Identification and characterization of the first fragment hits for SETDB1 Tudor domain

Article abstract of DOI:10.1016/j.bmc.2019.07.020

SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harb

Full text of DOI:10.1016/j.bmc.2019.07.020

Accepted Manuscript
Identification and Characterization of the First Fragment Hits for SETDB1 Tu-
dor Domain
Pavel Mader, Rodrigo Mendoza-Sanchez, Aman Iqbal, Aiping Dong, Elena
Dobrovetsky, Victoria B. Corless, Sean K. Liew, Scott R. Houliston, Renato
Ferreira De Freitas, David Smil, Carlo C. Dela Sena, Steven Kennedy, Diego
B. Diaz, Hong Wu, Ludmila Dombrovski, Abdellah Allali-Hassani, Jinrong
Min, Matthieu Schapira, Masoud Vedadi, Peter J. Brown, Vijayaratnam
Santhakumar, Andrei K.Yudin, Cheryl H. Arrowsmith
PII:
S0968-0896(19)30870-3
https://doi.org/10.1016/j.bmc.2019.07.020
BMC 15004
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 May 2019
3 July 2019
10 July 2019
Please cite this article as: Mader, P., Mendoza-Sanchez, R., Iqbal, A., Dong, A., Dobrovetsky, E., Corless, V.B.,
Liew, S.K., Houliston, S.R., De Freitas, R.F., Smil, D., Sena, C.D., Kennedy, S., Diaz, D.B., Wu, H., Dombrovski,
L., Allali-Hassani, A., Min, J., Schapira, M., Vedadi, M., Brown, P.J., Santhakumar, V., K.Yudin, A., Arrowsmith,
C.H., Identification and Characterization of the First Fragment Hits for SETDB1 Tudor Domain, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.020
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.

Identification and Characterization of the
First Fragment Hits for SETDB1 Tudor
Domain
Leave this area blank for abstract info.
Pavel Mader,† Rodrigo Mendoza-Sanchez,# Aman Iqbal†, Aiping Dong,† Elena Dobrovetsky,† Victoria B.
Corless,# Sean K. Liew,# Scott R. Houliston,§ Renato Ferreira De Freitas,† David Smil,† Carlo C. Dela Sena,†
Steven Kennedy,† Diego B. Diaz,# Hong Wu,† Ludmila Dombrovski,† Abdellah Allali-Hassani,† Jinrong Min,†
Matthieu Schapira, †,‡ Masoud Vedadi,†,‡ Peter J. Brown,† Vijayaratnam Santhakumar,† Andrei K.Yudin,*,#
Cheryl H. Arrowsmith*,†,§
N N
N
O
N
H
5
SPR Kd = 3.5mM
1

Identification and Characterization of the First Fragment Hits for SETDB1 Tudor Domain
Pavel Mader,† Rodrigo Mendoza-Sanchez,# Aman Iqbal†, Aiping Dong,† Elena Dobrovetsky,† Victoria B. Corless,# Sean K. Liew,#
Scott R. Houliston,§ Renato Ferreira De Freitas,† David Smil,† Carlo C. Dela Sena,† Steven Kennedy,† Diego B. Diaz,# Hong
Wu,† Ludmila Dombrovski,† Abdellah Allali-Hassani,† Jinrong Min,† Matthieu Schapira, †,‡ Masoud Vedadi,†,‡ Peter J. Brown,†
Vijayaratnam Santhakumar,† Andrei K.Yudin,*,# Cheryl H. Arrowsmith*,†,§
†Structural Genomics Consortium, University of Toronto, Toronto, Canada
#Department of Chemistry, University of Toronto, Toronto, Canada
‡Department of Pharmacology and Toxicology, University of Toronto, Toronto, Canada
§Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Canada
ABSTRACT
SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified
in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In
addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone
sequences containing both methylated and acetylated lysines, and likely contributes to its localization on
chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have
identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem
Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and
analogues and the biophysical characterization of key compounds. These confirmed small molecule
fragments will inform the development of potent antagonists of SETDB1 interaction with histones.
INTRODUCTION
Post-translational modifications (PTMs) such as methylation, phosphorylation, acetylation, ubiquitination,
and SUMOylation of histone proteins impact gene expression by altering chromatin compaction or
2

recruiting effector proteins.1-3 Methylation of histones occurs predominantly at the lysine and arginine
sidechains of histone 3 (H3K4, H3K9, H3K27, H3K36, H3K79 and H3R2) and histone 4 (H4K20 and
H4R3). Unlike some other PTMs, methylation of a lysine or arginine sidechain does not change overall
charge but results in subtle alterations in size and hydrophobicity, which can influence chromatin packing.4
Transcriptional activation or repression is dependent on the position and degree of methylation5 and
aberrant methylation of histones can result in dysregulation of cell growth and development of cancer.6
Many of the histone methyl transferases have been extensively studied and selective inhibitors have been
effective in preclinical models.7-9
Human SETDB1 is a histone-lysine methyltransferase (HMT), which specifically trimethylates histone
H3 lysine 9 (H3K9me3). It is deemed central in the silencing of specific euchromatic genes.10 Recent
studies have also suggested that SETDB1 could be an oncogene. Its overexpression is crucial to the growth
of non-small and small cell lung cancer cell lines and has been reported in various human cancers.11-13
Overexpressed SETDB1 increases H3K9me3 leading to alterations in gene expression.14
SETDB1 contains a tandem tudor domain (TTD), a methyl-DNA binding domain (MBD), and a SET
domain.15 The MBD of SETDB1 promotes direct binding to methylated DNA sites and mediates
trimethylation of H3K9 in a site-directed fashion. The SET domain catalytically “writes” the epigenetic
methyl mark, which then goes on to recruit proteins belonging to the Heterochromatin Protein 1 (HP1)
family to control gene repression.10 Tudor domains are associated with the recognition of different
methylation levels of lysine and arginine residues and the TTD of SETDB1 has been associated with the
recognition of di- and trimethylation states of H3K9.16,17 To the best of our knowledge, and despite the
evident therapeutic importance of SETDB1, no antagonists of this protein have been reported until
recently.18 We successfully crystallized the truncated tandem tudor domain of SETDB1 in complex with a
histone 3 peptide dimethylated at lysine 9 and acetylated at lysine 14 (H3K9me2K14ac).19 The co-crystal
3

structure shows that H3K9me2K14ac interacts with the protein at two interconnected pockets within the
TTD: a small pocket where the dimethyl lysine (Kme2) binds (hereafter called the Kme binding pocket) and
a relatively larger and deeper pocket engaged by the acetyl lysine (the Kac pocket) (Figure 1). We used
SiteMap (Schrödinger, New York) to evaluate the druggability of the peptide binding site. Two distinct sites
were automatically identified, corresponding to the Kac and the Kme3 binding cavities, with druggability
scores (DScores) of 0.92 and 075 respectively, placing the Kme site clearly in the "undruggable" category,
and the Kac site at the boundary of the "difficult" and "druggable" categories.
4

Figure 1. Crystal structure of SETDB1-TTD in complex with H3K9me2K14ac peptide
(NKQTARK(Me2)STGGK(Ac)APRKQR: Not all amino acids are resolved) (PDB: 6BHD).
We used a crystal soaking fragment screening approach to identify small-molecule ligands occupying the
Kme and/or Kac pockets of SETDB1-TTD. Crystal structures revealed binding modes that could enable
structure-guided hit optimization toward the development of SETDB1 antagonists.
RESULTS AND DISCUSSIONS
Hit Identification. In order to identify small molecule antagonists of SETDB1-TTD, we screened a
fragment library of 252 compounds by soaking the apo crystals of SETDB1-TTD with pools of 3
compounds and identified a single fragment hit (1). Co-crystal structure of 1 with SETDB1-TTD revealed
that it binds in the Kme pocket (Figure 2). However, fragment 1 did not show any HSQC shift (>2ppm) at
concentrations as high as 10 mM, indicating very weak binding.
As a major part of the H3K9me2K14ac peptide occupies and forms hydrogen bonding interactions in and
around the Kac pocket, fragments binding in the Kac pocket are likely to have higher ligand efficiency and
can potentially be optimized to improve affinity. To test this hypothesis, we selected and soaked 5
constrained analogues of known bromodomain inhibitors20 synthesized in our laboratory (Supplementary
Figure 1). Bromodomain inhibitors are known acetyl lysine mimics21 and could potentially bind to the Kac
pocket of the SETDB1-TTD. Out of 5 fragments, one fragment (2) was found to bind to SETDB1-TTD
occupying the TTD Kac pocket (Figure 2). Binding of 2 to SETDB1-TTD was also confirmed by HSQC
NMR at 5mM (Supplementary Figure 2). In order to explore the SAR of these hits and assess the feasibility
of improving their activity, a small set of analogs was synthesized as described according to the schemes
described in the chemistry section and tested by HSQC NMR and/or by SPR.
5

Figure 2. Superimposed crystal structures of the SETDB1 TTD in complex with 1 (PDB: 5KE2) and 2
(PDB: 6AU2) occupying the Kme and Kac pockets, respectively.
Chemistry
The tricyclic triazole 2 was prepared from commercially available 2-aminobenzyl alcohol (11), which was
reacted with ethylchloroacetate (12) in the presence of sodium hydride to give the lactam. Treatment of the
lactam with Lawesson’s reagent led to the corresponding thioamide 13, which was subsequently condensed
with acethydrazide (14) to give the desired tricycle 2 in 6% yield overall yield (Scheme 1).
Scheme 1a
6

S
N
N
O
NH2 OH
O
HN
a
O
NH2
Me
b
N
+
Cl
Me
N
H
+
O
EtO
12
11
14
13
2
aReagents and conditions: (a) (1) NaH, Toluene, 80 ºC; (2) Lawesson`s reagent, THF, 60 ºC (b) n-BuOH,
100 ºC, 6% overall yield.
N-Derivatized tricycle 4 was synthesized by the route outlined in Scheme 2. Diamine 15 was refluxed
with ethylchloroacetate in toluene in the presence of triethylamine to obtain 16 as the major product. The
crude product was selectively Boc-protected at the secondary amine to furnish 17. The resulting material
was carried directly through to intramolecular cyclization mediated by potassium tert-butoxide to give the
lactam 18 in 35% isolated yield over three steps after recrystallization. Treatment of the cyclic amide with
Lawesson’s reagent yielded thioamide 19, which was then refluxed with acethydrazide for three days to
furnish the triazole 20 in 80% yield after recrystallization. Finally, the Boc group was deprotected under
acidic conditions and the resulting secondary amine was capped with acetyl chloride to give compound 4.
Scheme 2a
O
NH2
NH2
NH2 NH2
HN
OEt
OEt
c
NBoc
18
a
b
N
N
H
Boc
O
O
17
15
16
S
N
N
N N
N
N
N N
N
e
d
HN
O
NBoc
f
Me
Me
Me
g
NBoc
N
NH
Me
19
20
21
4
aReagents and conditions: (a) ethylchloroacetate, TEA, Toluene, 100 ºC (b) Boc2O, cat.DMAP, THF, rt (c)
KOtBu, THF, 0 ºC, 35% over 3 steps (d) Lawesson’s reagent, THF, 60 ºC, 53% (e) acethydrazide, n-BuOH,
100 ºC, 80% (f) 1.25 M HCl in MeOH, 50 ºC, 97% (g) AcCl, TEA, DMF, rt, 54%
7

Triazoles 5-8 were prepared according to Scheme 3. Diamine 15 was treated with Boc anhydride followed
by acetic anhydride to give the Boc-protected acetamide 25, which was treated with Lawesson’s reagent to
furnish thioamide 26. Triazole 27 was obtained in moderate yield by treating 26 with acethydrazide under
microwave irradiation. Deprotection of the Boc group was accomplished with hydrochloric acid in
methanol and the free amine was treated with acetic anhydride or with methanesulfonyl chloride in the
presence of base to yield the corresponding acetamide 5 and sulfonamide 7, respectively. After the
deprotection of triazole 27 under the same conditions, the free amine of the crude product was treated with
triphosgene followed by methylamine to give 6. The corresponding triazole carboxylic acid 8 was prepared
by reacting 28 with acethydrazide.
Scheme 3a
8

O
NH2
NH2
Me
NH
a
c
b
NH2
NHBoc
NHBoc
25
15
24
S
N N
N N
N
e
Me
NH
d
Me
Me
Me
Me
NHBoc
N
O
NHBoc
26
N
Me
H
5
27
f
g
N N
N N
N
Me
Me
Me
Me
N
O
O
O
Me
S
N
N
H
N
Me
H
H
6
7
N N
N
N
O
Me
h
Me
Me
O
COOH
8
28
aReagents and conditions: (a) Boc2O, TEA, THF, rt, 99% (b) Ac2O, DCM, rt, 89% (c) Lawesson’s reagent,
THF, 60 ºC, 64% (d) acethydrazide, n-BuOH, 100 ºC, 47% (e) (1) 1.25 M HCl in MeOH, 50 ºC, 65%; (2)
Ac2O, TEA, MeCN, rt, 65% (f) (1) 1.25 M HCl in MeOH, 50 ºC; (2) triphosgene, TEA, DCM, rt; (3)
MeNH2, MeOH, rt (g) (1) 1.25 M HCl in MeOH, 50 ºC; (2) Methane sulfonyl chloride, TEA, MeCN, rt (h)
acethydrazide, EtOH, reflux, 35%
para-Substituted triazole analogues 9a-e were synthesized from intermediate 25 (Scheme 4).
Halogenation of 25 with NBS and NCS gave 29a and 29b, respectively. Following the same protocol for
converting 25 into 5 (Scheme 4), the corresponding halogenated intermediates 29a and 29b were converted
to 9a and 9b. Palladium-catalyzed cyanation of 9a gave 9c, and Suzuki coupling of 9a with the
corresponding boronic acids gave 9d and 9e.
Scheme 4a
9

S
O
O
Me
NH NHBoc
Me
NH NHBoc
Me
NH NHBoc
c
b
a
25
X
X
29a
29b
X = Br 88%
X = Cl 52%
30a
30b
X = Br 83%
X = Cl 60%
N N
N N
N
Me
Me
NHBoc
Me
Me
N
d
O
N
Me
H
X
X
31a
31b
X = Br 90%
X = Cl 56%
9a
9b
X = Br 77%
X = Cl 87%
N N
N
N N
Me
Me
O
e
f
Me
Me
9a
N
O
N
Me
H
N
Me
H
9d
9e
R = Me 70%
R = Cyclopropyl 58%
R
9c
36%
CN
aReagents and conditions: (a) NBS or NCS, 10% triphenylphosphine sulfide, CHCl3, rt (b) Lawesson`s
reagent, THF, 50 ºC (c) acethydrazide, n-BuOH, 100 ºC (d) (1) 1.25 M HCl in MeOH; (2) Ac2O, TEA,
MeCN, rt (e) 10% Pd(OAc)2, K4Fe(CN)6, i-PrOH, DMF, 140 ºC (f) 10% Pd(OAc)2, RB(OH)2, K2CO3,
dioxane / water, 100 ºC
Synthesis of the triazole fragment-linked molecule 10 began by condensing intermediate 28 with
intermediate 32 to give the corresponding triazole carboxylic acid 33. The alcohol 34, which was obtained
by the reduction of the carboxylic acid 33, was converted into the corresponding phthalimide 35 under
Mitsunobu conditions. Treatment of 35 with hydrazine to remove the phthalimide group followed by
acetylation and deprotection of the Boc group gave intermediate 37.
Scheme 5a
10

N N
N
N N
N
NHBoc
BocHN
BocHN
N
O
Me
H
N
Me
Me
OH
a
b
+
H2N
O
COOH
O
32
33
28
34
N N
N
N N
N
N N
BocHN
H2N
BocHN
Me
Me
e
c
d
Me
O
N
N
NHAc
37
NHAc
36
35
O
aReagents and conditions: (a) EtOH, reflux, 57% (b) LAH, THF, 54% (c) DIAD, PPh3, phthalimide, THF,
rt, 96% (d) (1) hydrazine, MeOH, reflux; (2) N-acetyl-N-methoxyacetamide, MeOH, rt, 51% (e) TFA, 67%
The peptide intermediate 40 for the synthesis of 10 was synthesized from peptide 38, which was in turn
obtained using a standard 2-chlorotrityl resin protocol. Hydrogenolysis of the Cbz group of 38 followed by
dimethylation via reductive amination of the lysine side chain with formaldehyde gave peptide 40 in 85%
yield over two steps (Scheme 6). Coupling of the acid 40 with the amine 37 was followed by deprotection
of Boc and tBu groups with TFA to give 10 (Scheme 7).
Scheme 6a
O
O
O
H
N
O
O
BocHN
NH2
OH
O
O
H
N
N
H
N
H
b
BocHN
OH
a
O
O
N
H
N
H
O
O
O
39
38
NHCbz
OH
N
N
O
O
H
H
O
Me
H2N
N
N
O
O
H
N
N
N
H
N
H
Me
BocHN
OH
NHAc
O
O
N
H
N
H
c
HO
O
O
O
40
N
10
Me
Me
N
11

aReagents and conditions: (a) Pd/C, H2, AcOH, EtOH, quant. (b) HCHO, NaBH(OAc)3, MeOH, 85% (c)
(1) Compound 37, EDC.HCl, DCM, rt; (2) TFA, DCM, rt, 30%
Biochemical evaluation and SAR of the fragments occupying Kme pocket. In the co-crystal structure of
the fragment hit 1 with SETDB1-TTD, the phenyl ring of the tetrahydroisoquinoline core occupies the
aromatic cage formed by W358, W363 and F379 and the carbonyl group makes hydrogen bonding with
D382 (Figure 3a). However, 1 did not show any significant shift (>2ppm shift) in the HSQC NMR of
SETDB1-TTD at concentrations up to 10 mM, indicating that 1 interacts very weakly with the Kme pocket.
Superimposed structures of SETDB1-TTD in complex with 1 and H3K9me2K14ac within the binding
pocket (Figure 3b) indicate that the fragment could be grown towards the Kac pocket, which could
potentially pick up additional interactions with the protein and thus improve affinity. We purchased a
library of 19 amides (Supplementary figure 3) and synthesized 3 amides, extending fragment 1 in the
desired direction. Ligand-protein interactions were assessed using HSQC NMR as a first pass screen and
considered as significant shift if at least one peak showed >2ppm shift. None of the 22 analogs tested
showed HSQC NMR shift at 10 mM. However, we were able to obtain the co-crystal structure of 3 with
SETDB1-TTD, wherein the tetrahydroisoquinoline core of 3 occupies the aromatic cage of the Kme pocket,
and the amide extends towards the Kac pocket (Figure 3c). No electron density was observed for the furan
group, indicating suboptimal interactions with the protein. As none of the analogs of 1 showed any
significant activity, we turned our focus toward identifying fragments that occupy the Kac pocket.
12

Figure 3. Crystal structure of SETDB1 TTD in complex with fragments. (a) Binding mode of 1 in the
aromatic cage of Kme pocket. (b) Superimposed structures of 1 (grey) (PDB: 5KE2) with the co-crystal
structure of H3K9me2K14ac (orange) (PDB: 6BHD). (c) Binding mode of 3 in the aromatic cage of the
Kme pocket (PDB: 5KE3). (d) Chemical Structure of 3.
Biochemical evaluation and SAR of fragments occupying the Kac pocket. In the co-crystal structure of
SETDB1-TTD in complex with 2, the triazole ring of 2 participates in -stacking interactions with F332
and its phenyl ring is buried in the narrow pocket, forming hydrophobic interactions with I333, G300, F296,
and C329. Both the nitrogen atom in the triazole ring and the oxygen atom in the seven membered ring form
hydrogen bonds with two water molecules rather than mimicking the acetyl group of the H3K9me2K14ac
13

as we anticipated (Figure 4a). At 10 mM, 2 showed a significant HSQC NMR shift (>2ppm shift).
Replacing O with N-Acetyl group for further branching, the simple acetamide derivative 4 showed a
significant HSQC NMR shift (Supplementary Figure 2), but neither compound showed significant binding
by ITC (not shown). In the H3K9me2K14ac co-crystal structure, the acetyl group of K14ac forms hydrogen
bonding interactions with G300 and Y389, both of which are found in the vicinity of 2. We therefore
synthesized the corresponding open chain analogue 5 to allow acetyl group to be flexible and to introduce
H-bond donor so that the NH-acetyl group can make optimal H-bond and obtained a co-crystal structure. As
expected, the N-acetyl group of 5 formed hydrogen bonds with G300 and Y389 (Figure 4b). A slight shift in
the scaffold binding pose compared with 2 produced an additional hydrogen bond between the trizole ring
of 5 and the side-chain of R394, but this also leads to reduced interactions at the I333 hydrophobic cavity
and loss of -stacking with F332 (Figure 4c). Compound 5 showed a significant HSQC NMR shift at 5 mM
(Supplementary Figure 2) and displayed KD values in low millimolar ranges by SPR and ITC (Kd=3.5 mM)
(Supplementary Figure 4).
14

e
N
N
N
N
N N
N
O
N
N
O
N
O
N
H
4
2
5
15

Figure 4. (a) Binding mode of 2 in the Kac pocket. (b) Binding mode of 5 in the Kac pocket (PDB: 6AU3).
(c) Superimposed structures of 2 (yellow) with 5 (grey) (d) ITC data of 5 (e) Chemical Structures of 2, 4
and 5.
We also synthesized the corresponding urea 6, sulfonamide 7, and carboxylic acid 8 (Figure 5). All three
compounds were predicted to form additional or alternate hydrogen bonds with the protein, however, only
the sulfonamide analog 7 showed a significant HSQC NMR shift at 5 mM (Supplementary Figure 2) and
affinity comparable to 5 by SPR. (Supplementary Figure 4) (Kd = 4mM – 17mM).
N N
N
N N
N
N N
N
O
O
O
S
NH2
N
N
OH
H
H
O
6
7
8
Figure 5. (a) Chemical Structures of 6-8.
Despite forming a number of hydrogen bonding interactions with the protein, 5 showed only weak binding
by ITC and SPR, which is likely due to incomplete occupation of the large pocket that is occupied by the
native peptide H3K9me2K14ac. We therefore decided to grow the fragment 5 into the pocket occupied by
the peptide. We initially focused on simple substitutions at the para position of the phenyl ring of 5 to
rescue hydrophobic interactions observed between 2 and I333. However, none of the para-substituted
analogues showed significant improvement of binding compared to 5 as measured by SPR (Table 1 and
Supplementary Figure 4).
16

Table 1. SPR data of substituted analogues of 5. Binding of compounds 5, 9a, 9b, 9c and 9d to SETDB1
TTD was confirmed by SPR with Kd values mostly in single digit milimolar.
N N
N
O
N
H
R2
R1
Compound
R1
H
R2
Kd (mM)
3.5
5
CH3
CH3
CH3
CH3
CH3
CH3
9a
9b
9c
9d
9e
10
Br
Cl
2.7
2.5
CN
Me
10
7.7
Cyclopropyl
H
N/D
Peptidea 1.27
N/D=Not determined (linear response), detailed
structure in figure 10c
Next, we linked fragment 5 with a portion of the H3K9me2K14ac peptide that occupies the desired Kme
pocket and linker region to obtain compound 10. The co-crystal structure indicates that 10 recapitulated the
binding pose of 5 in the Kac pocket and the binding pose of H3K9me2K14ac in the Kme pocket and linker
(Figure 6). Compound 10 showed a significant HSQC NMR shift, however displayed weak binding by SPR
(SPR Kd = 1.27 mM), despite occupying and making interactions in the major part of the pocket occupied
by the native peptide. This may be because the interactions in the cavities occupied by the native peptide,
but not occupied by 10, may be important for potency. This weak activity may reflect conformational strain
17

associated with binding of the chemical series explored here. Structural dynamics of the TTD may also
impact accessibility of the binding pocket.
C
OH
N
N
O
O
H
N
H
N
Me
H2N
N
N
H
N
H
NHAc
O
O
HO
Me
N
10
Me
Me
Figure 6. (a) Binding mode of 10 (PDB: 6BPI) (b) Overlay of the structures of 10 (green), 5 (purple) with
the H3K9me2K14ac peptide (orange). (c) Chemical structure of 10.
Conclusion
We have identified the first fragment hits occupying the peptide binding pocket of SETDB1 TTD, which
demonstrates its ligandability. Fragments occupying the Kac pocket form interactions with the protein. The
most promising binding interactions were observed in compound 5, which included four hydrogen bonds
18

and multiple hydrophobic interactions, however showed only weak binding by SPR and poor ligand
efficiency of 0.19. Compound 10, a hybrid of fragment 5 and a histone peptide capable of spanning both
Kme and Kac pockets, did not show significant binding, suggesting that cavities occupied by A15-R17 of
the native peptide may be important for potency. Future compounds exploiting this portion of the binding
site will help further define the structural chemistry of the SETDB1 TTD and map the road towards potent
and selective SETDB1 chemical probes.
EXPERMENTAL SECTION
Protein expression and purification. The expression construct for N-terminal His6-tagged SETDB1 Tudor
domain (residues 197 – 403) subcloned into pET28a-MHL vector was transformed into BL21(DE3)-
pRARE2 cells. The cultures were grown in Terrific Broth medium (TB) in the presence of 50 µg/mL
kanamycin and 34 µg/mL chloramphenicol at 37 °C. For NMR analyses, the cultures were grown in auto-
induction M9 minimal medium containing 1.2 g/l 15N-ammonium chloride (Sigma 299251) as the sole
nitrogen source using an auto-induction method. For SPR experiments, the expression construct was N-
terminally biotinylated SETDB1 Tudor domain (residues 197 – 403) subcloned into pET28BIOH_LIC
vector and growth was done as usual but supplemented with 1 mg/ml of biotin. The overexpression of
SETDB1 Tudor was induced by addition of isopropyl-1-thio-D-galactopyranoside (IPTG), final
concentration 0.5 mM, and incubated overnight at 16 °C. Next day, the cells were harvested by
centrifugation at 12,227 × g (10 min, 4 °C). Details of the purification peocedures are provided in the
supporting information.
Protein crystallization. Purified SETDB1 was crystallized using hanging drop vapor diffusion method at
20 °C by mixing 1.5 µl of the protein solution at 5 mg/ml with 1.5 µl of the reservoir solution containing
19

25% PEG3350, 0.2 M LiSO4, 0.1 M Bis-Tris, pH 6.5. Matured SETDB1 crystals were transferred to the
soaking solution containing 27% PEG3350, 0.2 M LiSO4, 0.1 M Bis-Tris, pH 6.5, 50 mM of the ligand and
0.5% DMSO. Soaking time varied from 24 to 48 hours.
Biophysical assays. Isothermal titration calorimetry (ITC) for 5 was performed at 25 °C using Nano ITC
instrument (TA Instruments, USA). The cell was loaded with 80 µM SETDB1 (Tudor domain) in 20 mM
HEPES pH 7.5 and 250 mM NaCl. The syringe was loaded with 100 mM compound 5 dissolved in the
same buffer (pH was checked and no DMSO was used). 25 injections of 2 µL volumes were applied at 180
s intervals. “Nano Analyze” software package (supplied by the manufacturer) was used for data analysis.
The data was fit to a one binding site model.
SPR assay. SETDB1 Tudor Domain (amino acids 197-403, theoretical MW24099.93) was captured onto an
SA Chip (~6500 RU). Buffer: HBS-EP, 5% DMSO. All compounds were run with a contact time of 30 s
and dissociation time of 20 s at 25 μL/min. Two blank cycles were run between each compound cycle, and a
regeneration cycle of 60 s injection of buffer at 40 μL/min was run after each cycle.
NMR spectroscopy. 1H-15N HSQC-TROSY spectra were collected at 298 K on 500 MHz Bruker Avance
spectrometer equipped with 1.7 mm micro cryoprobe. Experiments were performed at 5 mM ligand
concentrations with 190 µM 15N labeled SETDB1 in 20 mM HEPES pH 7.5 and 250 mM NaCl, 2.5%
DMSO, and 10% D2O. Negative controls contained 2.5% DMSO and no compound.
General Chemistry. All solvents acetonitrile, dimethylformamide (DMF), tetrahydrofuran (THF),
dichloromethane (DCM), and trifluoroacetic acid (TFA) were of reagent grade quality, purchased from
commercial sources and used as received. Flash column chromatography was carried out using Silicycle
230- 400 mesh silica gel. Thin-layer chromatography (TLC) was performed on Macherey Nagel precoated
20

glass-backed TLC plates (SIL G/UV254, 0.25 mm) and visualized using a UV lamp (254 nm) followed by
KMnO4, ninhydrin or iodine stain. Reported Rf values are dependent on ambient humidity. NMR spectra
were recorded at 25 °C on Bruker Advance III 400 MHz spectrometer, Varian Mercury 400, Agilent DD2
500, or Agilent DD2 600 instrument. Recorded shifts for protons are reported in parts per million ( scale)
downfield from tetramethylsilane and are referenced to residual protons in the NMR solvents (CDCl3: 7.26
or CD3CN:  1.94, centre line). Chemical shifts for carbon resonances are reported in parts per million (
scale) downfield from tetramethylsilane and are referenced to the carbon resonances of the solvent (CDCl3:
 77.16 or CD3CN:  1.32, centre line). Data are represented as follows: chemical shift  in ppm,
multiplicity (s singlet, d doublet, t triplet, q quartet, m multiplet, br broad), coupling constant J in Hz and
integration. High resolution mass spectra were obtained on a VG 70-250S (double focusing) mass
spectrometer at 70 eV or on an ABI/Sciex Qstar mass spectrometer with ESI source, MS/MS and accurate
mass capabilities or on JEOL AccuTOF-DART instrument.
All the test compounds synthesized showed ≥95% pure as determined by HPLC/MS. Low-resolution
mass spectra (ESI) were collected on an Agilent Technologies 1200 series HPLC paired to a 6130 Mass
Spectrometer. Compounds were resolved on Phenomenex’s Kinetex 2.6u C18 50x4.6mm column at room
temperature with a flow of 1 mL/min. The gradient consisted of eluents A (0.1% formic acid in double
distilled water) and B (0.1% formic acid in HPLC-grade acetonitrile). Method A: A linear gradient starting
from 5% of B to 95% over 15 min at a flow rate of 1.0 mL/min. Method B: A linear gradient starting from
5% of B to 95% over 4.00 min at a flow rate of 1.0 mL/min. Stays constant at 95% for 1 min and then
returns to 5% over 0.5 min. Method C: Stays constant at 5% of B for 1.00 min at a flow rate of 1.0 mL/min,
followed by a linear gradient to 95% over 8.0 min. Stays constant at 95% of B for 1.00 min and then returns
21

to 5% B over 1.00 min. Compound 2 (Method A); Compounds 4, 9c, 9d (Method B); Compounds 6-8, 9a,
9b, 9e (Method C).
Synthetic procedures. General procedure A: synthesis of thioamides. Amide (1 equiv) was dissolved in
THF (0.1 M) and Lawesson’s reagent (0.6 – 0.75 equiv) was added. The reaction was heated to 60 ºC for 2
– 4 hours until complete by TLC analysis. Excess silica gel was added to the stirred solution and was
allowed to cool to room temperature while stirring. The solvent was evaporated in vacuo and the product
dry loaded onto silica gel. The silica-loaded crude product was loaded onto a silica column and the product
carefully eluted (0% to 30% EtOAc in hexanes ) as a yellow band following the initial dark byproduct band.
General procedure B: Boc deprotection and acetylation of amines. The Boc-protected amine (1 equiv)
was dissolved in 1.25 M HCl in methanol (15 equiv) and the solution was heated to 50 ºC overnight. The
reaction achieved full conversion as gauged by LC/MS analysis and the solvent was evaporated under
reduced pressure. The residue was dissolved in acetonitrile (0.2 M) and Amberlite IRA-67 was added to
free-base the amine followed by addition of acetic anhydride (1.5 equiv). The reaction was allowed to stir
for 6 – 12 hours and after removal of the solvent, crude products were purified by reverse phase or normal
phase column chromatography.
General procedure C: Synthesis of triazoles from thioamides. Thioamide (1 equiv) was added to a
microwave vial followed by acethydrazide or formic hydrazide (4 – 5 equiv). n-Butanol (0.5 M) was added
and the vial capped and heated at 100 ºC under normal microwave irradiation for 24 – 36 hours until the
starting material was consumed as gauged by LC/MS analysis. The solvent was evaporated in vacuo and the
hydrazide sublimed from the reaction mixture under high vacuum at 100 ºC. The crude product was
subsequently purified by reverse phase column chromatography or by recrystallization.
22

General procedure D: Synthesis of para-halogenated N-acylanilines. N-acylaniline (1 equiv), NBS/NCS
(2 equiv) and triphenylphosphine sulfide (0.1 equiv) were dissolved in chloroform (0.05 M) and stirred for 1
(NBS) to 2 (NCS) days until the starting material was fully consumed. The solvent was evaporated in vacuo
and the crude product was redissolved in EtOAc, washed subsequently with water, saturated sodium
thiosulfate, water and brine and dried over sodium sulfate. The crude product was then purified by column
chromatography.
1-methyl-4H,6H-benzo[e][1,2,4]triazolo[3,4-c][1,4]oxazepine (2). 2-aminobenzylalcohol (11) (500 mg,
4.06 mmol) was combined with sodium hydride (195 mg, 4.87 mmol) in toluene (8.1 mL) and added with
ethylchloroacetate (12) (0.435 mL, 4.06 mmol). The solution was heated to 80 ºC for 1 hour, cooled to room
temperature, diluted with EtOAc and worked up with 10% aqueous HCl. The organic layer was washed
with water, brine, and dried over sodium sulfate. After passing through silica column the crude lactam (74.2
mg, ~11%, pale yellow solid (Rf (1:1 EtOAc:hexanes): 0.33) was carried through to the next step. Crude
lactam (73.7 mg, 0.45 mmol) was dissolved in toluene (4.52 mL) and Lawesson’s reagent (183.1 mg, 0.45
mmol) was added. The solution was heated to 60 ºC for 5 hours, concentrated and passed through a silica
gel plug (Rf (2:5 EtOAc:hexanes): 0.64). The crude thioamide product 13 (0.092 g, 0.52 mmol) was mixed
in n-butanol (2.2 mL) and acethydrazide (14) (76.3 mg, 1.03 mmol) was added. The solution was heated to
120 ºC for four days, the solvent removed in vacuo and the crude product was purified by column
chromatography. Yield: 48.0 mg, 53%, pale yellow crystals. Rf (1:19 MeOH:EA): 0.1. 1H NMR (500 MHz,
CDCl3)  7.60 (d, J = 7.9, 7.4, 1.7 Hz, 1H), 7.55 (ddd, J = 7.6, 1.8, 0.5 Hz, 1H), 7.49 (dt, J = 7.5, 1.2 Hz,
1H), 7.39 (dd, J = 8.0, 0.9 Hz, 1H), 4.66 (s, 2H), 4.46 (s, 2H), 2.62 (s, 3H). 13C NMR (126 MHz, CDCl3) 
151.8, 150.9, 134.4, 131.9, 131.1, 130.4, 129.3, 122.6, 67.1, 59.2, 11.9. IR (neat) (cm-1): 2928, 2864,
휈
23

1526, 1495, 1462, 1423, 1380, 1087, 878, 770. HRMS (DART) m/z: [M + H]+ calcd for C11H12N3O
202.0980, found 202.0980.
tert-butyl 2-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepine-4-carboxylate (18). In a flame dried
round-bottom flask, 2-aminobenzylamine (10 g, 82 mmol) was dissolved in toluene (410 mL, 0.2 M).
Triethylamine (12.6 mL, 90 mmol) and ethylchloroacetate (9.2 mL, 86 mmol) were added and the solution
was stirred under reflux at 110 °C for 22 hours, until complete as indicated by TLC and LC/MS. The
reaction mixture was cooled to room temperature and water (410 mL) was added. The organic layer was
separated and washed with water, followed by brine, dried over sodium sulfate and concentrated to yield
ethyl (2-aminobenzyl)glycinate (16) which was used in the subsequent step without further purification.
1
Yield: 15.8 g, 93%, brown/orange oil. Rf (EtOAc): 0.51. H NMR (300 MHz, CDCl3)  7.27 – 7.15 (m,
1H), 7.13 – 7.07 (m, 1H), 7.06 – 7.00 (m, 1H), 6.69 – 6.67 (m, 1H), 4.24 – 4.17 (m, 2H), 3.79 (s, 2H), 3.40
(s, 2H), 1.31 – 1.27 (m, 3H). 13C NMR (100 MHz, CDCl3)  172.7, 146.9, 130.3, 128.7, 123.2, 117.8,
115.8, 60.9, 52.1, 49.8, 14.3. IR (neat) (cm-1): 3436, 3342, 2976, 1730, 1617, 1495, 1459, 1192, 1131,
휈
749. The crude product 16 (15.8 g, 75.9 mmol) was dissolved in THF (760 mL) followed by the addition of
Boc2O (21.8 mL, 94.8 mmol) and a catalytic amount of DMAP. The solution was allowed to stir for 20
hours until reaction was complete by TLC analysis. The THF was removed under reduced pressure, and the
residue redissolved in EtOAc, washed with saturated sodium bicarbonate, brine, and dried over sodium
sulfate. The volatiles were removed in vacuo and the crude product 17 (Rf (1:3 EtOAc:hexanes): 0.7) was
dissolved in THF (760 mL) and cooled to 0 ºC. Potassium tert-butoxide (8.94 g, 79.7 mmol) was added
portionwise until the reaction was complete by TLC. The solvent was evaporated, the crude redissolved in
EtOAc, washed with saturated sodium bicarbonate, then brine, then dried over sodium sulfate. Compound
18 was purified by recrystallization from hot EtOAc. Yield: 7.6 g, 35% (3 steps), white solid. Rf (1:3
24

1
EtOAc:hexanes): 0.2. 1:1 Mixture of rotamers. H NMR integrations adjusted to reflect whole number
protons of each rotamer in solution. 1H NMR (500 MHz, CDCl3)  8.10 (br. s, 1H), 7.92 (br. s, 1H), 7.37 –
7.25 (m, 3H), 7.22 – 7.16 (m, 1H), 7.16 – 7.03 (m, 3H), 6.92 (d, J = 7.9 Hz, 2H), 4.55 (br. s, 2H), 4.48 (br.
13
s, 2H), 4.42 (br. s, 2H), 4.29 (br. s, 2H), 1.44 (br. s, 9H), 1.37 (br. s, 9H). C NMR (126 MHz, CDCl3) δ
171.1, 170.3, 154.4, 153.9, 136.3, 130.2, 129.6, 128.8, 128.6, 127.9, 125.0, 123.9, 120.3, 119.9, 81.1, 80.8,
51.6, 51.2, 49.9, 48.9, 28.2. IR (neat) (cm-1): 3215, 3061, 3003, 1681, 1661, 1590, 1385, 1366, 1166,
휈
1132, 752. HRMS (DART) m/z: [M + H]+ calcd for C14H22N3O3 263.1396, found 263.1399.
tert-butyl 2-thioxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepine-4-carboxylate (19). Synthesized
according to general procedure A. Scale: 7.62 mmol. Yield: 1.13 g, 53%, pale yellow solid. Rf (EtOAc):
1
0.93. H NMR (300 MHz, CDCl3)  9.50 (s, 1H), 7.34 (dt, J = 7.6, 1.6 Hz, 2H), 7.24 (d, J = 7.5 Hz, 1H),
7.04 – 6.99 (m, 1H), 4.53 (m, 4H), 1.46 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 200.6, 154.3, 130.8, 129.3,
127.2, 121.1, 81.3, 56.2, 48.5, 28.5. IR (neat) (cm-1): 3239, 2982, 1659, 1488, 1417, 1362, 1153, 1119,
휈
753. HRMS (ESI) m/z: [M + H]+ calcd for C14H19N2O2S 279.1167, found 279.1167.
tert-butyl
1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diazepine-5(6H)-carboxylate
(20).
Thioamide 19 (0.6 g, 2.16 mmol) and acethydrazide (0.24 g, 3.23 mmol) were dissolved in n-butanol, and
stirred at 110 °C for 3 days. The reaction mixture was concentrated and acethydrazide was sublimed from
the reaction mixture at 110 ºC under high vacuum. The crude was recrystallized from hot EtOAc followed
1
by recrystallization from methanol/diethylether. Yield: 516 mg, 80%, white solid. H NMR (500 MHz,
CDCl3)  7.63 – 7.51 (m, 2H), 7.50 – 7.43 (m, 1H), 7.36 (d, J = 7.9 Hz, 1H), 4.58 (br. s, 2H), 4.37 (br. s,
2H), 2.58 (s, 3H), 1.50 (br. s, 9H). 13C NMR (126 MHz, CDCl3) δ 153.9, 151.2, 150.2, 133.5, 131.7, 130.9,
129.6, 129.4, 123.0, 81.1, 45.6, 40.8, 28.4, 11.8. IR (neat) (cm-1): 2979, 1693, 1497, 1384, 1259, 1156,
휈
862, 762. HRMS (DART) m/z: [M + H]+ calcd for C16H21N4O2 301.1665, found 301.1664.
25

1-(1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diazepin-5(6H)-yl)ethan-1-one (4). Synthesized
according to general procedure B. Scale: 0.33 mmol. Yield: 43.6 mg, 54%, white solid. Rf (1:19
1
MeCN:DCM): 0.26. 1:2 mixture of rotamers. Methyl integral of 3 corresponds to major rotamer. H NMR
(500 MHz, CDCl3)  7.66 – 7.34 (m, 6H), 4.74 –4.44 (m, 5H), 4.36 (s, 1H), 2.61 (s, 3H), 2.59 (s, 1.5H),
2.28 (s, 1.5H), 2.20 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 169.4, 168.9, 151.5, 151.5, 150.4, 149.7, 133.7,
133.5, 132.1, 130.7, 130.6, 130.5, 130.3, 123.0, 129.7, 129.6, 123.7, 123.1, 48.9, 44.4, 42.5, 40.3, 22.2,
22.1, 12.0, 11.9. IR (neat) (cm-1): 3493, 3003, 1627, 1608, 1500, 1417, 1235, 1220, 776. HRMS (DART)
휈
m/z: [M + H]+ calcd for C13H15N4O 243.1246, found 243.1236.
tert-butyl (2-acetamidobenzyl)carbamate (25). 2-Aminobenzylamine (3.0 g, 24.6 mmol) was dissolved
in 50 mL of dry THF. Triethylamine (3.8 mL, 27.2 mmol) was added to the solution followed by Boc2O
(5.4 g, 24.6 mmol). The mixture was stirred overnight at room temperature under a N2 atmosphere. The
reaction mixture was poured into a 10% citric acid solution (25 mL) and extracted with EtOAc (2 x 50 mL).
The combined organics were washed with brine (25 mL), dried over MgSO4, filtered and concentrated to
give 24 as a white solid in 99% yield (5.41 g, 24.3 mmol). In a flame dried round-bottom flask, tert-butyl(2-
aminobenzyl)carbamate 24 (1.5 g, 6.75 mmol) was dissolved in DCM (13.5 mL) and acetic anhydride (0.7
mL, 7.43 mmol) was added to the solution at 0 ºC and allowed to warm to room temperature. After 2 hours,
the reaction reached completion by TLC. The reaction mixture was diluted with dichloromethane and
quenched with saturated sodium bicarbonate solution. The solution was washed with brine and dried over
magnesium sulfate. The solvent was removed in vacuo and the product purified by column chromatography.
Yield: 1.58 g, 89%, white solid. Rf (1:1 EtOAc:Hexanes): 0.52. 1H NMR (400 MHz, CDCl3)  9.41 (s, 1H),
8.24 (d, J = 8.2 Hz, 1H), 7.31 (ddd, J = 8.5, 7.4, 1.7 Hz, 1H), 7.14 – 7.11 (m, 1H), 7.03 (dt, J = 7.4, 1.3 Hz,
1H), 5.07 (s, 1H), 4.27 (d, J = 6.8 Hz, 2H), 2.24 (s, 3H), 1.45 (s, 9H). 13C NMR (125 MHz, CDCl3)  169.5,
26

157.4, 137.2, 130.4, 129.2, 186.2, 124.0, 122.4, 80.9, 42.0, 28.5, 24.6. IR (neat) (cm-1): 3339, 3288, 2982,
휈
1689, 1670, 1527, 1489, 1287, 1255, 1164, 748. HRMS (ESI) m/z: [M + Na]+ calcd for C14H20N2NaO3
287.1366, found 287.1365.
tert-butyl (2-ethanethioamidobenzyl)carbamate (26). Synthesized using general procedure A. Scale:
1
2.74 mmol. Yield: 494 mg, 64%, pale yellow solid. Rf (1:1 EtOAc:Hexanes): 0.8. H NMR (400 MHz,
CDCl3)  8.17 (d, J = 8.1 Hz, 1H), 7.39 – 7.35 (m, 1H), 7.25 (m, 1H), 7.23 – 7.21 (m, 1H), 4.18 (s, 2H),
13
2.82 (s, 3H), 1.42 (s, 9H). C NMR (125 MHz, CDCl3)  210.5, 157.6, 137.7, 132.3, 130.5, 128.3, 127.2,
126.7, 81.1, 41.2, 35.5, 28.5. IR (neat) (cm-1): 3273, 2928, 1669, 1609, 1530, 1365, 1288, 1151, 1139,
휈
851, 743, 701. HRMS (ESI) m/z: [M + H]+ calcd for C14H21N2O2S 281.1318, found 281.1330.
N-(2-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)benzyl)acetamide (5). Synthesized according to general
1
procedure B. Scale: 0.314 mmol. Yield: 50 mg, 65%, white solid. Rf (1:9 MeOH:EtOAc): 0.2. H NMR
(500 MHz, CDCl3)  7.70 – 7.64 (m, 1H), 7.53 (t, J = 7.6, 1H), 7.46 – 7.42 (m, 1H), 7.09 – 7.06 (m, 1H),
4.18 – 4.08 (m, 2H), 2.20 (s, 6H), 2.00 (s, 3H). 13C NMR (126 MHz, CDCl3)  170.4, 151.5, 136.3, 132.2,
130.9, 130.6, 129.1, 127.8, 39.7, 22.9, 11.0. IR (neat) (cm-1): 3362, 3253, 2992, 1687, 1666, 1542, 1512,
휈
1279, 1270. HRMS (ESI) m/z: [M + H]+ calcd for C13H17N4O 245.1397, found 245.1397.
1-(2-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)benzyl)-3-methylurea (6). After Boc deprotection of 27 using
general procedure B, the free amine (0.182 mmol) was stirred with triethylamine (43.7 µL, 0.313 mmol) in
dichloromethane (1.25 mL) and stirred for 30 minutes. In a separate vessel, triphosgene (18.6 mg, 0.063
mmol) was dissolved in dichloromethane (1.25 mL) and cooled to 0 ºC. The amine solution was added
dropwise to the triphosgene solution and the mixture was warmed to room temperature and stirred for 30
minutes. The solution was cooled to 0 ºC and 1.25 M methylamine in methanol (156 µL, 1.25 mmol) was
added and stirred for 1 hour. When the reaction was complete by LC/MS analysis, the solvent was removed
27

in vacuo and the product purified using reverse phase column chromatography. Yield: 11.8 mg, 25%, white
1
solid. H NMR (399 MHz, CD3OD)  7.66 – 7.57 (m, 2H), 7.55 – 7.47 (m, 1H), 7.28 (d, J = 7.7 Hz, 1H),
3.97 (s, 2H), 2.67 (s, 3H), 2.21 (s, 6H). 13C NMR (100 MHz, CD3OD)  153.5, 139.2, 132.7, 132.0, 130.5,
130.0, 129.3, 111.4, 40.5, 27.0, 10.6. IR (neat) (cm-1): 3260, 1690, 1545, 1495, 1416, 1242, 786. HRMS
휈
(DART) m/z: [M + H]+ calcd for C13H18N5O 260.1511, found 260.1510.
N-(2-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)benzyl)methanesulfonamide (7). After Boc deprotection of
27 using general procedure B, free amine (1.0 equiv., 48 mg, 0.237 mmol) and triethylamine (1.2 equiv.,
39.7 L, 0.285 mmol) were dissolved in anhydrous dichloromethane (2.4 mL, 0.1 M) in an oven-dried 2-
dram vial equipped with magnetic stir bar and rubber septum. The resulting mixture was cooled to 0 ºC and
then methanesulfonyl chloride (1.2 equiv., 22 L, 0.285 mmol) was added. After stirring at 0 ºC for 1 hour,
the reaction was warmed to room temperature over 2 hours and then quenched with 5 mL of a saturated
solution of sodium bicarbonate. The organic layer was separated, and the aqueous layer was extracted with
dichloromethane (3x5 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and
solvent was removed in vacuo. The crude residue was then purified by reverse-phase flash-chromatography
using a 5-100% MeCN gradient in H2O with 1% of formic acid. The fractions containing desired product
were pooled and lyophilized. Yield: 35%, white solid. 1H NMR (400 MHz, CD3OD)  2.10 - 2.30 (m, 6H),
2.86 (s, 3H), 3.95 (s, 2H), 7.32 (d, J = 7.7 Hz, 1H), 7.54 - 7.62 (m, 1H), 7.62 - 7.69 (m, 1H), 7.75 (d, J = 7.7
Hz, 1H), 8.46 (s, sulfonamide NH under deuterium exchange). 13C NMR (126 MHz, CD3OD)  153.6,
137.1, 133.2, 132.2, 132.0, 130.8, 129.6, 43.4, 40.0, 10.8; HRMS (DART) m/z: [M + H]+ calcd for
C12H17N4O2S 281.1072, found 281.1070.
21
2-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)benzoic acid (8). Anhydride 28 (1.0 g, 6.21 mmol, 1.1 equiv.)
and acetylhydrazide (418 mg, 5.64 mmol, 1.0 equiv.) were refluxed in EtOH (56 mL, 0.1M) for 3 h.22 The
28

reaction was cooled down to r.t. and then dry loaded on celite. The crude was purified using C18
functionlized silica gel using a gradient from 5:95 MeCN:H2O to 1:1 MeCN:H2O. The fractions containing
1
the desired product were lyophilized. Yield: 27%, white solid. H NMR (500 MHz, DMSO-d6)  13.38 (s,
1H), 8.05 (dd, J = 7.8, 1.6 Hz, 1H), 7.78 (dt, J = 7.7, 1.7 Hz, 1H), 7.68 (dt, J = 7.6, 1.3 Hz, 1H), 7.49 (dd, J
= 7.8, 1.3 Hz, 1H), 2.01 (s, 6H). 13C NMR (126 MHz, DMSO-d6)  165.8, 150.5, 133.6, 133.0, 131.5,
130.3, 129.6, 129.6, 10.6. HRMS (DART) m/z: [M + H]+ calcd for C11H12N3O2 218.0930, found 218.0930.
tert-butyl (2-acetamido-5-bromobenzyl)carbamate (29a). Synthesized according to general procedure
D. Scale: 1.89 mmol. Yield: 569 mg, 88%, white solid. Rf (1:3 EtOAc:hexanes): 0.42. 1H NMR (500 MHz,
CDCl3)  9.51 (br. s, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.40 (dd, J = 8.8, 2.4 Hz, 1H), 7.29 – 7.26 (m, 1H), 5.09
(br. s, 1H), 4.22 (d, J = 6.9 Hz, 2H), 2.22 (s, 3H), 1.45 (s, 9H). 13C NMR (126 MHz, CDCl3)  169.5, 157.5,
136.4, 133.1, 132.0, 130.2, 124.0, 116.2, 81.2, 41.5, 28.5, 24.6. IR (neat) (cm−1): 3360, 3251, 2988, 1686,
휈
1666, 1539, 1514, 1278, 1270, 1163, 641. HRMS (DART) m/z: [M + H]+ calcd for C14H20BrN2O3 343.0665,
found 343.0664.
tert-butyl (2-acetamido-5-chlorobenzyl)carbamate (29b). Synthesized according to procedure D. Scale:
1.89 mmol. Yield: 295 mg, 52%, white solid. Rf (1:2 EtOAc:hexanes): 0.35. 1H NMR (500 MHz, CDCl3) 
9.50 (s, 1H), 8.21 (d, J = 8.8 Hz, 1H), 7.26 (dd, J = 8.8, 2.5 Hz, 1H), 7.11 (d, J = 2.5 Hz, 1H), 5.10 (s, 1H),
13
4.22 (d, J = 6.8 Hz, 2H), 2.23 (s, 3H), 1.45 (s, 9H). C NMR (126 MHz, CDCl3) δ 171.2, 157.5, 135.8,
130.2, 130.0, 129.0, 128.6, 123.7, 81.2, 77.2, 41.6, 28.5, 24.5. IR (neat) (cm-1): 3357, 3254, 2982, 1665,
휈
1542, 1515, 1365, 1277, 1162, 932, 837. HRMS (DART) m/z: [M + H]+ calcd for C14H20ClN2O3 298.1084,
found 298.1082.
tert-butyl (5-bromo-2-ethanethioamidobenzyl)carbamate (30a). Synthesized according to general
1
procedure A. Scale: 23.3 mmol. Yield: 6.93 g, 83%, pale yellow powder. Rf (1:3 EtOAc:hexanes): 0.5. H
29