Discovery of inhibitors of the mitotic kinase TTK based on N-(3-(3-sulfamoylphenyl)-1H-indazol-5-yl)-acetamides and carboxamides

Article abstract of DOI:10.1016/j.bmc.2014.06.027

TTK kinase was identified by in-house siRNA screen and pursued as a tractable, novel target for cancer treatment. A screening campaign and systematic optimization, supported by computer modeling led to an indazole core with key sulfamoylphenyl and acetamido moieties at positions 3 and 5, respectively, establishing a novel chemical class culminating in identification of 72 (CFI-400936). This potent inhibitor of TTK (IC₅₀ = 3.6 nM) demonstrated good activity in cell based assay and selectivity against a panel of human kinases. A co-complex TTK X-ray crystal structure and results of a xenograft study with TTK inhibitors from this class are described.

Full text of DOI:10.1016/j.bmc.2014.06.027

Accepted Manuscript
Discovery of Inhibitors of the Mitotic Kinase TTK Based on N-(3-(3-Sulfa-
moylphenyl)-1H-indazol-5-yl)- Acetamides and Carboxamides
Radoslaw Laufer, Grace Ng, Yong Liu, Narendra Kumar B. Patel, Louise G.
Edwards, Yunhui Lang, Sze-Wan Li, Miklos Feher, Don E. Awrey, Genie
Leung, Irina Beletskaya, Olga Plotnikova, Jacqueline M. Mason, Richard
Hodgson, Xin Wei, Guodong Mao, Xunyi Luo, Ping Huang, Erin Green, Reza
Kiarash, Dan Chi-Chia Lin, Marees Harris-Brandts, Fuqiang Ban, Vincent
Nadeem, Tak W. Mak, Guohua J. Pan, Wei Qiu, Nicolay Y. Chirgadze, Henry
W. Pauls
PII:
S0968-0896(14)00473-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.06.027
BMC 11664
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 April 2014
6 June 2014
14 June 2014
Please cite this article as: Laufer, R., Ng, G., Liu, Y., Patel, N.K.B., Edwards, L.G., Lang, Y., Li, S-W., Feher, M.,
Awrey, D.E., Leung, G., Beletskaya, I., Plotnikova, O., Mason, J.M., Hodgson, R., Wei, X., Mao, G., Luo, X.,
Huang, P., Green, E., Kiarash, R., Lin, D.C-C., Harris-Brandts, M., Ban, F., Nadeem, V., Mak, T.W., Pan, G.J.,
Qiu, W., Chirgadze, N.Y., Pauls, H.W., Discovery of Inhibitors of the Mitotic Kinase TTK Based on N-(3-(3-
Sulfamoylphenyl)-1H-indazol-5-yl)- Acetamides and Carboxamides, Bioorganic & Medicinal Chemistry (2014),
doi: http://dx.doi.org/10.1016/j.bmc.2014.06.027
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Discovery of Inhibitors of the Mitotic Kinase TTK
Based on N-(3-(3-Sulfamoylphenyl)-1H-indazol-5-yl)-
Acetamides and Carboxamides
a
Radoslaw Laufer*, Grace Ng, Yong Liu, Narendra Kumar B. Patel, Louise G. Edwards, Yunhui
a
a
a
a
a
a
a
a
a
a
Lang, Sze-Wan Li, Miklos Feher, Don E. Awrey, Genie Leung, Irina Beletskaya, Olga Plotnikova,
a
a
a
a
a
a
Jacqueline M. Mason, Richard Hodgson, Xin Wei, Guodong Mao, Xunyi Luo, Ping Huang, Erin
a
a
Green, Reza Kiarash, Dan Chi-Chia Lin, Marees Harris-Brandts, Fuqiang Ban, Vincent Nadeem,
a
a
a
a
a
a
b
b
Tak W. Mak, Guohua J. Pan, Wei Qiu, Nicolay Y. Chirgadze, Henry W. Pauls*
,a
a
Campbell Family Institute for Breast Cancer Research, Therapeutics Group, University Health
Network, 101 College St. W, Toronto, Ontario, M5G 1L7, Canada
b
Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Hospital,
University Health Network, 610 University Ave, Toronto, Ontario M5G 2C4, Canada
Corresponding authors. e-mail: rlaufer@uhnresearch.ca, Tel.: +1-416-581-7572; e-mail:
hpauls@uhnresearch.ca, Tel.: 416-581-7620.
Abstract
TTK kinase was identified by in-house siRNA screen and validated as a tractable, novel target for cancer
treatment. A screening campaign and systematic optimization, supported by computer modeling led to
indazole core with key sulfamoylphenyl and acetamido moieties at positions 3 and 5, respectively,
establishing a novel chemical class culminating in identification of 72 (CFI-400936). This potent inhibitor
of TTK (IC50 = 3.6 nM) demonstrated good potency in cell based assay and selectivity against a panel of
1

human kinases. A co-complex TTK X-ray crystal structure and results of a xenograft study with the
TTK inhibitor are described.
Keywords
Anticancer, antimitotic agents; indazolyl benzenesulfonamide; mitotic kinase; Monopolar Spindle 1
kinase; Mps1; Tyrosine Threonine Kinase ,TTK.
1. Introduction
1
Tyrosine Threonine Kinase (TTK) also known as Monopolar Spindle 1 (Mps1), or Phosphotyrosine-
Picked Threonine Kinase (PYT) is a conserved dual-specificity, cell cycle-regulated kinase, essential for
2
genomic integrity during cell division through establishment and maintenance of the mitotic checkpoint.
3
-12
TTK is overexpressed in a variety of solid cancers , and elevated levels of TTK correlate with high
1
0
7,8,9
histological grade in tumors and poor patient outcome.
Overexpression of TTK may promote tumor
1
0,13,14
initiation, and survival of genomically unstable and aneuploid cancer cells.
Beyond mitosis, TTK has
1
5
16
roles in development and several signaling pathways, including Hedgehog/Gli1 and EGFR. Also,
several lines of evidence implicate TTK in DNA damage signaling. TTK controls DNA damage responses
1
7
and genome stability by phosphorylating the Bloom syndrome helicase (BLM) , CHK2 in the G2/M
1
8,19
20
checkpoint
and c-ABL after oxidative stress. Taken together, these observations point to TTK as a
potential target for therapeutics for solid tumors. In efforts to validate this hypothesis we have
performed a series of in vitro and in vivo experiments: RNAi-mediated depletion of TTK in cancer cells
disabled the mitotic checkpoint, caused gross chromosome segregation errors resulting in aneuploidy,
2
1
induced cell death, and suppressed the growth of tumor xenografts in mice.
The interest in TTK as a potential target for the discovery of novel therapeutics is underscored by the
number of TTK inhibitors that have been disclosed since the commencement of our study. Small
2
2,23
24
25,26
The
2
molecule TTK inhibitors (Chart 1) include reversine , its analog MPI-0479605, NMS-P715,
2
7
28,29
30,31
MPS1-IN-1, AZ 3146
, the Genentech and Cancer Research UK inhibitors (CCT251455).

striking feature in all of these inhibitors is the common occurrence of a key N-phenylpyrimidin-2-amine
3
2,33
motif highlighted in blue in Chart 1. The Shionogi-1
inhibitor incorporates a variation on this theme.
Reversine is also an inhibitor of Aurora B; analoging efforts demonstrated that the inclusion of a
substituent on the aniline ring (R = Me) served to abrogate this activity. It’s notable that the subsequent
analogs with the N-phenylpyrimidin-2-amine motif all incorporate a substituent at this position. After
3
4
completion of the research presented in this manuscript, Kusakabe et al. have disclosed a unique series
3
5
of indazole-based inhibitors. The work, stimulated by the structure of a nonselective kinase inhibitor,
3
6
SP600125, explored a set of C5 and C6 substitutions of the indazole ring and culminated in the
synthesis of potent TTK inhibitors exemplified by the structure of Shionogi-2 (
Chart 1) with undisclosed in vivo activities. Despite the plethora of reports on TTK inhibitors,
apparently, none as yet, has advanced to the clinic, and the discovery of new efficacious TTK inhibitors
presents a challenge and the promise of new approaches to cancer therapy.
The results of our lead generation and subsequent optimization efforts are described in the body of this
publication. We began with a focused screening approach that included virtual hits from commercial
collections and our in-house library of kinase inhibitors. One chemotype that was identified by our
medium throughput assay was indazole, a well precedented class of ATP-competitive protein kinase
inhibitors. The initial synthetic work was driven by two objectives, namely, a desire to identify new
chemotypes for subsequent elaboration, and to increase the potency of the series. Subsequent lead
optimization efforts on the C-3 sulfamoylphenyl compounds that resulted, established the acetamido
moiety at position 5 but not 6, as being distinctive and pivotal to potent TTK inhibition. Further
optimization of this novel series, together with structural work, corroborated the binding hypothesis and
gave potent and selective TTK inhibitors, with the desired in vitro and in vivo anticancer activity.
2. Results and discussion
2
.1 Screening and Lead Finding.
3

Our lead finding strategy began with an internal library of focused kinase inhibitors whose assembly
3
7
37
was previously described and supplemented by a series of PLK4 inhibitors and intermediates. In
addition, virtual screening hits against a TTK model, based on the known TTK X-ray structure (PDB
2
7
3
CEK vide infra), were obtained. The combined collection (~13,000 compounds) was screened on a
medium throughput robotics workstation using an indirect ELISA TTK assay. Hits from several
chemical series (over 75% inhibition at the screening dose of 10 µM) were validated as follows: the
active compounds were clustered into chemical series; their purity was checked by LCMS and IC50
values were determined. Selected compounds from series of interests were subjected to a modeling
exercise in the TTK active site.
2
.1.2 Molecular modeling.
The first publicly available TTK X-ray structure appeared during our initial lead finding efforts (2.3 Å
2
7
resolution, PDB code 3CEK) which we modified to render it suitable for molecular modeling. The
structure is characterized by a bound pentaethyleneglycol (PEG) unit wound around Lys553 with
hydrogen bonds to the positively charged side chain amino group. This PEG is situated in a volume
where it would prevent ligands from binding into the kinase active site. The X-ray structure was
modified by removing the PEG; molecular dynamics simulations of the active site indicated that it
remained largely unchanged. This result implied that the PEG unit did not significantly influence the
conformation of the TTK binding site. Hence, this modified X-ray structure was utilized for the virtual
screen of available commercial collections and modeling new synthetic targets going forward. This
model was later superseded by a co-crystal structure with a ligand from this series (vida infra). The
modified 3CEK structure was useful in describing the binding pose of our ligands but had limitations: a
somewhat flat SAR was predicted for structural changes in the region close to the putative activation
loop.
2
.1.3. Lead Finding
4

Of the series identified by these exercises, 3- substituted-indazoles, represented by compounds 1-3
Scheme 1), held the most promise based on such criteria as: micromolar TTK inhibition, acceptable
ligand efficiency, predictable binding mode and ease of chemical synthesis. This particular motif could
(
3
6
also be recognized in the Jun N-terminal kinases inhibitor, SP600125; an anthrapyrazole, and at the
3
8
time, the only known inhibitor of TTK. Besides improving TTK binding, the challenge presented was
to generate a previously unexplored chemotype. Given the propensity of 3-vinylic indazoles 1 to inhibit
3
7
PLK4, we felt that 3-aryl indazoles 3 represented a better opportunity for the development of novel and
selective inhibitors of TTK. We conducted molecular docking exercises, based on the known 3CEK
2
7
crystal structure, to overlay indazole hit 3 and a previously described VEGFR inhibitor, GW-786034
(
pazopanib). The latter was reported as a low submicromolar inhibitor of TTK in a large kinase panel
3
9
screen study reported by Karaman et al. From the results of the overlay presented in Figure 1 it is clear
that the sulfonamide group in GW-786034 is engaged in a bonding interaction with the hinge residue
Gly605. We hypothesized that a similar interaction would be maintained if a sulfonamide group was
grafted to the meta position of 3-phenyl group to yield indazoles 4 and 5. In our model, the indazole
core makes two anticipated H-bonds to the hinge region and the sulfonamide group captures additional
interactions with polar functions of the TTK active site.
We were pleased to see that the meta-benzenesulfonamides 4 and 5 inhibited TTK in the single digit
micromolar range, about 5-7 fold more potent than the unsubstituted parent, compound 3 (Table 1). In
contrast, the para-substituted regioisomer (6) was an order of magnitude less active than the meta
isomer. Bioisosteric replacement of the 3-benzensulfonamide with a meta-benzamide or meta-benzylic
amine (Table 1, compounds 7 and 8), reduced potency by up to an order of magnitude, underscoring the
importance of the meta sulfonamide group for optimal binding. Our interest in this chemotype was
enhanced by the paucity of literature reports on the indazole-based benzenesulfonamides, the later patent
4
0
by Kusakabe et al notwithstanding.
5

2
.2 Inhibitor Design. To corroborate the nature of the binding of 3-(1H-indazol-3-
yl)benzenesulfonamides, a kinetic analysis of compound 4 was performed (Supplementary material). This
study showed that compound 4 displays kinetics consistent with direct competition for ATP binding in
the active site of TTK with an apparent K of 3 µM. Inspection of the binding modes of compound 4 and
i
analogs with various substituents on the indazole ring convinced us that substitution at position C7 of the
indazole ring would not be tolerated. On the other hand, capturing additional interaction with the
enzyme should be possible by substituting positions C4, C5 and C6. Accordingly, a limited SAR study of
C4-C6 substitutions of the indazole core was undertaken; the data is summarized in Table 2. Soon after
we initiated this work the substrate used in the TTK assay was changed from MBP to a recombinantly
4
2b
produced protein construct containing residues 1-275 of TTK (referred to herein as TTK-N) and the
data in Tables 2-6 was obtained using the latter condition. In head to head comparison, this change
improved the robustness of the assay and the absolute value of the TTK inhibition was effected but the
relative potency was not.
Although phenyl rings are tolerated at all three positions there was no advantage to substitution with
these hydrophobes (compounds 9-11). In stark contrast, there was a discernible trend favoring C-5
substitutions involving polar linkers. For the C5 and C6 amide matched pairs; it was observed that C5-
isomers were markedly more active against TTK (14/15, and 16/17). For example, compound 16 was an
order of magnitude more potent than the parent sulfonamide, 4, whereas the C6 regioisomer 17 was
significantly less active. These results focused the subsequent lead optimization work around the C5
substitution.
2
.3 Chemical Synthesis. Halogenation of indazoles followed by a palladium-catalyzed cross coupling
reaction comprise the key events in the synthesis of all of the indazolyl benzenesulfonamides described
herein (Scheme 2). For example 1H-indazol-5-amine (19) was, after incorporation of N-Boc group,
subjected to a halogenation followed by Suzuki-Miyaura arylation and a deprotection step to provide
aniline 21. This versatile intermediate was used in reactions with a variety of electrophiles in preparation
of ureas, sulfonamides, sulfamides, amides and carbamates (23). In the same way, indazole carboxamides
6

2
6 were synthesized from methyl 1H-indazole-5-carboxylate 24 in a sequence consisting of halogenation
followed by Pd-catalyzed cross-coupling reaction and an amide formation. Alternatively, alkylations of
H-indazol-5-ol (27) followed by halogenation and arylation steps allowed for preparation of ethers of
1
type 29. α Amino acids used in the preparation of acetamides 22 were most conveniently prepared via a
Petasis borono-Mannich reaction utilizing a mixture of arylboronic acid, dialkyl amine and glyoxylic
4
1
acid. In most cases, the α-substituted amides were tested as racemates. In selected cases, optical
resolution by chiral HPLC, distereomeric recrystallization, or chiral synthesis provided the required
optically active materials.
2
.5 Lead optimization. Lead optimization efforts were focused on appending hydrophobic, aryl
terminal groups to the C5 position of the indazole via a variety of linkers Table 3. To this end, a
comprehensive set of compounds was synthesized consisting of ethers (30, 31), acetamides (32, 33),
sulfonamides (34, 35), ureas (36, 37, 38), sulfamides (39, 40) and carboxamides (41, 42, 43).
Acetamides, ureas and carboxamides proved to be potent TTK inhibitors and the most active analogs in
each class i.e. 33, 37 and 42, respectively, inhibited HCT116 cell growth in the single digit micromolar
range. To gauge the suitability of these series for extensive lead optimization efforts we measured their
plasma exposure in mice upon IP dosing at 25 mg/kg. Acetamide 33 attained micromolar levels (AUC0-
.
last 0.72 µg h/mL) whereas urea 37 could not be detected; this was attributed to the compounded effects
of reduced solubility and permeability of the latter (Table 6).
The SAR (Tables 2 & 3) points to the importance of the linker in conferring optimal activity. To better
understand these effects, we masked the potential hydrogen bond donors by introducing an N-methyl
group in the urea linker (38) and amide linker (43). This modification resulted in a loss in enzymatic
activity as illustrated by the matched pairs 41 and 37 and was especially significant for the urea. A loss of
activity was also observed on replacement of the proximal nitrogen in the urea 36 with carbon (entry 45)
and by isomeric displacement of the amide linker (entry 44). In toto, these observations pointed to the
significance of the presence and position of the amide and/or urea functionality, perhaps in the formation
of an H-bond interaction or by enforcing a binding conformation.
7

Based on these results, subsequent efforts focused exclusively on the two types of the amide-
linked compounds (Table 4). Carboxamide and acetamide congeners were effective inhibitors of TTK; in
many cases the activities for the matched pairs 33/42 (Table 3); 50/49 and 61/62 were remarkably similar.
In other matched sets one or other of these linkers was more effective: acetamide 51 was about 4-fold
more potent than carboxamide 52 whereas carboxamide 55 was appreciably more active than acetamide
5
4. We noted that the optimal distance from the hydrophobe to the indazole attachment was 4 bonds
e.g. acetamides 32, 33 and carboxamide 42). Shorter distances of 3 bonds (16) and longer distances of
bonds (46) were less effective. The nature of the terminal hydrophobe was also probed; the pyridines
were less effective than phenyl, whereas thiophenes was generally more active (entries 47, 48, 32 and
3). Ortho mono and o,o-disubstitutions of the terminal ring were found to be greatly advantageous in
terms of the biochemical as well as antiproliferative properties, as exemplified by compounds 49, 50 and
1.
With the exception of α-gem-disubstitution (examples 53, 54), α-substitution by small side chains such
(
5
3
5
as alkyls, alkoxides, and amines led to a significant improvement in cellular GI50’s and also to lesser
extent in enzyme IC50’s (examples 32 and 56, 63, 67 in Table 5). An α-amino modification was
particularly beneficial as illustrated by the results for matched pairs: i-Pr/NMe
pentyl/pyrrolidinyl (60 /70) and c-hexyl/piperidinyl (61 /71). The 2-ethylphenyl (72) and thiophen-3-yl
73) alpha-amines emerged as the two most potent molecules from this group with IC50‘s of 3.6 and 2.9
2
, (57/67) c-
(
nM and GI50’s of 0.1 and 0.07 µM respectively. The sense of chirality of the α-substituent did not appear
to have great significance as illustrated by matched racemates and enantio-enriched pairs: 63 /64 /65 and
6
7 /68 /69 (Table 5). These results were consistent with the molecular modeling findings that aromatic
and alkylamino groups occupy a large hydrophobic pocket and engage in relatively non-specific
interactions.
2
.6 X-ray structure
8

During the course of this work we obtained an X-ray co-complex crystal structure of the potent TTK
inhibitor 32 and the TTK kinase domain at 2.2 Å resolution (PDB 4JT3, Figure 2). The kinase is in a
DFG-in conformation with the inhibitor binding into the ATP binding site of the enzyme. Only part of
the activation loop is resolved and a PEG unit is not seen in the electron density (Figure 4 supplementary
material). It should be noted that the published TTK structures available during the course of this work
4
2
were characterized by the presence of a PEG unit in the ATP binding site. More recently, TTK/small
3
1,33,35
molecule co-complex structures have been reported wherein the PEG unit is absent.
The salient
features of our co-complex X-ray structure (Figure 2) include extensive hydrophobic contacts between
ligand and protein (Val539, Met602, Ile531), two hydrogen bonds to hinge residues (Glu603 and
Gly605), as well as two hydrogen bonds of the sulfonamide nitrogen to the backbone carbonyls of
Gly605 and Asn606. The carbonyl of the acetamido group of inhibitor 32 makes a hydrogen bond to
Lys553 (N-O 2.83 Ǻ); this result explains the preference for amide linkers at this position. This
interaction was not always seen in the models based on the 3CEK structure (wherein a bound PEG unit
was wound around Lys553) and represented a significant advance in our understanding of the SAR.
However, no direct H-bond to the amide NH is observed suggesting that the N-Me capping of the
nitrogen reduces potency through a conformational or steric effect. Inspection of the co-complex X-ray
structure (Figure 2) reveals a larger packet at the position alpha to the amide carbonyl. Modeling
indicated that α-substituents would result in compounds with increased hydrophobic interactions as
represented by the more potent inhibitors of Tables 4 and 5.
2
.7 In vitro profile and kinase selectivity. Selected TTK inhibitors, 32, 63 and 73 were profiled
against a panel of 55 kinases (Millipore, Dundee, Scotland) at a screening concentration of 1 µM. This
data is presented as a heat map (Figure 3) and of the 55 kinases tested only a handful are appreciably
inhibited at the screening concentration of 1 µM (Supplementary Material). In this panel, our TTK
inhibitors display a selectivity profile somewhat more selective than sorafenib and less than imatinib, two
9

clinically useful kinase inhibitors. In addition, IC50s were determined for compounds 72 and 73 against a
panel of mitotic kinases (Table 6). Four kinases were inhibited at the sub-micromolar level namely
PLK4, AuroraA, Aurora B and CHK2 with IC50’s of 0.083, 0.40, 0.48, and 0.42 µM and 0.089, 0.9,
0
.72, and 0.1 µM for compounds 72 and 73, respectively. Thus, 72 and 73 are selective inhibitors of
TTK being at least 1.5 orders of magnitude more potent against the target vis a vis relevant mitotic
kinases. The compounds also exhibited a desirable cytochrome P450 inhibition profile with IC50 values
approximately 3 orders of magnitude greater than the TTK IC50 (Table 6).
Three cancer cell lines (HCT116, MDA-MB-468, A549) were chosen to monitor the growth inhibitory
effects of these compounds. These lines are well-characterized, encompass some of the major human
cancer types (breast, colon, lung) and thus demonstrate the spectrum of cellular activities achievable with
our inhibitors. TTK is overexpressed in each of these cancer cell lines relative to their normal tissue
3
counterparts. As a group these compounds had variable effects on cell growth inhibition; GI50s were in
the sub-micromolar range for the HCT116, MDA-MB-468 and A549 cell lines (Table 5). In particular,
compound 72, attenuates cell growth of HCT116 cells with GI50 of 0.10 µM. Compound 72 also inhibits
the cell growth of A549, MD-MB-468, OVCAR-3 andSW620 cancer cell lines with GI50’s of 0.16, 0.66,
1
.3, and 0.21, respectively.
A correlation between TTK inhibition and GI50 values is presented in Figure 4; there is a clear
2
relationship between the two values (pGI50 vs pIC50 slope = 0.81, R = 0.59). In an attempt to improve
the correlation a limited subset of physicochemical parameters were evaluated; of these, logP (a
2
surrogate for cell permeability) had the greatest effect and improved the correlation to an R of 0.72.
Note that the outliers present in the direct correlation (compounds 32, 33 and 39) are eliminated by the
introduction of the second variable, logP. Besides statistical variation, the remaining variance observed
may indicate that additional unrecognized factors such as for example, binding kinetics and transporter
affinity, contribute to the observed cell growth inhibition. The results support the hypothesis that TTK
inhibition is a driver of cell growth inhibition. The graph also allows us to estimate the average shift
between the two values (TTK IC50 and HCT116 GI50) which is about 1.5 log units. A shift of this kind
1
0

3
1,35
is often seen for anti-proliferative kinase inhibitors including TTK inhibitors,
although the
magnitude is variable, depending not only on the compound properties but also the specific methods
used to determine the two values. That being said, it would appear that the inhibitors from this series
are less permeable than compounds with more drug-like properties.
2
.8 Cell cycle analysis. To verify the mechanism of action of inhibitors 72 and 73, we examined their
ability to override the mitotic checkpoint in nocodazole-arrested cells, and analyzed their effect on cell
4
3
cycle progression. Similarly to TTK silencing by RNAi, inhibitors 72 and 73 promoted mitotic
checkpoint bypass with phospho-histone H3 EC50 values of 0.52 µM and 0.42 µM, respectively. Cell
cycle analysis showed that both inhibitors caused massive aneuploidization of cells at 1 µM and cell death
as indicated by sub-G1 cells (Figure 5) recapitulating the TTK RNAi phenotype in HCT116 cells. The
polyploidy phenotype, typical of Aurora B inhibitors, was not observed. On the other hand, a much
weaker TTK inhibitor 74 (TTK IC50 0.054 µM, HCT116 GI50 0.78 µM) did not induce any change in the
cell cycle up profile to the concentration of 2.5 µM. These data support that TKK inhibitors 72 and 73
are acting on target in the HCT116 cell line.
2
.9 In vivo efficacy. In anticipation of an in vivo efficacy experiment, the plasma levels of compound
7
2 were determined in mice at 25 mg/kg IP and PO dosing. Upon IP dosing a Cmax of 4.8 µg/mL and
.
.
AUC of 3.0 µg h/mL were obtained as compared to a Cmax of 0.13 µg/mL and AUC of 0.14 µg h/mL for
PO dosing. The result for IP dosing corresponds to a plasma level of about 6 µM, well above the GI50 in
HCT116 cell line and accordingly IP was selected as the preferred route of administration. Figure 6
shows the results of the xenograft experiment using an HCT116 mouse xenograft model. At 15 mg/kg
4
4
daily IP dose, 5-fluorouracil (5-FU), a literature standard, achieved a 46 % reduction in tumor volume
compared to the negative control. Upon IP dosing of the TTK inhibitor 72 at 12.5 and 25 mg/kg QD,
the compound demonstrated a dose dependent tumor growth inhibition (TGI) of 10 % and 66 %,
respectively. In the 12.5 mg/kg BID arm we observed a TGI of 46% i.e. comparable to the positive
control 5-FU. One mouse was lost at 25 mg/kg QD but there was no significant weight loss in the
remaining animals or in any of the other arms of the study.
1
1

3
Conclusions
TTK was identified by an in-house siRNA screen and supporting in-house and literature validation
efforts as a novel target for anti-cancer drug discovery. A screening campaign led to identification of the
3
-arylindazole core for subsequent validation work. Substitution of the indazole core with key
sulfamoylphenyl and amide moieties at positions 3 and 5 respectively established a novel class of TTK
inhibitors. Systematic optimization of the 3-(1H-indazol-3-yl)benzenesulfonamide series, supported by
computer modeling and X-ray co-crystal structure, yielded inhibitors with nanomolar activity against
TTK. Selected inhibitors from this series recapitulate the phenotype of TTK silencing RNAi on HCT116
colon cancer cells.
Representative inhibitors were selected for additional studies. Compounds 72 and 73 are potent against
TTK (IC50 = 3.6 and 2.9 nM respectively); the later shows selectivity against a broad panel of kinases.
TTK inhibitor 72 demonstrated a dose dependent inhibition of HCT116 tumor growth, comparable to the
standard of care 5-fluorouracil. The data reported herein, on this new class of antimitotic agents,
illustrates the potential of these novel TTK inhibitors for cancer therapy. However the series suffers
from some shortcomings; lack of oral exposure being among them. Further efforts detailing the
optimization of physicochemical properties and in vivo performance, culminating in and the identification
of a development candidate, will be disclosed in due course.
4
Experimental
4
.1 Computational Methods.
Modified 3CEK Structure: The 3CEK structure was modified prior to use in virtual screening
experiments. The bound PEG ligand of the first chain was removed and the active site of the resulting
structure was subjected to molecular dynamics simulations. In a 10 ns simulation the binding site
remained largely unchanged; this version was subsequently handled as an apo-structure and used for
virtual screening experiments. Limited molecular dynamics simulation of the 3CEK structure was
4
5
46
performed using MOE with the OPLS-AA force field, using the GB/VI continuum solvation model.
Subsequently, the conformation of Lys553 was modified by using the Rotamer Explorer in MOE,
1
2

followed by the local energy minimization of the side chain, the rest of the structure being held frozen in
place.
4
7
Docking Experiments: Most of the molecule handling was performed from within the MOE software.
4
8
First ligand pKa values were determined using ADMET Predictor.
Molecules were protonated,
deprotonated according to their predicted predominant protonation state at pH 7 and if predicted pKa
values fell between 6-8, all relevant protonation states were simultaneously considered. All relevant
4
9
stereoisomers were generated using the stergen setting in Corina.
This was followed by a
conformational analysis using the Conformation Import in MOE (up to 20 conformers in a 1 kcal/mol
energy window) to generate multiple input copies in agreement with our previous docking
5
0,51
recommendation
followed by the minimization of these structures using the MMFF94x forcefield in
MOE The modified protein structures were first prepared using the Protonate3D process in MOE, which
assigns relevant ionization states for the residues and finds the most favorable positions for hydrogens.
5
2
For the purposes of Glide docking, the Protein Preparation wizard in the Schrodinger suite was utilized
5
3
next. Molecules were docked using the Glide XP software.
Hinge hydrogen bond constraints to
Glu603 and Gly605 were applied in all cases to ensure correct top-scoring ligand poses.
4
4
.2 Chemistry
.2.1 General Experimental Methods. Commercially available starting materials, reagents, and
solvents were used as received. In general, anhydrous reactions were performed under an inert
atmosphere such as nitrogen or Ar. Microwave reactions were performed with a Biotage Initiator
microwave reactor. In most cases, the yields were not optimized. Reaction progress was generally
monitored by TLC using Merck SiO
by LCMS (Bruker Exquire 4000). Flash column chromatographic purification of intermediates or final
products was performed using 230-400 mesh SiO 60 from EMD chemicals. Preparative TLC SiO plates
2
plates with visualization by UV at 254 nm, by analytical HPLC or
2
2
with l mm thickness were purchased from SiliCycle Inc. Final products were also purified by preparative
reverse-phase HPLC on a Varian PrepStar model SD-1HPLC system with a Varian MonoChrom 10u C-
1
8 reverse-phase column using a gradient of about 5-30 % MeCN/ H O + 0.05 % TFA to 70-100%
2
1
3

MeCN/H O + 0.05% TFA over a 20-40-min period at a flow rate of 30-50 mL/min. Fractions containing
2
the desired material were concentrated and lyophilized to obtain the final products. Nuclear magnetic
resonance spectra were recorded on a Bruker AV 400 spectrometer using Bruker 5 mm probe PABBO
BB-1H/D Z-GRD Z108618/0004. Chemical shifts were reported in parts per million (ppm) from
tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet
(
q), multiplet (m), and broad (br). Low resolution mass spectra (MS ESI) were obtained using a Bruker
Esquire 4000 spectrometer under electric spray ionization conditions. High resolution mass spectra
HRMS) were obtained by a Waters Acquity UPLC coupled with a Waters Synapt G2 time-of-flight
(
mass spec, using an ESI source. Compounds in DMSO solutions (5 mg/mL) were diluted to 0.01
mg/mL; 5uL was injected on to a Waters BEH C18 1.7 um 2.1x100 mm column equilibrated at 10%
MeCN (solvent B) in H O (solvent A) spiked with 0.1 % formic acid at a flow rate of 0.5 mL/min.
2
Bound analytes were eluted by a linear gradient of 10 to 95 % B, between 1 and 8 min. The mass
spectrometer was calibrated using formate clusters; leucine enkephalin was used as a”lock mass" for
additional, real-time, accurate- mass correction. The purity of all compounds was determined by (A)
LCMS (Phenomenex Gemini C18 column, 50 mm x 4.6 mm, 3µm), with gradient of 10-90% MeCN in
H
2
O (with 0.1% formic acid in each mobile phases), with UV detection at λ = 254 or 214 with an Agilent
1
100 Series; or (B) analytical HPLC (Varian MonoChrom C18 column, 100 mm x 4.6 mm, 10 µm), with
gradient of 10-90% MeCN in H
2
O (with 0.1% TFA in each mobile phases), with UV detection at λ =
®
2
54 and 214 or 325 nm with a Varian ProStar 335 LC detector. PorapPak Rxn CX refers to a
commercial cation-exchange resin available from Waters.
Optical Rotations were measured at the sodium D-line (589.44 nm) using an AA-55 Polarimeter from
Optical Activity Ltd with a 2.5x100 mm unjacketed stainless steel tube at given sample concentrations (c,
units of g/100mL). Chiral supercritical fluid chromatography (SFC) analyses were performed at Lotus
Separations, LLC, using Chiralpak AS-H (25 x 0.46 cm) 30% i-BuOH(0.1% DEA)/CO , 100 bar 3
2
mL/min, detecting at 220 and 254 nm. Compound names were generated using the software built into
ChemBioDraw Ultra version 12.0.
1
4

Method A (amide coupling)
A DMF solution of 3-(5-amino-1H-indazol-3-yl)benzenesulfonamide 2,2,2-trifluoroacetate (1.0
o
equiv), DIPEA (N,N-diisopropylethylamine, 3 equiv) and RCO
2
H (1.05 equiv) at 0 C was treated with
TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate) (1.05 equiv) added in
one portion. The reaction was stirred allowing slowly to warm to rt. After several hours or overnight
stirring the crude reaction was purified directly by preparative HPLC.
Alternatively, a DMF solution of 3-(3-sulfamoylphenyl)-1H-indazole-5-carboxylic acid (1.0 equiv),
o
DIPEA (3 equiv) and RR’NH (1.05 equiv) at 0 C or rt was treated with TBTU (1.05 equiv) added in
one portion. The reaction was stirred allowing slowly to warm to rt. After several hours or overnight
stirring the crude reaction was purified directly by prepHPLC.
Method B (iodination)
o
To a cooled (0 C) DMF solution indazole (1.0 equiv) and K
2
CO
3
or KOH (~3 equiv) was added I (2-
2
4
equiv) in one portion. The reaction was stirred with cooling or rt for several h and then was treated
with xs 10 % aq NaHSO and subsequently diluted with H O. In the majority of examples a filtration
and washing (H O) of the precipitate provided the desired material with the required purity.
3
2
2
Method C (Suzuki-Miyaura cross coupling)
A mixture of 3-iodo-1H-indazole (1.0 equiv), ArB(OH)
2
or ArB(OR’) (1.2 equiv ), xs base (typically
2
3
-4 equiv, Na
PdCl (PPh
DMF/H
2
CO
3
, K
2
CO
3
, NaHCO
3
, Cs
2
CO
3
or KF) and palladium catalyst (0.05 equiv, Pd(PPh ) ,
3
4
2
3
)
2
or PdCl
2
(dppf)) in solvents (DME/H
2
O, DME/H
2
O/EtOH, PhMe/EtOH/H O or
2
o
2
O) was degassed with Ar and heated sealed in a microwave reactor (110-130 C, 1 h). The
crude material after passing through Celite using MeOH to rinse the pad was purified by preparative
HPLC or flash chromatography on SiO
2
.
Method D (Petasis borono-Mannich reaction)
To a mixture of arylboronic acid (5 mmol) and glyoxylic acid monohydrate (460 mg, 5 mmol)
in CH
2
Cl (25 mL) was added dialkylamine (5 mmol). The resulting mixture was stirred overnight at rt.
2
After evaporation of solvents, it was used as crude or purified by column chromatography.
1
5

4
.2.2 Synthesis of 3-(5-amino-1H-indazol-3-yl)benzenesulfonamide (21). tert-Butyl 1H-indazol-5-
ylcarbamate. A DMF (15 mL) solution of 1H-indazol-5-amine (1.0 g, 7.5 mmol) and DIPEA (2.0 mL,
1
1 mmol) was treated with Boc
2
O (1.7 g, 7.7 mmol) (50 % added in one portion as a solid, and 50 %
o
as a solution in anh DMF (1 mL)) at 0 C. The reaction was stirred with the cooling for 1.5 h and then
allowed to warm slowly to rt overnight. Later it was diluted with H
2
O to ~ 100 mL. A tan precipitate
was collected by filtration, washed with H
2
O and dried to afford the product as a light tan solid (1.7 g,
1
9
4 %). H NMR (400 MHz, DMSO-d
6
) δ ppm 12.90 (s., 1 H), 9.27 (s, 1 H), 7.94 (s, 1 H), 7.87 (br.s, 1
+
H), 7.41 (d, J = 8.80 Hz,1 H), 7.33 (d, J = 9.2Hz, 1 H), 1.47 (s, 9 H). MS ESI 233.9 [M + H] , calcd
+
for [C12
H
15
N
3
O
2
+ H] 234.1 .
o
tert-Butyl 3-iodo-1H-indazol-5-ylcarbamate. To a cooled (0 C) DMF (30 mL) solution t-butyl 1H-
indazol-5-ylcarbamate (1.6 g, 6.9 mmol) and K CO (3.8 g, 27.6 mmol) was added I (1.8 g, 7.1 mmol)
in one portion. The reaction was stirred with cooling for 3 h and then treated with 10 % aq NaHSO (50
mL) and subsequently with H O (150 mL). A filtration and washing (H O) of the precipitate provided
crude material which after purification by flash chromatography (SiO , 70 g, 0 to 6 % MeOH in DCM)
2
3
2
3
2
2
2
and recrystallization from EtOAc/hexanes yielded tert-butyl 3-iodo-1H-indazol-5-ylcarbamate as an
1
off-white solid (1.9 g, 78 %). H NMR (400 MHz, acetone-d
6
) δ ppm 12.54 (br. s., 1 H), 8.51 (br. s., 1
+
H), 7.87 (br.s., 1 H), 7.24 - 7.66 (m, 2 H), 1.51 (s, 9 H); MS ESI [M + H] 359.9, calcd for
+
[
C
12
H14IN
3
O
2
+ H] 360.0.
tert-butyl 3-(3-sulfamoylphenyl)-1H-indazol-5-ylcarbamate
t-Butyl 3-iodo-1H-indazol-5-ylcarbamate (100 mg, 0.28 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzenesulfonamide (118 mg, 0.42 mmol) and KF (50 mg, 0.86 mmol) in DMF (2
mL) and H
2
O (0.7 mL) were degassed by evacuation and refill with Ar (3x). Pd(PPh
3
4
) (16 mg, 0.014
mmol) was then added and the degassing was repeated. The reaction mixture was heated (sealed,
o
microwave reactor) at 120 C for 1.5 h. The crude material was concentrated under reduced pressure
and then purified by flash chromatography (SiO
2
, 50 g, 0 to 10 % MeOH in DCM) to provide tert-butyl
1
3
-(3-sulfamoylphenyl)-1H-indazol-5-ylcarbamate as a white solid (76 mg, 70 %). H NMR (400 MHz,
1
6

CD
3
OD) δ ppm 8.48 (s, 1 H), 8.21-8.14 (m, 2 H), 7.94 (d, J =8.0 Hz, 1 H), 7.72 (t, J =7.8 Hz, 1 H),
+
7
.52 (d, J=9.2 Hz,1 H), 7.42 (d, J=9.2 Hz, 1 H), 1.55 (s, 9H); MS ESI [M + H] 389.2 (100), calcd for
+
[
C
18
H
20
N
4
O
4
S + H] 389.2.
3
-(5-Amino-1H-indazol-3-yl)benzenesulfonamide.
A
suspension
of
tert-butyl
3-(3-
sulfamoylphenyl)-1H-indazol-5-ylcarbamate (0.56 g, 1.4 mmol) in DCM (30 mL) was treated with
o
o
TFA (10 mL) at 0 C. The reaction mixture was stirred for 2 h at 0 C and then at rt for 1 h.
Concentration under reduced pressure and drying in vacuo afforded the title compound as a light beige
1
solid (TFA salt, 0.58 g, quant). H NMR (400 MHz, CD
3
OD) δ ppm 8.47 (s, 1 H), 8.17 (d, J = 8.28
Hz, 1 H), 8.02 (s, 1 H), 7.98 (d, J = 7.53Hz, 1 H), 7.70 - 7.78 (m, 2 H), 7.42 (d, J = 9.29 Hz, 1 H). MS
+
+
ESI [M + H] 288.9, calcd for [C13
H
12
N
4
O
2
S + H] 289.1. HPLC: 95A% at 254 nm.
4
.2.3 (E)-3-(2-(Pyridin-4-yl)vinyl)-1H-indazole-6-carbonitrile (1).
To a suspension of 4-
(
(
chloromethyl)pyridine hydrochloride (3.28 g, 20 mmol) in PhH (50 mL) was added 40 % aq NaOH
1.35 mL). The resulting mixture was sonicated for 10 min and filtered. The residue was treated with
additional PhH (10 mL), sonicated and filtered. The combined PhH layers were dried (Na
2
SO ) to give a
4
solution of 4-(chloromethyl)pyridine in PhH.
To a solution of diethyl phosphate (3.03 g, 22 mmol) in PhH (35 mL) was added freshly cut Na (510
o
o
mg, 22 mmol). The resulting mixture was refluxed (oil temp. 90 C) for 30 min then cooled to 0 C. The
solution of 4-(chloromethyl)pyridine in PhH obtained above was added dropwise to this solution via
o
dropping funnel over 10 min. After addition, the resulting mixture was refluxed for 3 h (oil temp. 100 C)
and LC-MS indicated the completion of reaction. After cooling to rt, the insoluble white precipitate
(
NaCl) was filtered off and rinsed with PhH (20 mL). The filtrate was concentrated and dried under high
o
vacuum to give 3.50 g colorless oil which was redissolved in DMF (25 mL). It was cooled to 0 C and
treated with t-BuOK (3.36 g, 30 mmol) portion wise over 2 min; the reaction turned dark reddish brown.
o
54
After stirring for 2 min at 0 C, a solution of 3-formyl-1H-indazole-6-carbonitrile (1.71 g, 10 mmol) in
DMF (15 mL) was added dropwise by pipette over 5 min. After addition, the resulting mixture was
o
stirred for 40 min at 0 C before quenching with ice, 2 M aq HCl, H
2
O and adjusted pH to about 8 using
1
7

NaHCO
3
. The resulting precipitate was collected by filtration and rinsed with H
2
O, then dried under high
) δ
1
vacuum to give the title compound (2.016 g, 82%) as a beige solid. H NMR (400 MHz, DMSO-d
6
1
7
3.90 (s, 1 H), 8.60- 8.54 (m, 2 H), 8.43 (d, J = 8.0 Hz, 1 H), 8.20 (s, 1 H), 7.88 (d, J = 16.8 Hz, 1 H),
+
+
.73-7.67 (m, 2 H), 7.60-7.52 (m, 2 H); MS ESI 247.0 [M + H] , calcd for [C15
H
10
N + H] 247.1.
4
+
HRMS (ESI) m/z calcd for [C15
H N
10 4
+ H] 247.0984 , found 247.0985; HPLC: >99A% at 214 nm.
4
.2.4 Synthesis of 3-(Pyridin-3-yl)-1H-indazole-6-carbaldehyde (2).
trimethylsilyl)ethoxy)methyl)-1H-indazole-6-carbaldehyde and 3-iodo-2-(2-(trimethylsilyl)ethyl)-2H-
indazole-6-carbaldehyde. To a stirred suspension of 3-iodo-1H-indazole-6-carbaldehyde (3.01 g, 11.1
3-Iodo-1-((2-
(
mmol) and TBAB (36 mg, 0.11 mmol) in CH
2
Cl
2
(70 mL) and 50 % aq. KOH (20 mL) was added,
o
dropwise, (2-(chloromethoxy)ethyl)trimethylsilane (2.3 mL, 13.3 mmol) at 0 C. The stirring was
o
continued at 0 C for 3 h. The solution was then diluted with CH
2
Cl
2
(200 mL), the layers were
) and concentrated
under reduced pressure. The resulting residue was purified by column chromatography (SiO , 100 %
CH Cl ) to give of the N-1 isomer (higher eluting spot, 2.88 g, 65 %) and the N-2 isomer (lower eluting
spot, 757 mg, 17 %). H NMR N-1 isomer: (400 MHz, CDCl
J = 8.4 Hz, 1 H), 7.65 (d, J = 8.4 Hz, 1 H), 5.82 (s, 2 H), 3.60 (m, 2 H), 0.91 (m, 2 H), -0.042 (s, 9 H);
separated and the organic phase was washed with brine (2 x 100 mL), dried (MgSO
4
2
2
2
1
3
) δ 10.18 (s, 1 H), 8.11 (s, 1 H), 7.81 (d,
+
+
MS ESI 425.0 [M + Na] , calcd for [C14
H19IN
2
O
2
Si + Na] 425.02. N-2 isomer : (400 MHz, CD
3
OD)
1
0.09 (s, 1 H), 8.31 (s, 1 H), 7.62 (m, 2 H), 5.91 (s, 2 H), 3.71 (m, 2 H), 0.92 (m, 2 H), -0.039 (s, 9 H);
+
+
MS ESI 425.0 [M + Na] , calcd for [C14
-(Pyridin-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole-6-carbaldehyde. A mixture of 3-
iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole-6-carbaldehyde (100 mg, 0.25 mmol), pyridin-3-
ylboronic acid (37 mg, 0.30 mmol), PdCl (PPh (17 mg, 0.025 mmol) and 2 M aq Na CO (0.13 mL,
H
19IN
2
O
2
Si + Na] 425.0.
3
2
3
)
2
2
3
0
.25 mmol) in DME/H
2
O/EtOH (1.4 mL/0.4 mL/0.2 mL) was degassed by evacuation and refilling with
o
Ar. The reaction mixture was sealed and heated with stirring under microwave irradiation at 125 C for 2
h. The crude reaction mixture was concentrated under reduced pressure. The crude product was purified
by flash chromatography on SiO using 20-40% EtOAc/hexanes as eluent to give the desired product as a
2
1
8

1
white solid (70 mg, 79 %). H NMR (400 MHz, CDCl
3
) δ ppm 10.16 (s, 1 H), 9.22 (d, J =1.8 Hz, 1 H),
8
.67 (dd, J =4.8, 1.5 Hz, 1 H), 8.25 (ddd, J = 7.9, 1.9, 1.8 Hz, 1 H), 8.16 (s, 1 H), 8.10 (d, J = 8.3Hz, 1
H), 7.81 (dd, J = 8.3, 1.0 Hz, 1 H), 7.45 (dd, J = 7.9, 4.9 Hz, 1 H), 5.87 (s, 2 H), 3.64 (t, J = 8.5 Hz, 2
H), 0.92 (t, J = 8.3Hz, 2 H), -0.07 (s, 9 H).
3
-(Pyridin-3-yl)-1H-indazole-6-carbaldehyde.
trimethylsilyl)ethoxy)methyl)-1H-indazole-6-carbaldehyde (70 mg, 0.20 mmol) in THF (10 mL) was
treated with TBAF (1.0 mL of a 1 M in THF) and refluxed for 24 h. The solvent was removed and the
residue re-dissolved into EtOAc. The organics were washed with H O (2x), brine (2x) and then dried
MgSO ). The solvent was removed and the solid was triturated with EtOAc to give the title compound
as a pale yellow powder (5 mg, 11 %). H NMR (400 MHz, CD
1.8 Hz, 1 H), 8.61 (dd, J =4.8, 1.5 Hz, 1 H), 8.47 (dt, J = 8.0, 2.3Hz, 1 H), 8.19 - 8.24 (m, 2 H), 7.81
A
solution
of
3-(pyridin-3-yl)-1-((2-
(
2
(
4
1
3
OD) δ ppm 10.15 (s, 1 H), 9.19 (d, J
=
+
+
(
d, J = 9.5 Hz, 1 H), 7.63 (dd, J = 8.2, 5.1Hz, 1 H); MS ESI 224.0 [M + H] , calcd for [C13
24.07. HPLC: > 99A% at 214 nm.
.2.5 3-Phenyl-1H-indazole (3). The title compound was synthesized according to the General
Method C, utilizing iodo-1H-indazole (Sinova Inc., 40 mg, 0.16 mmol), phenylboronic acid (31 mg, 0.25
H
9
N
3
O + H]
2
4
mmol), Na
2
CO
3
(106 mg, 1.0 mmol), PdCl
2
dppf*CH
2
Cl
2
(12 mg, 0.015 mmol) in EtOH (5 mL) and H O
2
o
(
1 mL). After heating, sealed in microwave reactor for 3 h at 120 C under Ar the reaction was filtered
using MeOH to rinse the vial and the filter. The filtrate was concentrated and purified by flash
chromatography (SiO
2
, 0-40 % EtOAc in hexanes) to afford the desired product as a pale yellow solid
) δ ppm 10.65 (br. s., 1 H), 8.06 (d, J = 8.0 Hz, 1 H),
1
(
22.7 mg, 71 %). H NMR (400 MHz, CDCl
3
8
.00-8.40 ( m, 2 H), 7.52-7.58 (m, 2 H), 7.40-7.48 (m, 3 H), 7.22-7.28 (m, 1 H); MS ESI 195.2 [M +
+
+
H] , calcd for [C13
.2.6 3-(1H-indazol-3-yl)benzenesulfonamide (4). The title compound was synthesized according to
the General Method C, utilizing iodo-1H-indazole (Sinova Inc., 30 mg, 0.12 mmol), 3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (42 mg, 0.15 mmol), K CO (34 mg, 0.25
mmol), DME (1 mL), EtOH (1 mL), H O (0.5 mL) and Pd(PPh (14 mg, 0.012 mmol). The degassed
H
10
N
2
+ H] 195.1. HPLC: 97.3 A% at 254 nm, 98.3 A% at 300 nm.
4
2
3
2
)
3 4
1
9

o
solution was sealed and heated in a microwave reactor at 120 C for 1 h. Purification by preparative
1
HPLC provided the title compound as a white solid (21 mg, 63 %). H NMR (400 MHz, CD
3
OD) δ
ppm 8.51 (s, 1 H), 8.20 (d, J = 7.8 Hz, 1 H) 8.09 (d, J = 8.3Hz, 1 H), 7.94 (d, J = 8.0 Hz, 1 H), 7.70 (t,
J = 7.8 Hz, 1 H), 7.60 (d, J = 8.5 Hz, 1 H), 7.46 (t, J = 7.4 Hz, 1 H), 7.27 (t, J = 7.2Hz, 1 H); MS ESI
+
+
2
74.0 [M + H] , calcd for [C13
H
11
N
3
O S + H] 274.1. HPLC: 98A% at 254 nm.
2
4
.2.6 3-(4-Chloro-1H-indazol-3-yl)benzenesulfonamide (5). To a stirred mixture of 4-chloro-3-
iodo-1H-indazole (0.18 g, 0.65 mmol) and TBAB (4 mg, 0.01 mmol) in CH Cl (20 mL) and 50 % aq.
KOH (20 mL) was added, dropwise, (2-(chloromethoxy)ethyl)trimethylsilane (0.13 g, 0.78 mmol) at 0
2
2
o
o
C. The stirring was continued at 0 C for 70 min and then at rt for 3 h. The solution was then diluted
with CH Cl and brine. The layers were separated and the organic phase was washed (H O, brine) , dried
Na SO ) and concentrated under reduced pressure. The resulting residue was purified by column
chromatography (SiO , 0-6 % EtOAc in hexanes) to give of 2:1 mixture of 4-chloro-3-iodo-1-((2-
trimethylsilyl)ethoxy)methyl)-1H-indazole and 4-chloro-3-iodo-2-((2-(trimethylsilyl)ethoxy)methyl)-2H-
2
2
2
(
2
4
2
(
+
+
indazole as a pale yellow gum (0.23g, 85 %). MS ESI 408.9 [M + H] , calcd for [C13
09.0. The mixture of isomers (100 mg, 0.24 mmol) was subjected to Suzuki-Miyaura coupling
following the method described for 3-(1H-indazol-3-yl)benzenesulfonamide (4) using 3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (76 mg, 0.27 mmol), Cs CO (0.16 g, 0.50
mmol), Pd(PPh (14 mg, 0.012 mmol) in H O (1 mL) and DME (4 mL). After 40 min of microwave
heating at 100 C under Ar, the reaction was portioned between EtOAc and H O, the organic layer was
washed (satd aq NaHCO , brine), dried (Na SO ) and concentrated under reduced pressure. The residue
was purified by preparative TLC (SiO
H18ClIN
2
OSi+H]
4
2
3
3
)
4
2
o
2
3
2
4
2
, using 5:1 hexanes:EtOAc) to afford 3-(4-chloro-1-((2-
1
(
trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)benzenesulfonamide as colorless gum (69 mg, 64 %), H
NMR (400 MHz, CDCl ) δ ppm 8.29 (t, J =1.50 Hz, 1 H), 7.99 (dq, J = 8.00, 0.80 Hz, 1 H), 7.94 (dt, J
3
=
7.50, 1.50 Hz, 1 H), 7.61 (t, J = 7.80 Hz, 1 H), 7.56 (dd, J = 8.53, 0.75 Hz, 1 H), 7.38 (dd, J = 8.41,
7
.40 Hz, 1 H), 7.23 (dd, J = 7.53, 0.75 Hz, 1 H), 5.78 (s, 2 H), 4.97 (br. s. 2 H), 3.61 (dd, J = 8.78, 7.78
+
Hz, 2 H), 0.91 (t, J = 8.30 Hz, 2 H), -0.05 (s, 9 H).; MS ESI 438.1 [M + H] , calcd for
2
0

+
[
(
(
C
19
H24ClN
3
O
3
SSi+ H] 438.1 An oven-dried round bottom was charged with 3-(4-chloro-1-((2-
trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)benzenesulfonamide (34 mg, 0.078 mmol), and CH Cl
(0.1 mL, 0.8 mmol) was added dropwise and the reaction
2
2
.
5 mL) under an atmosphere of N
2
. BF
Cl
3
OEt
2
was stirred at rt for 2.5 h. The CH
2
2
was removed under reduced pressure. A mixture of EtOH ( 4
o
mL) and 2 M aq HCl (1 mL) was added and the reaction was heated at 50 C for 1 h. The reaction was
cooled to rt and concentrated under reduced pressure. The residue was purified by preparative HPLC to
1
give 3-(4-chloro-1H-indazol-3-yl)benzenesulfonamide as a white powder (15 mg, 63 %). H NMR (400
MHz, CD
3
OD) δ ppm 8.23 (t, J =1.50 Hz, 1 H), 7.99 (dq, J = 7.80, 1.10 Hz, 1 H), 7.90 (dq, J = 7.80,
1
1
.00 Hz, 1 H), 7.65 (t, J = 7.80 Hz, 1 H), 7.55 (dq, J = 8.30, 0.70 Hz, 1 H), 7.39 (dd, J = 8.30, 7.30 Hz,
+
+
H), 7.20 (dd, J = 7.30, 0.70 Hz, 1 H); MS ESI 308.0 [M + H] , calcd for [C13
H10ClN
3
O S+ H] 308.0..
2
+
HRMS (ESI) m/z calcd for [C13
H
10ClN
3
O
2
S+ H] 308.0261, found 308.0263; HPLC: 98A% at 254 nm.
4
.2.7 4-(4-Chloro-1H-indazol-3-yl)benzenesulfonamide (6). 4-chloro-3-iodo-1H-indazole (30 mg,
.12 mmol) was subjected to Suzuki-Miyaura coupling following the method described for 3-(1H-
indazol-3-yl)benzenesulfonamide (4) using 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzenesulfonamide (Frontier Scientific, 46 mg, 0.16 mmol) and Pd(PPh
Na CO
0
3
)
4
(12 mg, 0.010 mmol) in aq
o
2
3
( 1M, 0.2 mL) and DMF (0.8 mL). After heating in a microwave reactor for 1 h at 120 C, the
reaction mixture was cooled to rt, diluted with MeOH and filtered through a plug of Celite. The filtrate
was concentrated under reduced pressure and purified by preparative HPLC to afford the title compound
1
as a white powder (14.4 mg, 29 %). H NMR (400 MHz, CD
3
OD) δ ppm 8.00 (d, J = 8.53Hz, 2 H), 7.85
(
d, J = 8.50 Hz, 2 H), 7.55 (dd, J = 8.30, 0.50 Hz, 1 H), 7.39 (dd, J = 8.41, 7.40 Hz, 1 H), 7.20 (dd, J =
+
+
7
2
.30, 0.75 Hz, 1 H); MS ESI 308.1 [M + H] , calcd for [C13
H10ClN
3
O S + H] 308.0. HPLC: 99A% at
2
54 nm.
4
.2.8 3-(4-Chloro-1H-indazol-3-yl)benzamide (7). 4-Chloro-3-iodo-1H-indazole (30 mg, 0.12
mmol) was subjected to Suzuki-Miyaura coupling following the method described for 3-(1H-indazol-3-
yl)benzenesulfonamide (4) using (3-carbamoylphenyl)boronic acid (26 mg, 0.16 mmol) and Pd(PPh (12
)
3 4
2
1

mg, 0.010 mmol) in aq Na
2
CO (1 M, 0.2 mL) and DMF (0.8 mL). After heating in a microwave reactor
3
o
for 1 h at 120 C, the reaction mixture was cooled to rt, diluted with MeOH and filtered through a plug
of Celite. The filtrate was concentrated under reduced pressure and purified by preparative HPLC to
1
afford the title compound as a white powder (13 mg, 44 %). H NMR (400 MHz, CD
3
OD) δ ppm 8.21 (t,
J =1.50 Hz, 1 H), 7.97 (dq, J = 8.00, 1.00 Hz, 1 H), 7.86 (dq, J = 7.50, 0.80 Hz, 1 H), 7.58 (t, J = 7.50
Hz, 1 H), 7.54 (dd, J = 8.30, 0.50 Hz, 1 H), 7.38 (dd, J = 8.30, 7.30 Hz, 1 H), 7.19 (dd, J = 7.30, 0.50
+
+
Hz, 1 H);MS ESI 272.0 [M + H] , calcd for [C14
H10ClN
3
O + H] 272.0. HPLC: 98A% at 254 nm.
4
.2.9 (3-(4-Chloro-1H-indazol-3-yl)phenyl)methanamine (8). 4-Chloro-3-iodo-1H-indazole (30
mg, 0.12 mmol) was subjected to Suzuki-Miyaura coupling following the method described for 3-(1H-
indazol-3-yl)benzenesulfonamide (4) using (3-(aminomethyl)phenyl)boronic acid hydrochloride (Frontier
Scientific, 30 mg, 0.14 mmol) and Pd(PPh
3
)
4
(14 mg, 0.01 mmol), K
2
CO
3
(50 mg, 0.36 mmol) in EtOH
o
(
1 mL), DME (1 mL) and H
2
O (0.5 mL). After heating in a microwave reactor for 1 h at 120 C, the
reaction mixture was cooled to rt, diluted with MeOH and filtered through a plug of Celite. The filtrate
was concentrated under reduced pressure and purified by preparative HPLC to afford the title compound
1
as a white powder (TFA salt, 22 mg, 49 %). H NMR (400 MHz, CD
3
OD) δ ppm 7.76 (m, 2 H), 7.53 -
7
2
.58 (m, 3 H), 7.39 (dd, J = 8.30, 7.30 Hz, 1 H), 7.19 (dd, J = 7.40, 0.63Hz, 1 H), 4.21 (s, 2 H);MS ESI
+
+
41.0 [M – NH
2
] , calcd for [C14
H12ClN
3
- NH ] 241.0. HPLC: 99A% at 254 nm.
2
4
.2.10 Synthesis of 3-(4-(2,6-Difluorophenyl)-1H-indazol-3-yl)benzenesulfonamide (9). 4-Chloro-
1
-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole. SEMCl (0.65 mL, 3.6 mmol) was added dropwise at 0
o
C to mixture of 4-chloro-1H-indazole (0.50 g, 3.3 mmol) and TBAB (10.5 mg, 0.03 mmol) in aq KOH
50 %, 40 mL) and DCM (60 mL). The reaction was stirred with cooling for 2 h and at rt for another 2
h before it was diluted with DCM. The layers were separated and the organic phase washed (H O,
) and concentrated under reduced pressure. The residue was purified by flash
(
2
brine), dried (Na
2
SO
4
chromatography (SiO
2
, 0-6 % EtOAc in hexanes) to afford 4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-
1
1
1
H-indazole as a pale yellow oil (0.69 g, 75 %). H NMR (400 MHz, CDCl
3
) δ ppm 8.10 (d, J =1.00 Hz,
H), 7.48 (d, J = 8.28 Hz, 1 H), 7.33 (dd, J = 8.28, 7.53 Hz, 1 H), 7.18 (dd, J = 7.53, 0.50 Hz, 1 H),
2
2

+
5
.74 (s, 2 H), 3.52 (s, 2 H), 0.85 - 0.91 (m, 2 H), -0.07 (s, 9 H); MS ESI 282.9 [M + H] , calcd for
+
[
C
13
H19ClN
2
OSi + H] 283.1.
4
-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole.
mixture of 4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole (107 mg, 0.38 mmol), KOAc (110
mg, 1.1 mmol), B pin (286 mg, 1.1 mmol) and Pd dba (6.8 mg, 0.007 mmol) in anh. dioxane ( 2mL)
A
2
2
2
3
was degassed with Ar. S-Phos (6.2 mg, 0.02 mmol) was added and the degassing was repeated. The
o
reaction mixture was heated sealed under microwave irradiation at 110 C for 90 min. The reaction was
then concentrated under reduced pressure filtered through a plug of two equal layers: one of SiO and
2
one of Celite using hexanes then 5:1 hexanes:EtOAc. The filtrate was concentrated under reduced
pressure to afford 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-
1
1
H-indazole as an off white solid (130 mg, 75 %). H NMR (400 MHz, CD
3
OD) δ ppm 8.32 (d, J =1.00
Hz, 1 H), 7.79 (d, J = 8.50 Hz, 1 H), 7.66 (dd, J =6.80, 0.80 Hz, 1 H), 7.47 (dd, J = 8.50, 7.00 Hz, 1 H),
.77 (s, 2 H), 3.54 (t, J = 7.80 Hz, 2 H), 1.42 (s, 12 H), 0.84 (t, J = 7.80 Hz, 2 H), -0.09 (s, 9 H); MS
5
+
+
ESI 375.1 [M + H] , calcd for [C19
-(2,6-difluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole.
,3,2-dioxaborolan-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole (130 mg, 0.35 mmol) was
subjected to Suzuki-Miyaura coupling following the method described for 3-(1H-indazol-3-
yl)benzenesulfonamide (4) using 2-bromo-1,3-difluorobenzene (81 mg, 0.42 mmol) and Pd(PPh (40
O (1 mL) and DME (4 mL). After heating in a
H31BN
2
O
3
Si + H] 375.2.
4
4-(4,4,5,5-tetramethyl-
1
)
3 4
mg, 0.038 mmol) in Cs
2
CO
3
( 342 mg, 1.0 mmol), H
2
o
microwave reactor for 1 h at 100 C, the reaction mixture was cooled to rt, diluted with EtOAc and
washed (2 M aq NaOH, H
2
O, H
2
O-brine 1:1, brine). The organic layer was dried (Na
2
SO
4
) and
concentrated under reduced pressure. Purification by flash chromatography (SiO
2
, 0-10 % EtOAc in
hexanes) afforded 4-(2,6-difluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole as a pale
1
yellow gum (121 mg, 97 %). H NMR (400 MHz, CDCl
3
) δ ppm 7.85 (d, J =1.00 Hz, 1 H), 7.66 (d, J =
8
.53 Hz, 1 H), 7.53 (dd, J = 8.41, 7.15 Hz, 1 H), 7.35 - 7.46 (m, 1 H), 7.29 (d, J = 7.03 Hz, 1 H), 7.05 -
2
3

7
.10 (m, 2 H), 5.80 (s, 2 H), 3.57 - 3.63 (m, 2 H), 0.88 - 0.94 (m, 2 H), -0.05 (s, 9 H); MS ESI 361.0 [M
+
+
+
H] , calcd for [C19
H
22
F
2
N OSi + H] 361.1.
2
4
-(2,6-Difluorophenyl)-1H-indazole. 4-(2,6-difluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
.
indazole (121 mg, 0.34 mmol) in anh DCM ( 5mL) was treated with BF
3
OEt (0.4 mL, 3.2 mmol) at rt.
2
The reaction was sealed and stirred at for 2.5 h before being concentrated under reduced pressure. The
o
residue was heated in EtOH (4 mL) and aq HCl (2 M, 1 mL) at 50 C overnight. The reaction mixture
was concentrated under reduced pressure and purified by flash chromatography (SiO
DCM) and later preparative TLC (SiO 1 mm, 2 % MeOH in DCM, 2x) to afford 4-(2,6-difluorophenyl)-
H-indazole as a white solid (55 mg, 70 %);MS ESI 231.0 [M + H] , calcd for [C13
2
, 0-2 % MeOH in
2
+
+
1
H
8
F
2
N + H] 231.1.
2
4
-(2,6-Difluorophenyl)-3-iodo-1H-indazole
-(2,6-difluorophenyl)-1H-indazole (48 mg, 0.21 mmol) was iodinated following method B using
4
powdered KOH (44 mg, 0.8 mmol), I
2
(106 mg, 0.4 mmol) in DMF ( 5mL) to afford 4-(2,6-
1
difluorophenyl)-3-iodo-1H-indazole as a white solid (57 mg, 77 %). H NMR (400 MHz, CD
3
OD) δ
+
ppm 7.11 (d, J = 8.53 Hz, 1 H), 6.90 - 6.99 (m, 2 H), 6.45 - 6.55 (m, 3 H);MS ESI 357.0 [M + H] ,
+
calcd for [C13
H
7
F
2
IN
2
+ H] 357.0.
3
-(4-(2,6-Difluorophenyl)-1H-indazol-3-yl)benzenesulfonamide. 4-(2,6-difluorophenyl)-3-iodo-1H-
indazole (20 mg, 0.06 mmol) was subjected to Suzuki-Miyaura coupling following General Method C
using 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (Frontier Scientific, 33 mg, 0.1
mmol), DME (1.3 mL), H
2
O (0.3 mL), NaHCO
3
(20 mg, 0.24 mmol) and Pd(PPh
3
4
) (6.8 mg, 0.006
mmol). Purification by prep TLC (SiO
2
1 mm, 10 % MeOH-DCM) followed by prep HPLC afforded 3-
1
(
(
(
4-(2,6-difluorophenyl)-1H-indazol-3-yl)benzenesulfonamide as a white solid (3 mg, 14 %). H NMR
400 MHz, CD
3
OD) δ ppm 7.80 (s, 1 H), 7.71 - 7.74 (m, 1 H), 7.69 (dd, J = 8.50, 0.50 Hz, 1 H), 7.55
dd, J = 8.50, 7.00 Hz, 1 H), 7.18 - 7.25 (m, 3 H), 7.15 (d, J = 7.00 Hz, 1 H), 6.73 (dd, J = 8.30, 7.80
+
+
Hz, 2 H); MS ESI 386.2 [M + H] , calcd for [C19
H
13
F
2
N
3
O
2
S + H] 386.1. HPLC: 95A% at 254 nm.
4
.2.11 Synthesis of 3-(5-Phenyl-1H-indazol-3-yl)benzenesulfonamide (10). 5-Phenyl-1H-indazole
2
4

Following General Method C, tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole-
-carboxylate (Ryan Scientific, 110 mg, 0.32 mmol), PhBr (50 mg, 0.32 mmol), Cs CO (291 mg, 0.89
O (0.75 mL) was heated sealed in a
1
2
3
mmol) and Pd(PPh
3
)
4
(17 mg, 0.016 mmol) in DME (3 mL), H
2
o
microwave reactor at 120 C for 1.5 h. The reaction was concentrated and purified by prep TLC (SiO
2
,
1
0 % MeOH in DCM). The material was stirred than with TFA (0.5 mL) in DCM ( 6 mL) at rt for 2.5 h.
The reaction concentrated under reduced pressure and the crude 5-phenyl-1H-indazole was used directly
1
in following step. H NMR (400 MHz, CDCl
3
) δ ppm 13.08 (br. s., 1 H), 8.52 (s, 1 H), 8.07 (s, 1 H),
8
.00 (dd, J = 9.00, 1.50 Hz, 1 H), 7.90 (d, J = 9.00 Hz, 1 H), 7.61 - 7.67 (m, 2 H), 7.52 (t, J = 7.53Hz, 2
+
+
H), 7.38 - 7.48 (m, 1 H); MS ESI 194.9 [M + H] , calcd for [C13
H
10
N
2
+ H] 195.1.
3
-Iodo-5-Phenyl-1H-indazole. 5-Phenyl-1H-indazole (59 mg, 0.30 mmol) was iodinated following
General method B to afford purification by prep TLC (SiO
2
, 2 % MeOH in DCM) 3-iodo-5-phenyl-1H-
OD) δ ppm 7.67 (dd, J = 8.66,
1
indazole as a light beige solid (81 mg, 83 %). H NMR (400 MHz, CD
3
1
.63Hz, 1 H), 7.59 - 7.64 (m, 3 H), 7.52 (dd, J = 8.66, 0.63Hz, 1 H), 7.43 (t, J = 7.65 Hz, 2 H), 7.32 (t,
+
+
J = 7.28 Hz, 1 H); MS ESI 320.9 [M + H] , calcd for [C13
-(5-Phenyl-1H-indazol-3-yl)benzenesulfonamide. 3-Iodo-5-phenyl-1H-indazole (40 mg, 0.12 mmol)
was subjected to Suzuki-Miyaura coupling following General Method C using 3-(4,4,5,5-tetramethyl-
,3,2-dioxaborolan-2-yl)benzenesulfonamide (Frontier Scientific, 53 mg, 0.18 mmol), DMF (1.0 mL),
H
9
IN
2
+ H] 321.0.
3
1
H
2
O (0.4 mL), Cs
2
CO
3
(122 mg, 0.37 mmol) and Pd(PPh
3
)
4
(7.2 mg, 0.0068 mmol). Purification by prep
1
HPLC afforded 3-(5-phenyl-1H-indazol-3-yl)benzenesulfonamide as a white solid (10 mg, 24 %). H
NMR (400 MHz, CD
3
OD) δ ppm 8.54 (s, 1 H), 8.23 (d, J = 7.78 Hz, 1 H), 8.20 (s, 1 H), 7.96 (d, J =
7
.53Hz, 1 H), 7.65 - 7.77 (m, 5 H), 7.46 (t, J = 7.65 Hz, 2 H), 7.34 (t, J =6.80 Hz, 1 H); MS ESI 350.1
+
+
[
M + H] , calcd for [C19
H
15
N
3
O
2
S + H] 350.1. HPLC: 99A% at 254 nm.
4
.2.12 Synthesis of 3-(6-Phenyl-1H-indazol-3-yl)benzenesulfonamide (11). 3-(6-bromo-1H-
indazol-3-yl)benzenesulfonamide. 6-bromo-3-iodo-1H-indazole (70 mg, 0.22 mmol), was subjected to
Suzuki-Miyaura coupling following General Method C using 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2
-yl)benzenesulfonamide (Frontier Scientific, 64 mg, 0.23 mmol), DME (1.2 mL), H O (0.3 mL),
2
2
5

NaHCO
3
(55 mg, 0.65 mmol) and Pd(PPh
3
4
) (25 mg, 0.02 mmol). After heating sealed under Ar in a
microwave reactor at 120o for 1 h, the reaction was cooled to rt, diluted with MeOH and filtered
through Celite. The filtrate was concentrated and purified by preparative HPLC to afford 3-(6-bromo-
1
1
H-indazol-3-yl)benzenesulfonamide as a white solid (19 mg, 25 %). H NMR (400 MHz, CD
3
OD) δ
ppm 8.48 (t, J =1.50 Hz, 1 H), 8.18 (dt, J = 7.91, 1.19 Hz, 1 H), 8.00 (d, J = 8.53Hz, 1 H), 7.95 (dq, J =
.78, 1.00 Hz, 1 H), 7.80 (d, J =1.00 Hz, 1 H), 7.70 (t, J = 7.91Hz, 1 H), 7.38 (dd, J = 8.78, 1.76 Hz, 1
7
+
+
H); MS ESI 351.9/353.9 [M + H] , calcd for [C13
-(6-Phenyl-1H-indazol-3-yl)benzenesulfonamide. 3-(6-bromo-1H-indazol-3-yl)benzenesulfonamide
19 mg, 0.053 mmol), was subjected to Suzuki-Miyaura coupling following General Method C using
H10BrN
3
O
2
S + H] 352.0/354.0.
3
(
PhB(OH)
Pd(PPh
2
(10 mg, 0.082 mmol), DMF (1.6 mL), H
2
O (0.3 mL), Cs
2
CO
3
(54 mg, 0.16 mmol) and
o
3
)
4
(8 mg, 0.008 mmol). After heating sealed under Ar in a microwave reactor at 120 C for 3 h,
the reaction was diluted with DMSO, filtered and purified by preparative HPLC to afford 3-(6-phenyl-
1
1
H-indazol-3-yl)benzenesulfonamide as a white solid (7 mg, 37 %). H NMR (400 MHz, CD
3
OD) δ
ppm 8.54 (t, J =1.60 Hz, 1 H), 8.22 (ddd, J = 7.80, 1.80, 1.30 Hz, 1 H), 8.14 (dd, J = 8.50, 0.50 Hz, 1
H), 7.95 (ddd, J = 7.80, 1.76, 1.00 Hz, 1 H), 7.78 (q, J =0.80 Hz, 1 H), 7.70 - 7.74 (m, 3 H), 7.56 (dd, J
=
8.53, 1.51Hz, 1 H), 7.48 (t, J = 7.30 Hz, 2 H), 7.38 (tt, J = 7.30, 1.30 Hz, 1 H); MS ESI 350.1 [M +
+
+
+
H] , calcd for [C19
H
15
N
3
O
2
S + H] 350.1. HRMS (ESI) m/z calcd for [C19
H
15
N
3
O
2
S+ H] 350.0963, found
3
50.0959;HPLC: 99A% at 254 nm
.2.13 Synthesis of 3-(4-Methoxy-1H-indazol-3-yl)benzenesulfonamide (12). 3-Iodo-4-methoxy-
H-indazole. 4-Methoxy-1H-indazole (Sinova Inc., 60 mg, 0.40 mmol) was iodinated following General
(206 mg, 0.81 mmol) in DMF (1 mL). A
4
1
Method B, using powdered KOH (91 mg, 1.6 mmol) and I
2
standard work up followed by purification by flash chromatography (SiO
2
, 0-2 % MeOH in DCM)
1
afforded 3-iodo-4-methoxy-1H-indazole as a off white solid (100 mg, 90 %). H NMR (400 MHz,
CD
3
OD) δ ppm 7.29 (t, J = 8.03Hz, 1 H), 7.07 (d, J = 8.53Hz, 1 H), 6.52 (d, J = 7.53Hz, 1 H), 3.93 (s,
+
+
3
H); MS ESI 274.8 [M + H] , calcd for [C
8
H
7
IN O + H] 275.0.
2
2
6

3
-(4-Methoxy-1H-indazol-3-yl)benzenesulfonamide. 3-iodo-4-methoxy-1H-indazole (70 mg, 0.25
mmol), was subjected to Suzuki-Miyaura coupling following General Method C using 3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (Frontier Scientific, 86 mg, 0.30 mmol), DMF
(
1.5 mL), H
2
O (0.4 mL), Cs
2
CO
3
(165 mg, 0.51 mmol) and Pd(PPh
3
4
) (15 mg, 0.014 mmol). After
o
heating sealed under Ar in a microwave reactor at 120 C for 2 h, the reaction was diluted with DMSO,
filtered and purified by preparative HPLC to 3-(4-methoxy-1H-indazol-3-yl)benzenesulfonamide as a
1
white solid (14 mg, 18 %). H NMR (400 MHz, CD
3
OD) δ ppm 8.53 (s, 1 H), 8.09 (d, J = 7.78 Hz, 1
H), 7.91 (d, J = 7.78 Hz, 1 H), 7.60 (t, J = 7.78 Hz, 1 H), 7.35 (t, J = 8.00 Hz, 1 H), 7.13 (d, J = 8.28
+
Hz, 1 H), 6.62 (d, J = 7.53Hz, 1 H), 3.92 (s, 3 H); MS ESI 304.0 [M + H] , calcd for [C14
H
13
N
3
3
O S +
+
H] 304.1. HPLC: 99A% at 254 nm.
4
.2.14 3-(5-Methoxy-1H-indazol-3-yl)benzenesulfonamide (13).
synthesized according to the General Method C, utilizing 3-iodo-5-methoxy-1H-indazole (Sinova Inc.,
0 mg, 0.15 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (54 mg, 0.19
mmol), Cs CO (143 mg, 0.44 mmol), DMF (1mL), H O (0.25 mL) and Pd(PPh (8 mg, 0.007 mmol).
The title compound was
4
2
3
2
)
3 4
o
The degassed solution was sealed and heated in a microwave reactor at 120 C for 1.5 h. Purification by
1
preparative HPLC provided the title compound as a white solid (12 mg, 27 %). H NMR (400 MHz,
CD
3
OD) δ ppm 8.46 (s, 1 H), 8.15 (d, J = 7.78 Hz, 1 H), 7.93 (d, J = 7.78 Hz, 1 H), 7.70 (d, J = 8.03Hz
1
H), 7.50 (d, J = 9.03Hz, 1 H), 7.38 (d, J =1.76 Hz, 1 H), 7.13 (dd, J = 9.03, 2.26 Hz, 1 H), 3.89 (s, 3
+
+
H); MS ESI 304.0 [M + H] , calcd for [C14
H
13
N
3
O S + H] 304.1. HRMS (ESI) m/z calcd for
3
+
[
C
14
H
13
N
3
O
3
S+ H] 304.0756, found 304.0756; HPLC: 99A% at 254 nm.
4
.2.15 Synthesis of N-(3-(3-Sulfamoylphenyl)-1H-indazol-5-yl)acetamide (14). N-(1H-indazol-5-
yl)acetamide. A DMF (2 mL) solution of Ac O (82 mg, 0.80 mmol) N,N-dimethylpyridin-4-amine (5.5
2
mg, 0.045 mmol) and DIPEA (0.26 mL, 1.5 mmol) was treated 1H-indazol-5-amine (0.10 g, 0.75 mmol)
o
added in one portion at 0 C. The reaction was stirred with cooling for 3 h then treated with H
2
O (10
O. Trituration
) δ
mL) and concentrated under reduced pressure and then quenched by addition of xs H
2
1
with DCM provided the title compound as a red solid (0.11 g, 84 %). H NMR (400 MHz, DMSO-d
6
2
7

ppm 12.94 (br. s., 1 H), 9.91 (s, 1 H), 8.11 (s, 1 H), 8.00 (s, 1 H), 7.45 (d, J = 9.00 Hz, 1 H), 7.37 (d, J
+
+
=
8.53Hz, 1 H), 2.05 (s, 3 H); MS ESI 175.8 [M + H] , calcd for [C
9
H
9
N
3
O + H] 176.1.
N-(3-Iodo-1H-indazol-5-yl)acetamide. The title compound was synthesized according to the General
Method B, utilizing N-(1H-indazol-5-yl)acetamide (105 mg, 0.60 mmol ), I (304 mg, 1.2 mmol) and
2
1
K
2
CO
3
(330 mg, 2.4 mmol) in DMF (2 mL). A red solid (124 mg, 69 %). H NMR (400 MHz, DMSO-
d
6
) δ ppm 13.41 (s, 1 H), 10.03 (s, 1 H), 7.89 (s, 1 H), 7.42 - 7.50 (m, 2 H), 2.06 (s, 3 H).
3-(3-Sulfamoylphenyl)-1H-indazol-5-yl)acetamide. The title compound was synthesized according to
(
the General Method C, utilizing N-(3-iodo-1H-indazol-5-yl)acetamide (62 mg, 0.20 mmol), 3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (73 mg, 0.26 mmol), KF (23 mg, 0.40 mmol),
DMF (1.5mL), H
2
O (0.37 mL) and Pd(PPh
3
4
) (12 mg, 0.010 mmol). The degassed solution was sealed
o
and heated in a microwave reactor at 120 C for 2 h. Purification by preparative HPLC provided the title
1
compound as an off-white solid (29 mg, 44 %). H NMR (400 MHz, CD
3
OD) δ ppm 10.03 (s, 1 H),
8
.47 (s, 1 H), 8.35 (br. s., 1 H), 8.16 (d, J = 7.03Hz, 1 H), 7.94 (d, J = 8.28 Hz, 1 H), 7.70 (t, J = 7.78
+
Hz, 1 H), 7.56 (d, J = 8.50 Hz, 1 H), 7.51 (d, J = 9.00 Hz, 1 H), 2.17 (s, 3 H); MS ESI 331.2 [M + H] ,
+
calcd for [C15
H
14
N
4
O
3
S + H] 331.1. HPLC: 99A% at 254 nm.
4
.2.16 Synthesis of N-(3-(3-sulfamoylphenyl)-1H-indazol-6-yl)acetamide (15). N-(3-iodo-1H-
o
indazol-6-yl)acetamide. To a cold (0 C) DMF (10 mL) solution of DIPEA (1.3 mL, 7.5 mmol) and
Ac O (0.40 g, 3.9 mmol) and DMAP (14 mg, 0.11 mmol) was added 1H-indazol-6-amine (0.50 g, 3.7
2
mmol) in one portion. The reaction was stirred at the temperature for 3.5 h then aq NaOH (2 M, 2 mL)
was added and stirring was continued at rt for 1 h. The solvent was removed under reduced pressure,
the residue was taken into DCM, filtered, rinsed with DCM and subsequently with H
2
O (10 mL) to
+
afford N-(1H-indazol-6-yl)acetamide as a white solid (0.56 g, 87 %); MS ESI 175.8 [M + H] , calcd for
+
[
C
9
H
9
N
3
O + H] 176.1. N-(1H-indazol-6-yl)acetamide (0.56 g, 3.2 mmol) was iodinated using General
Method B using I
2
(0.97 g, 3.8 mmol) and K
2
CO
3
(0.88 g, 6.4 mmol) in DMF (10 mL) to afford after
filtration, N-(3-iodo-1H-indazol-6-yl)acetamide as a light yellow solid (0.37 g, 38 %). MS ESI 301.8 [M
+
+
+
H] , calcd for [C
9
H
8
IN O + H] 302.0.
3
2
8

N-(3-(3-Sulfamoylphenyl)-1H-indazol-6-yl)acetamide. N-(3-iodo-1H-indazol-6-yl)acetamide (62 mg,
.21 mmol) was subjected to Suzuki-Miyaura coupling following the General Method C using 3-
0
(
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (70 mg, 0.24 mmol), KF (35 mg,
0
.60 mmol), DMF (1 mL), H
2
O (0.25 mL) and Pd(PPh
3
)
4
(12 mg, 0.010 mmol). The degassed solution
o
was sealed and heated in a microwave reactor at 120 C for 2 h. Purification by preparative HPLC
1
provided the title compound as a white solid (27 mg, 39 %). H NMR (400 MHz, CD
3
OD) δ ppm
1
7
3
0.12 (br. s., 1 H), 8.52 (s, 1 H), 8.22 (s, 1 H), 8.18 (d, J = 7.03Hz, 1 H), 8.00 (d, J = 8.53Hz, 1 H),
.94 (d, J = 7.78 Hz, 1 H), 7.70 (t, J = 8.00 Hz, 1 H), 7.19 (d, J = 8.03Hz, 1 H), 2.20 (s, 3 H); MS ESI
+
+
31.0 [M + H] , calcd for [C15
H
14
N
4
O S + H] 331.1. HPLC: 95A% at 254 nm.
3
4
.2.17 Synthesis of N-(3-(3-Sulfamoylphenyl)-1H-indazol-5-yl)thiophene-2-carboxamide (16).
A. N-(1H-Indazol-5-yl)thiophene-2-carboxamide. A DMF (5 mL) solution of 1H-indazol-5-amine
o
(
0.67 g, 5.0 mmol) and Et
3
N (0.84 mL, 6.0 mmol) an 0 C was treated dropwise with thiophene-2-
carbonyl chloride (0.53 mL, 5.0 mmol) in DMF ( 1 mL). The reaction was stirred with cooling for 1 h
and then quenched by addition of xs H
2
O. The collected by filtration solid was washed with H O to
2
1
provide the title compound as a yellow solid (0.73 g, 57 %). H NMR (400 MHz, CD
3
OD) δ ppm 8.11
(
s, 1 H), 8.04 (s, 1 H), 7.92 (d, J =3.51Hz, 1 H), 7.73 (d, J =5.02Hz, 1 H), 7.61 (d, J = 8.50 Hz, 1 H),
+
7
.55 (d, J = 9.00 Hz, 1 H), 7.20 (t, J =5.00 Hz, 1 H); MS ESI 243.9 [M + H] , calcd for [C12
H
9
3
N OS +
+
H] 244.0.
N-(3-Iodo-1H-indazol-5-yl)thiophene-2-carboxamide was synthesized according to the General
Method B, utilizing N-(1H-indazol-5-yl)thiophene-2-carboxamide (0.75 g, 3.1 mmol ), I
mmol) and K CO
400 MHz, CD
2
(1.6 g, 6.2
1
2
3
(1.3 g, 9.3 mmol) in DMF (11 mL). An off-white solid (0.31 g, 27 %). H NMR
(
3
OD) δ ppm 7.94 (d, J =3.76 Hz, 1 H), 7.91 (s, 1 H), 7.75 (d, J =4.77 Hz, 1 H), 7.71 (dd,
+
J =2.01, 9.03 Hz, 1 H), 7.53 (d, J = 9.03 Hz, 1 H), 7.21 (t, J =4.39 Hz, 1 H); MS ESI 369.9 [M + H] ,
+
calcd for [C12
H
8
IN
3
OS + H] 370.0.
N-(3-(3-Sulfamoylphenyl)-1H-indazol-5-yl)thiophene-2-carboxamide.
synthesized according to the General Method C, utilizing N-(3-iodo-1H-indazol-5-yl)thiophene-2-
The title compound was
2
9