pNO2, Br, CO2Me)
Scheme 2. Alternative synthetic approaches to diamino-s-triazinylmethyl
benzoates, avoiding the use of ethyl glycolate.
Initial studies were focused on protection of ethyl glycolate
using an alcohol protecting group (6; X = OTIPS, OBn,
OCH2C6H4p-NO2) that could later be readily removed to reveal
the
{4-amino-6-(phenylamino)-[1,3,5]triazin-2-yl}methanol
intermediates 5a,b for conversion into the final products. The use
of the triisopropylsilyl (TIPS) or benzyl (Bn) protecting groups
was found not to be successful, with both these protecting groups
being unstable under the basic conditions of the triazine
cyclisation reaction (sodium ethoxide in ethanol under reflux),
and giving rise to complex product mixtures. The p-nitrobenzyl
derivative of ethyl glycolate was found to be unstable under the
strongly basic conditions of triazine formation, meaning that
potential direct access to final triazine products TZ8 and TZ9
could not be realised.
A more successful approach to the target trisubstituted
triazines involved the use of ethyl bromoacetate (6; X=Br) as the
biguanide cyclisation partner. For these reactions, we found that it
was necessary to use the free base of phenylbiguanide, obtained
by treatment of the hydrochloride salt with 25% sodium
methoxide in methanol, to give the best results (see
Supplementary Data). Reaction of the phenylbiguanide (3a,b) free
bases with ethyl bromoacetate in methanol gave the required 6-
Further development of our trisubstituted triazine-based lead
compounds was severely compromised by poor yielding and
unreliable synthetic procedures. Following the discovery of the
first selective E2 ubiquitin conjugating enzyme inhibitors of
Rad6B, it was imperative to develop a general and reliable
synthetic route to this target class of compounds. In this paper we
report the discovery and development of two new synthetic
approaches to Rad6B-inhibitory trisubstituted triazines, including
new structural analogues.
(bromomethyl)-N2-aryl-1,3,5-triazine-2,4-diamines
7a,b
in
moderate yields (39-43%) following chromatographic purification
(Scheme 3). Nucleophilic substitution of the bromo group using
4-nitrobenzoic acid promoted by tetrabutylammonium fluoride
(TBAF) in refluxing THF gave rise to the target triazines TZ8 and
TZ9 in high yields (79-86%) following column chromatography.
Although this alternative synthetic route using ethyl bromoacetate
as a key reagent was successful, the moderate yields for the
triazine formation step encouraged us to search for alternative
methods to deliver the target compounds in high yields and
quantities sufficienty for full biological characterization.
Our initial synthesis of Rad6-inhibitory triazines TZ8 and TZ9
was adapted from the method of Saczewski et al.,11 and was
accomplished in three chemical steps from commercially
available starting materials as outlined in Scheme 1.8a Reaction of
toluidine (1a) or aniline (1b) with dicyandiamide (2) with heating
under acidic conditions (3
M HCl) gave the required
phenylbiguanide hydrochlorides 3a,b in high yields (84% and
73%, respectively).18 Formation of trisubstituted triazines by
reaction of biguanides 3a,b with ethyl glycolate (4) to give the {4-
amino-6-(phenylamino)-[1,3,5]triazin-2-yl}methanol
NH2
NH2
intermediates 5a,b proved to be a low yielding and unreliable
synthetic step, severely compromising the quantity of pure
triazine intermediates 5a,b that could be obtained for conversion
into final compounds for anticancer testing. Following extensive
chromatographic purification, reaction of intermediates 5a,b with
4-nitrobenzoyl chloride under basic conditions provided the
required target triazines TZ8 and TZ9.
R
R
NO2
R
NH NH
(a)
(b)
N
N
N
N
O
Br
N
H
N
H
NH2
N
H
N
N
H
N
O
TZ 8 (R=Me)
TZ 9 (R=H)
3a (R=Me)
3b (R=H)
7a,b
aReagents and conditions: (a) BrCH2COOEt, MeOH (39-43 %); (b) 4-NO2C6H4COOH, TBAF, THF, reflux (79-86 %)
Scheme 3. Synthetic route to diamino-s-triazinylmethyl benzoates using ethyl
bromoacetate.

3
Further success in finding alternative synthetic routes to our
target triazines was achieved using dimethyl oxalate as the
biguanide reaction partner in place of the originally used ethyl
glycolate, as outlined in Scheme 4. Heating a mixture of
arylbiguanide free base 3a-c with excess dimethyl oxalate gave
the intermediate methyl 4-amino-6-(arylamino)-1,3,5-triazine-2-
carboxylates 8a-c in high yields (83-92%) following
recrystallisation from methanol. Reduction of the intermediate
triazine carboxylates 8a-c using lithium aluminium hydride in
anhydrous THF then gave the key {4-amino-6-(phenylamino)-
[1,3,5]triazin-2-yl}methanol intermediates 5a-c in moderate
yields following purification. Finally, esterification of the
hydroxymethyl intermediate using 3- or 4-nitrobenzoyl chlorides
gave the target triazines 9a-f, including TZ8 and TZ9, in good
yields following recrystallisation from methanol or acetonitrile.
Algerian Consulate (PhD studentship to G.B.). We thank Miss
Frances Harwood (MPharm student, Cardiff University) for
preliminary chemistry studies, and the EPSRC National Mass
Spectrometry Facility (Swansea, U.K.) for provision of accurate
mass spectrometric analysis.
Supplementary data
Supplementary data (experimental
procedures and
characterization data, including NMR and mass spectrometry
data for all isolated compounds) associated with this article can
be found in the online version.
References
1. Kisselev, A. F.; van der Linden, W. A.; Overkleeft, H. S. Chem.
Biol. 2012, 19, 99-115.
NH2
NH2
2. Burger, A. M.; Seth, A. Eur. J. Cancer 2005, 40, 2217-2229.
3. Husnjak, K.; Dikic, I. Ann. Rev. Biochem. 2012, 81, 291-322.
4. Wang, W.; Hu, Y. Med. Res. Rev. 2012, 32, 1159-1196.
5. Brahemi, G.; Kona, F. R.; Fiasella, A.; Buac, D.; Soukupovà, J.;
Brancale, A.; Burger, A. M.; Westwell, A. D. J. Med. Chem. 2010,
53, 2757-2765.
R
R
R
NH
NH
NH2
N
N
N
N
(a)
(b)
OMe
OH
N
H
N
H
N
H
N
N
H
N
O
5a-c
8a-c
3a (R=Me)
3b (R=H)
3c (R=OMe)
(c)
NH2
aReagents and conditions: (a) dimethyloxalate, MeOH, reflux (83-92 %);
(b) LiAlH4, THF (29-44%); (c) 3- or 4-nitrobenzoyl chloride, NEt3, CH2Cl2
(52-70 %)
6. Shekhar, M. P. V.; Lyakhovich, A.; Visscher, D. W.; Heng, H.;
Kondrat, N. Cancer Res. 2002, 62, 2115-2124.
R
N
N
O
N
H
N
7. Shekhar, M. P. V.; Gerard, B.; Pauley, R. J.; Williams, B. O.; Tait,
L. Cancer Res. 2008, 68, 1741-1750.
NO2
O
9a (R=Me, 4-NO2, TZ8)
9b (R=H, 4-NO2, TZ9)
8. a) Sanders, M. A.; Brahemi, G.; Nangia-Makker, P.; Balan, V.;
Morelli, M.; Kothayer, H.; Westwell, A. D.; Shekhar, M. P. V.
Mol. Cancer Therap. 2013, 12, 373-383; b) Kothayer, H.;
Elshanawani, A. A.; Abu Kull, M. E.; El-Sabbagh, O. I.; Shekhar,
M. P. V.; Brancale, A.; Jones, A. T.; Westwell, A. D. Bioorg.
Med. Chem. Lett. 2013, 23, 6886-6889.
9c (R=OMe, 4-NO2
9d (R=Me, 3-NO2
9e (R=H, 3-NO2
9f (R=OMe, 3-NO2
)
)
)
)
Scheme 4. Synthetic route to diamino-s-triazinylmethyl benzoates 9a-e using
dimethyloxalate.
9. Hiraishi, H.; Haruma, K.; Miwa, H.; Goto, H. Aliment.
Pharmacol. Therap. 2010, 31, 824-833.
10. Nozaki, S.; Maeda, M.; Tsuda, H.; Sledge, G. W. Breast Cancer
Res. Treat. 2004, 83, 195-199.
In conclusion, we have successfully developed two general
and reliable synthetic routes to trisubstituted diamino-s-triazines
bearing carbon-based substituents (methyl benzoates). This
development work facilitates the availability of lead compounds
TZ8 and TZ9 in sufficient quantities and purity for further
development, and provides a general route of access to new
compounds bearing this pharmacophore. The generality of the
new synthetic route is further illustrated by the preparation of
new analogues 9c-f for structure-activity relationship studies.
Further work on related trisubstituted s-triazines is continuing to
further elaborate the potential therapeutic activity of this novel
class of compounds.
11. Saczewski, F.; Bulakowska, A.; Bednarski, P.; Grunert, R. Eur. J.
Med. Chem. 2006, 41, 219-225.
12. Agarwal, A.; Srivastava, K.; Puri, S. K.; Chauhan, P. M. S.
Bioorg. Med. Chem. Lett. 2005, 15, 531-533.
13. a) Solankee, A.; Kapadia, K.; Ciric, A.; Sokovic, M.;
Doytchinova, I.; Geronikaki, A. Eur. J. Med. Chem. 2010, 45,
510-518; b) Patel, R. V.; Kumari, P.; Rajani, D. P.; Pannecouque,
C.; De Clercq, E.; Chikhalia, K. H. Fut. Med. Chem. 2012, 4,
1053-1065.
14. Dao, P.; Jarray, R.; Le Coq, J.; Lietha, D.; Loukaci, A.;
Lepelletier, Y.; Hadj-Slimane, R.; Garbay, C.; Raynaud, F.; Chen,
H. Bioorg. Med. Chem. Lett. 2013, 23, 4552-4556.
Acknowledgments
15. Dao, P.; Garbay, C.; Chen, H. Tetrahedron 2012, 68, 3856-3860.
16. Chen, H.; Dao, P.; Laporte, A.; Garbay, C. Tetrahedron Lett.
2010, 51, 3174-3176.
The authors are grateful to the Higher Education Ministry of
the Arab Republic of Egypt for the award of a Channel
Scholarship (to H.K.). We acknowledge the support of the
17. Shie, J.-J.; Fang, J.-M. J. Org. Chem. 2007, 72, 3141-3144.
18. LeBel, O.; Maris, T.; Duval, H.; Wuest, J. D. Can. J. Chem. 2005,
83, 615-625.