Nonclassical Phenyl Bioisosteres as Effective Replacements in a Series of Novel Open-Source Antimalarials

Article abstract of DOI:10.1021/acs.jmedchem.0c00746

The replacement of one chemical motif with another that is broadly similar is a common method in medicinal chemistry to modulate the physical and biological properties of a molecule (i.e., bioisosterism). In recent years, bioisosteres such as cubane and bicyclo[1.1.1]pentane (BCP) have been used as highly effective phenyl mimics. Herein, we show the successful incorporation of a range of phenyl bioisosteres during the open-source optimization of an antimalarial series. Cubane (19) and closo-carborane (23) analogues exhibited improved in vitro potency against Plasmodium falciparum compared to the parent phenyl compound; however, these changes resulted in a reduction in metabolic stability; unusually, enzyme-mediated oxidation was found to take place on the cubane core. A BCP analogue (22) was found to be equipotent to its parent phenyl compound and showed significantly improved metabolic properties. While these results demonstrate the utility of these atypical bioisosteres when used in a medicinal chemistry program, the search to find a suitable bioisostere may well require the preparation of many candidates, in our case, 32 compounds.

Full text of DOI:10.1021/acs.jmedchem.0c00746

Subscriber access provided by UNIV OF SOUTHERN QUEENSLAND
Article
Non-Classical Phenyl Bioisosteres as Effective
Replacements in a Series of Novel Open Source Antimalarials
Edwin Tse, Sevan D. Houston, Craig M. Williams, G. Paul Savage, Louis Rendina, Irene Hallyburton,
Mark Anderson, Raman Sharma, Gregory S Walker, R. Scott Obach, and Matthew H. Todd
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00746 • Publication Date (Web): 17 Jul 2020
Downloaded from pubs.acs.org on July 17, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 18
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Non-Classical Phenyl Bioisosteres as Effective Replacements in a
Series of Novel Open Source Antimalarials
Edwin G. Tse,[b] Sevan D. Houston,[c] Craig M. Williams,[c] G. Paul Savage,[d] Louis M. Rendina,[b]
Irene Hallyburton,[e] , Mark Anderson,[e] Raman Sharma,[f] Gregory S. Walker,[f] R. Scott Obach[f] and
Matthew H. Todd*[a]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[a] School of Pharmacy, University College London, London WC1N 1AX, United Kingdom
[b] School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia
[c] School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia
[d] CSIRO Manufacturing, Ian Wark Laboratory, Melbourne, Victoria 3168, Australia
[e] Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of
Dundee, Dundee DD1 5EH, United Kingdom
[f] Pfizer Inc., Groton, Connecticut 06340, USA
Keywords: Open Source Malaria, bioisostere, cubane, bicyclo[1.1.1]pentane, carborane
ABSTRACT: The replacement of one chemical motif with another that is broadly similar is a common method in medicinal
chemistry to modulate the physical and biological properties of a molecule (i.e. bioisosterism). In recent years, bioisosteres such as
cubane and bicyclo[1.1.1]pentane (BCP) have been used as highly effective phenyl mimics. Herein we show the successful
incorporation of a range of phenyl bioisosteres during the open source optimization of an antimalarial series. Cubane (19) and
closo-carborane (23) analogues exhibited improved in vitro potency against Plasmodium falciparum when compared to the parent
phenyl compound, however these changes resulted in a reduction in metabolic stability; unusually, enzyme-mediated oxidation was
found to take place on the cubane core. A BCP analogue (22) was found to be equipotent to its parent phenyl compound and
showed significantly improved metabolic properties. While these results demonstrate the utility of these atypical bioisosteres when
used in a medicinal chemistry program, the search to find a suitable bioisostere may well require the preparation of many
candidates, in our case, thirty-two compounds.
Our recent experiences in dealing with such issues are related to an
INTRODUCTION
antimalarial medicinal chemistry program. Malaria is one of the most
prevalent infectious diseases affecting low income countries, with 219
million cases reported worldwide in 2017, 2 million more cases than
the previous year. This translates to around 1200 deaths per day,
mostly involving young children.[6] With an increasing number of
reports of resistance to current treatment and prevention strategies,
new medicines must be discovered to combat the disease.[7,8] To help
address this, the Open Source Malaria (OSM) consortium was
created, with the aim of discovering new antimalarial medicines using
an inclusive community operating on open science principles, where
all data and experiments are shared in real time (for example every
experiment involved in the current study is freely available to view
online).[9] The most recent, fourth series examined by the consortium,
the so-called Series 4 triazolopyrazine antimalarials, originated from a
high-throughput screen performed at Pfizer in collaboration with the
Medicines for Malaria Venture (MMV). The series was then passed to
TCG Lifesciences for further optimization before being donated to the
OSM consortium in 2013.[10] The series has produced many
compounds with potent (<100 nM) activity against Plasmodium
falciparum, promising physicochemical properties and low toxicity
(Figure 1). Two compounds from the series were shown to have high
potency in an in vivo mouse model (full results to be reported
elsewhere).[11] While the potency of compounds in the series has been
frequently high, issues still remain, particularly with regards to poor
solubility and moderate clearance rates. As such, modification of the
Phenyl rings are ubiquitous in medicinal chemistry, appearing in all
manner of biologically-relevant molecules. Yet phenyl groups may
not always be the optimal motif: they are relatively non-polar, they
may be involved in - stacking interactions that can contribute to
low aqueous solubility, or have limited bioavailability and they can be
a metabolic liability.[1,2] Fortunately, there are several well-established
strategies for addressing solubility issues.[3,4] Perhaps the most
common strategy is to append charged or polar groups such as alcohol
or amine moieties to the parent compound. Such modifications will
typically reduce the hydrophobicity of the compound, but can often
dramatically worsen potency. Similar effects may be seen through the
use of heterocyclic ring replacements, but again these may alter the
potency of the desired compound.[5] Alternatively, modifications can
be made to alter the crystal packing of a compound by either
removing aromaticity via removing the phenyl rings or changing the
molecular geometry or topology. For example, replacement of a
phenyl ring by an alkyl group (linear, cyclic or caged) may help
improve solubility by eliminating - stacking interactions, and have
the added advantage of introducing further options for chemical
derivatization that may not be accessible with a phenyl ring. Again,
however, there is the caveat that these changes can heavily influence
the binding interactions between a drug and its ultimate biological
target.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 18
phenyl groups present in the structure was seen as an attractive
modifications
strategy for mitigating these issues. Careful consideration of such
1
Perhaps less immediately obvious to medicinal chemists is the use
of the boron-rich icosahedral clusters known as carboranes and their
potential application as phenyl and adamantyl bioisosteres.[28] One
prominent example is the closo-carboranyl derivative (closed form
with all carbon and boron vertices) of tamoxifen.[29] The resulting
carboranyl compounds showed high activity as anti-estrogen agents
and were even found to be more stable to degradation than tamoxifen
(C, Figure 2). Another example is that of asborin, a closo-1,2-
carborane derivative of aspirin.[30] Interestingly in this case the
pharmacological profile of the resulting carboranyl derivative was
changed drastically. Instead of acting as a selective cyclooxygenase
enzyme inhibitor (like aspirin), asborin was instead found to be a
potent inhibitor to the unrelated aldo/keto 1A1 reductase family.[31]
Other examples of carboranyl bioisosteres of polycycles such as
adamantanes can be found in medicinal chemistry, including their in
vivo application as CNS-modifying agents.[32]
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. The Series 4 triazolopyrazines possess promising
biological properties however further improvements to solubility and
metabolic stability are required.
was required as the series is in the lead optimization stage of
development and displays high sensitivity to functional group
changes.
The approach adopted was based on bioisosteres, a concept first
described by Harris Friedman in 1950 as “compounds or groups that
possess near-equal molecular shapes and volumes, approximately the
same distribution of electrons, and which exhibit similar physical
properties”.[12] The use of a bioisostere can be an extremely useful
strategy in medicinal chemistry programs for altering the physical and
biological properties of a compound.[13] Classical bioisosteres focus
on the use of structurally simple atoms, groups and ring equivalents
(e.g. replacement of a phenyl ring with a pyridine ring), while non-
classical bioisosteres may differ quite dramatically from the original
group.[14,15,16,17]
One of the more interesting non-classical phenyl bioisosteres is
cubane, a motif that has been used in medicinal chemistry projects
spanning a range of applications.[18,19,20] The size and shape of cubane
mimics the rotational volume and shape of a phenyl ring.[21] Several
biologically active molecules have been evaluated for their ability to
tolerate a cubane as a replacement for an aromatic ring, examining
changes in biological activity, solubility, metabolism, stability and
synthetic tractability (A, Figure 2).[21] The five compounds evaluated
included those with chemotherapeutic (Vorinostat), anesthetic
(Benzocaine) and neotropic (Leteprinim) applications. It was found
that four of the five showed equivalent or improved potency when the
phenyl ring was replaced with a cubane. In these four cases, the slight
increase in logP values with the cubane replacements (largest
difference of logP ~0.5) did not have a significant impact on
compound solubility. However, in the one case that led to decreased
potency, a much larger increase in logP (~1.4) was seen for the
cubane analogues that translated to a large reduction in solubility. A
more recent evaluation of a further five pharmaceuticals revealed a
mixed effect of the cubane replacement on both potency and
solubility.[22]
Figure 2. Examples of the use of phenyl bioisosteres. (A) Cubane
in the insecticide, diflubenzuron. (B) BCP in the LpPLA2
inhibitor, darapladib. (C) closo-1,2-carborane in the anti-estrogen
agent, tamoxifen.
While it is not unusual for a medicinal chemistry campaign to
explore select phenyl bioisosteres (typically cubane and BCP),[33,34] it
is much less common to see the wider range of possible bioisosteres
used in a single lead optimization program. In the case of the OSM
Series 4 triazolopyrazines, the changes required to achieve our goal
(improve the aqueous solubility and metabolic clearance parameters,
but not significantly change the potency) were not immediately clear,
in part because the project was a phenotypic drug discovery program
with no confirmed biological target. While the calculation of cLogP
values may give an indication of the potential aqueous solubility of a
compound, these values can only be used as a general guideline and
do not necessarily provide a good indication of bioavailability (note
that in this study cLogP was prioritised over other possibly relevant
parameters, such as molecular weight or total polar surface area; these
values may be found in the Supporting Information, though they
appear to show no correlation with experimental potency). This is
particularly notable for some of the phenyl bioisosteres that are
mentioned above, where the calculated logP values can be
inconsistent across different software platforms,[35] particularly where
the carborane motif is involved; in such cases it may be necessary to
measure solubility experimentally.[22] Accordingly, we investigated a
wide range of possible phenyl bioisosteres for Series 4, without
relying heavily on the calculated cLogP values as a guide.
In a similar manner to cubane, the bicyclo[1.1.1]pentane (BCP)
motif has recently found use as a non-classical phenyl ring
bioisostere.[23] While the size of the BCP motif does not match that of
benzene as closely as cubane (vide infra), its use as an effective
phenyl bioisostere has been demonstrated in a number of cases. For
example, the phenyl ring in a -secretase inhibitor currently in
development was replaced with the BCP motif resulting in a
compound with not only equipotent enzyme inhibition, but also
improved passive permeability, aqueous solubility and oral absorption
characteristics.[24] In another example, a BCP replacement in a
LpPLA2 inhibitor resulted in maintenance of potency while improving
the compound’s physicochemical properties (B, Figure 2).[25]
Increasing work has been done in recent years with the synthesis of
BCP building blocks allowing for more possibilities for its use as a
phenyl bioisostere.[26,27]
RESULTS AND DISCUSSION
2
ACS Paragon Plus Environment

Page 3 of 18
Journal of Medicinal Chemistry
Chemistry. The synthesis of the triazolopyrazine core was
Condensation with the appropriate aldehyde afforded the hydrazone
intermediate (2a–g), which was subsequently cyclised to give the
chlorinated triazolopyrazine core (3a–g). Final nucleophilic
displacement
achieved in three steps from the commercially available 2,6-
dichloropyrazine (Scheme 1). Initial displacement of a chlorine atom
with hydrazine hydrate gave 2-chloro-6-hydrazinylpyrazine (1).
1
2
3
4
Scheme 1. Preparation of Series 4 target compoundsa
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aSynthetic route to target compounds and subsequent derivatisation where applicable. Reaction conditions: i) N2H4•H2O, EtOH, 80 oC,
overnight; ii) aldehyde, EtOH, rt, overnight; iii) PhI(OAc)2, CH2Cl2, rt, overnight; iv) alcohol, 18-crown-6, PhMe, rt, overnight; v) TFA, CH2Cl2,
rt, overnight; vi) see SI; vii) CsF, EtOH, 80 oC, 25 h. (B) Aldehyde and alcohol building blocks were either commercially available or synthesized;
sat. het. = saturated heterocycle; hyd. cage = hydrocarbon cage; carb. isom. = closo-carborane isomer. (C) Target compounds.
of the chlorine atom with the appropriate alcohol produced the
desired target compounds 4–31. number of additional
Specifically, any chain length other than two carbon atoms (6)
resulted in reduced potency with complete inactivity seen with a
phenol side chain (4).
A
transformations were performed on select target compounds.
Deprotection of N-Boc compounds 14, 26 and 28 was carried out
using standard TFA conditions to give the corresponding free amine
compounds 32, 33 and 34, respectively. Compounds 16 and 19 were
used to generate late-stage biofunctionalized derivatives using dog or
rabbit liver microsomes (see Experimental). In the case of the
norbornene compound 16, oxidation of the norbornene double bond
led to a mixture of epoxides and E and/or Z diol compounds (35 and
36). In the case of the cubane compound 19, two metabolites were
isolated and were found to be hydroxylated on the cubane framework
(37 and 38). The reaction of compound 23 with CsF in EtOH gave the
hydrophilic nido-7,8-carboranyl derivative, as its cesium salt 39.
Surprisingly, when the saturated heterocyclic derivatives (9–14,
26–29 and 32–34) were evaluated, it was found that they were all
inactive with complete loss in potency compared to the parent phenyl
compound. This was seen in both the northwest and northeast cases.
The increased flexibility of these saturated heterocycles is presumably
contributing to this loss of activity.
Encouragingly, the comparable two- and three-methylene linker
cubane analogues 19 and 20 were only slightly less potent than the
phenyl compounds but also followed the trend of decreased potency
with longer chain length. The recovery of activity compared to the
saturated heterocyclic derivatives provides strong evidence of the
ability of cubane to mimic the rotational volume of a phenyl ring. It
was also verified that the mechanism of action had not changed
following these modifications: the potent bioisosteric compounds
showed activity in an ion regulation assay which is linked to the
malarial target PfATP4.[36,37]
In vitro Activity. All compounds were evaluated for in vitro
antiplasmodial activity against the 3D7 strain of P. falciparum. The
activities, indicated by their IC50 values, are summarized in the SI and
shown graphically in Figure 3. While the inherited dataset for OSM
Series 4 suggested an ether chain length of two methylene groups
between the heterocyclic core and the northwest pendant phenyl ring
was optimal, the specific compounds to prove this relationship were
absent from the dataset. In order to validate this hypothesis, a set of
compounds with an ether methylene chain length from zero to three
were evaluated, clearly showing that this separation of the phenyl
from the core was indeed important in order to maintain potency.
However, on the contrary the northeast cubane derivative 31 was
found to be completely inactive whereas the analogous phenyl
compound 30 remained potent. This surprising result suggests that the
ability of cubane to mimic a phenyl ring is reduced if the phenyl ring
possesses restricted rotation (as is assumed to be the case here), in
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 18
which case the volume occupied by the phenyl ring is not well
compounds, the remaining investigation was focused on the further
mimicked by the bulk of the cubane. Alternatively, the change may
have removed a key -stacking interaction. Considering that all
northeast phenyl replacements resulted in completely inactive
exploration of the northwest pendant phenyl ring.
1
2
3
4
The BCP derivative 22 was relatively well tolerated when
compared to the analogous benzylic ether compound 3 with only a
slight
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Heat map of in vitro potency. Color gradient: green = <1 M; orange = 1–2.5 M; red = >2.5 M. (A) Variations in potency with
northwest phenyl replacements. (B) Late-stage biofunctionalization compounds evaluated at lower concentrations than the other compounds. (C)
Variations in potency with northeast phenyl replacements.
decrease in activity. This loss in potency may be due to the smaller
size of the BCP motif, however it is noted that many examples of the
use of BCP as a phenyl bioisostere tend to result in more significant
changes to the metabolic and pharmacokinetic properties instead, as
will be discussed below.
isomers.[39] The more hydrophobic and less polar the closo-carborane
isomer is (as pertaining to the relative positions of the two carbon
atoms in each carborane cage and the resulting dipole moments), the
less potent the final compound was found to be.
In a similar manner to closo-1,2-carborane, the hydrophilic nido-
7,8-carborane cage (open form with one boron vertex removed) has
also shown potential as a phenyl bioisostere both in a carborane
variant of tamoxifen[40] and trimethoprim,[41] for example. In the latter
case, the nido form was found to be less toxic than the closo form,
however it also performed less usefully as a boron neutron capture
therapy (BNCT) agent, with poorer tumor retention and lower
selectivity ratios for boron distribution in tumor versus normal tissues.
In our case, the nido-7,8-carborane 39 was also found to be less
active, with its potency decreasing significantly when compared to the
closo-1,2-carborane 23. Selective removal of a single BH group from
the 3 (or 6) position of the closo-carborane cage affords a hydrophilic,
anionic species which would likely show diminished binding to a
hydrophobic receptor pocket.
The larger adamantyl derivatives were less well tolerated with the
one- and two-carbon chain compounds (17 and 18, respectively)
showing potencies approximately 2 to 9 times lower than the
corresponding phenyl compounds (5 and 6, respectively). This result
may be attributed to the larger volume of adamantane (vide infra)
compared to either a cubane or a rotating phenyl ring.[38] Other
hydrocarbon-caged derivatives including nopol, norbornene and
trishomocubane (15, 16 and 21, respectively) were better tolerated,
however these were still found to be less potent than the parent phenyl
compounds.
As a final investigation point, the closo-1,2-, 1,7- and 1,12-
carborane isomers were installed in place of the northwest phenyl
position. Unexpectedly, the closo-1,2- and 1,7-carborane isomers (23
and 24) showed potencies greater than not only all the other previous
derivatives but, more importantly, the parent phenyl compound. A
significant drop in activity was seen between the closo-1,7- and 1,12-
carborane isomers (24 and 25). The potency was observed to decrease
from closo-1,2- < 1,7- < 1,12-carborane. This trend is likely related to
the differences in polarity (and hydrophobicity) of the three carborane
With these encouraging results, select compounds were further
evaluated for a number of other properties including toxicity,
solubility and metabolic stability.
Toxicity. Having synthesized
a number of highly potent
compounds that possessed unusual motifs, it was important to check
4
ACS Paragon Plus Environment

Page 5 of 18
Journal of Medicinal Chemistry
for any associated toxicity. While a number of examples in the
observe preferential hydroxylation of the benzylic position in the
phenyl case but core hydroxylation for cubane. This is unexpected.
Experiments on enzymatic oxidation of methylcubane have indicated
that hydroxylation on the methyl group is favored over hydroxylation
on the cubane framework with only small amounts of the latter
products being identified.[46,47] If the cubane does become
hydroxylated, it is thought that such products are unstable and lead to
rearrangement to a ring-opened ketene product.[48] This possibly
explains why there are a small number of reports of hydroxycubanes
in the literature.[49,50] The present isolation of hydroxycubane
derivatives is therefore unusual.
literature show no significant increases in toxicity with cubane
bioisosteres,[42,43] carborane associated toxicity has been far less
studied. Encouragingly, when closo- and nido-carborane compounds
23 and 39 were evaluated for potential cytotoxicity in HepG2 cells,
both compounds were found to be inactive, with IC50 values of >10
M. Compounds 6, 19 and 23 were additionally evaluated for their
hERG activities with the phenyl compound 6 showing moderate
activity (IC50 = 7.41 M) and the cubane and carborane analogues
showing slightly greater potencies (IC50 = 4.26 M and 3.62 M,
respectively). While these values are not entirely desirable, further
hERG optimization in order to lower inhibition may be achieved
through further derivatization.
1
2
3
4
5
6
7
8
9
Comparison of Phenyl Bioisosteres. In literature reports where
phenyl bioisosteres have been used, a size comparison between the
bioisostere and the phenyl ring is often given. There is some variation
between these values. For example, in one report the diagonal C–C
atom distances for benzene and cubane have been quoted as the same
(2.70 Å)[51], while another report has quoted them as different (2.82 Å
and 2.72 Å for benzene and cubane, respectively).[34] Similar
disagreements have been seen for BCP comparisons as well (vide
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Metabolic and Physicochemical Properties. The reported effects
on metabolic and physicochemical properties of phenyl bioisosteric
replacement by cubane have been mixed. While a number of cases
have reported improvements in solubility following cubane
substitutions,[34] the trend for metabolic stability is less consistent.
There have been reports of both increased[21] and decreased[33]
metabolic stability as a result of cubane substitution. In the present
case, when compounds 6, 19 and 23 were evaluated for their
metabolic and physicochemical properties, they were found to
perform poorly compared to the parent phenyl compound (Table 1).
The solubility at pH 6.5 was low for 19 and 23 (<1.6 g/mL) when
compared to 6 (6.3 – 12.5 g/mL). Both 19 and 23 also exhibited high
clearance and short half-lives in human liver microsomes (HLM) and
mouse liver microsomes (MLM). In rat cryopreserved hepatocytes
(RCH), 19 and 23 showed intermediate and low degradation rates
with low clearance rates and short half-lives. It is likely that clearance
of the parent compound occurs via benzylic oxidation,[44] and that the
removal of this phenyl ring should slow down the rate of clearance
due to the absence of the benzylic position. Our results suggest that
the incorporation of cubane in place of phenyl has led to the cubane
becoming a metabolic liability. This appears to be in contrast to the
notion that cubane derivatives are more metabolically stable to
hydroxylation than the corresponding phenyl analogues (increased s-
character from the strong and hindered tertiary C–H bonds).[45] In our
case (vide infra), it appears that the metabolic ‘hotspot’ has shifted
onto the cubane itself. Encouragingly, the BCP derivative 22 was
found to possess significantly improved metabolic and
physicochemical properties compared to the parent phenyl compound.
While the solubilities were within the same range, 22 exhibited lower
clearance and shorter half-life values across human, mouse and rat
liver microsomes.
infra). For meaningful comparisons,
a unified approach was
preferable. We have therefore examined deposited crystal structure
files from The Cambridge Crystallographic Data Centre (CCDC), and
calculated the dimensions of key phenyl bioisosteres with UCSF
Chimera, allowing for a direct comparison (Table 2).
While the literature atom distances are, for the most part, consistent
with those calculated with UCSF Chimera, there is a notable increase
of about 10–15% between the calculated and
Table 2. Structural information of select phenyl
bioisosteres calculated using UCSF Chimera. Where they
exist, literature values are shown in parentheses. C–C and
H–H distances are indicated in purple and orange
respectively.
Structure
(CCDC
Identifier)
C–C
Distance
(Å)
H–H
Distance
(Å)
Surface
Area (Å2)a
Volume
(Å3)a
Benzene
(ABELUO)
2.77
4.63
89.7
70.1
Table 1. Metabolic and physicochemical properties of
select Series 4 phenyl bioisosteres compounds. The color
coding of the values indicates the performance of the
compound (green being more desirable).
(2.70[51]
;
(5.90[52]
)
(79.0[38]
)
2.79[21]
2.82[34]
;
)
Cubane
(CUBANE)
2.68
4.70
(4.88[53]
103
126
87.1
(2.70[51]
;
)
;
cLogP
a
LogD
(pH 7.4)
Solubility
(pH 6.5)
HLM
(CLint/T1/2
MLM
(CLint/T1/2)
2.72[21,34]
)
)
Adamantane
(ADAMAN08)
3.53
4.92
119
(6.36[54]
(134[52]
)
(128[52]
;
6
3.2
2.5
2.4
1.7b
3.7
4.3
3.6
4.4
6.3 – 12.5
<1.6
66/26
197/9
9/190
262/7
6.40[52]
)
136[38]
)
19
22
23
573/3
BCPb
(HEHRUJ)
1.88
(1.70[24]
;
1.85[34]
1.90[51]
4.78c
80.2d
61.4d
6.3 – 12.5
<1.6
26/66
;
)
249/7
>866/<2
b
acLogP values calculated using DataWarrior. Carboranes are
Norbornene
(HOBBOP)
2.89
4.64
5.26
101
135
84.8
127
not currently handled well by prediction software: when this value
is calculated with ChemDraw Professional the value is 4.0.
closo-1,2-
Carborane
(TOKGIJ)
3.23e
(148[38]
)
)
Late-Stage Biofunctionalization. Further light is shed on these
results by the isolation of the four metabolites (35–38), which were
found to be inactive. Similar metabolism of phenyl-substituted
compounds leads to oxidation at the benzylic position, as expected,
(these will be reported separately for this series) whereas for cubane
the hydroxylation occurred on the hydrocarbon cage instead, i.e. we
closo-1,7-
Carborane
(TOKGOP)
3.20e
5.12
134
126
(143[38]
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 18
carried out in silicone oil baths, controlled by temperature probe in
the oil bath.
closo-1,12-
Carborane
(TOKGUV)
3.06
4.85
136
127
(5.35[55]
)
(141[38]
)
1
2
3
4
5
6
7
8
Melting points (mp) were recorded on a Stanford Research Systems
o
OptiMelt at 1 C min-1 (capillaries ø = 1.5–1.6 mm, 90 mm) or a
aSurface areas and volumes were calculated using a model of the
solvent-excluded molecular surface with hydrogen atoms included. bR
o
Stuart SMP10 at 2 C min-1 (capillaries ø = 1.8–1.9 mm, 100 mm).
Infrared spectroscopy was carried out on
a Bruker Alpha-E
c
d
= terminal alkyne. Alkyne C–C distance. Surface area and volume
(attenuated total reflectance) without atmospheric compensation and
processed using OPUS 7.0 software. Samples were analysed neat.
Nuclear magnetic resonance spectroscopy was carried out at 300 K on
Bruker spectrometers: either AVANCE 200 (1H at 200 MHz),
AVANCE 300 (1H at 300 MHz, 13C at 75 MHz), AVANCE III 400
(1H at 400 MHz, 13C at 101 MHz) or AVANCE III 500 (1H at 500
MHz, 13C at 126 MHz). Spectra were processed using Bruker Topspin
or Mestrelab Research Mnova. Deuterated solvents (CDCl3, DMSO-
d6, CD3OD, acetone-d6) obtained from the Cambridge Isotope
Laboratories. 1H and 13C chemical shifts are reported in parts per
million (ppm) with respect to TMS at 0.00 ppm. The chemical shifts
of the spectra were calibrated to residual solvent peaks (1H: CHCl3
7.26 ppm, DMSO 2.50 ppm, MeOH 3.31 ppm, (CH3)2CO 2.05 ppm,
TMS 0.00 ppm; 13C: CHCl3 77.16 ppm, DMSO 39.52 ppm, MeOH
49.00 ppm, (CH3)2CO 39.52 ppm, TMS 0.00 ppm). 1H signal
multiplicity is reported as: singlet (s), doublet (d), triplet (t), quartet
(q), pentet (p) and combinations thereof, or multiplet (m). Broad
signals are designated broad (br). Coupling constants (J) are reported
calculated without alkyne substituents. eCarborane C–B distance.
literature values for surface area and volume. It remains largely
unclear as to which methods provide calculated values closest to those
measured from X-ray structures; accurate calculations would be
useful for calculating the dimensions of novel bioisostere motifs.
Most literature sources only provide a small selection of calculations
and specifically for volume calculations none take into account the
free rotation of the bioisostere. However, as these comparisons are
often made to show the relative dimensions, calculations performed
using the same method or program would be the most reliable, such as
in this case with UCSF Chimera. Nevertheless, it is interesting to note
how different the dimensions can be for phenyl and an effective
bioisostere. For example, the closo-carborane core was found
experimentally to be an excellent mimic, yet its volume is
significantly larger than that of a phenyl ring, meaning the functional
significance of bioisosteric size comparisons must be treated with
care.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
in Hertz (Hz). Integrals are relative.
= apparent when the
app
multiplicity was unexpected, e.g. coincidental or unresolved. Low
resolution mass spectrometry (m/z) was carried out on a Finnigan
quadrupole ion trap mass spectrometer using electrospray ionization
(ESI) or atmospheric-pressure chemical ionization (APCI). High
resolution mass spectrometry (HRMS) was performed on a Bruker 7T
FT-ICR using ESI or APCI. Positive and negative detection is
indicated by the charge of the ion, e.g. [M+H]+ indicates positive ion
detection.
CONCLUSION
While phenyl rings can be found in all manner of biologically-
important molecules, they may not always be the ideal motif for drug-
like molecules. By using classical and non-classical bioisostere
replacements, the biological properties of a compound may be altered
in dramatic ways. These changes may not always result in the desired
improvements and accurate predictions can be challenging. In
phenotypic projects like this one, which are now so common in early
stage drug discovery, rational modifications, such as the substitution
of a rotatable phenyl group with a cubane, may not lead to
improvement in the overall properties of the molecule given how
many other potential interactions may be involved before the
molecule reaches its intended target. As seen in this work, equivalent
substitutions for different phenyl rings in the same molecule can lead
to widely different potencies. In any given case, it may well be
necessary to explore a considerable range of possible bioisosteres
before arriving at an effective solution. In our case, we have identified
Purity of all compounds was >95% as determined by NMR
spectroscopy (provided for all compounds evaluated biologically).
2-Chloro-6-hydrazinylpyrazine (1) To a solution of 2,6-
dichloropyrazine (22.3 g, 150 mmol, 1 equiv.) in EtOH (428 mL, 0.35
M) was added hydrazine monohydrate (14.7 mL, 299 mmol, 2
equiv.). The reaction was heated to 80 oC until completion as
indicated by TLC. The solution was allowed to cool to rt and the
solvent was removed under reduced pressure. H2O and EtOAc were
added and the organic layer was separated. The aqueous layer was
extracted with EtOAc (3 ×) and the combined organic layers washed
with brine, dried (Na2SO4), filtered and concentrated under reduced
pressure to give 1 as fine yellow needles (19.7 g, 93%); Rf 0.12 (30%
a
series of novel closo-carborane containing compounds that
possessed potencies greater than that of the parent phenyl compound.
To get to this point required the synthesis of 30 variations of the
initial hit. More rational substitutions may be possible in cases where
the structures of the biological targets are known, particularly through
consideration of the volumes occupied by the various isosteres.
o
o
1
EtOAc in hexanes); mp 133–135 C (lit.[56] 136–139 C); H NMR
(300 MHz; CDCl3) δ: 8.13 (s, 1H), 7.89 (s, 1H), 6.38 (s, 1H), 3.86 (s,
2H); 13C NMR (75 MHz; CDCl3) δ: 156.4, 146.7, 132.4, 129.0.
Spectroscopic data matched those in the literature.[56]
General Procedure for the Synthesis of 2a–g. Compound 1 (1
equiv.) was stirred into EtOH (112 mM). Aldehyde (1 equiv.) was
added and the reaction stirred at rt until completion as indicated by
TLC. The solvent was removed under reduced pressure to give
compounds 2a–g which were carried forward without further
purification unless otherwise stated.
EXPERIMENTAL SECTION
General Information. Reagents were purchased from either
Sigma–Aldrich, Alfa Aesar, Acros, Merck, Fischer Scientific, Matrix
Scientific, Ajax or Fluorochem. Unless otherwise specified, the
reagents were used without further purification. Anhydrous
o
conditions: glassware was dried at >130 C for >12 h, assembled hot
(E)-2-Chloro-6-(2-(4-
and allowed to cool under a high vacuum where appropriate or purged
with inert gas. Anhydrous solvents were obtained from the PureSolv
system or by drying over activated 3 Å molecular sieves. Nitrogen gas
was dried over silica and calcium chloride. Argon gas was used as
acquired. The phrase in vacuo corresponds to ∼1 mbar on a Schlenk
(difluoromethoxy)benzylidene)hydrazinyl)pyrazine (2a). From 4-
(difluoromethoxy)benzaldehyde (1.92 mL, 14.5 mmol) and 1 (2.10 g,
14.5 mmol) to give 2a as light brown crystals (3.29 g, 76%); (3.29 g,
o
1
76%); Rf 0.63 (25% EtOAc in hexanes); mp 197–200 C; H NMR
(300 MHz, DMSO-d6) δ: 11.55 (s, 1H), 8.56 (s, 1H), 8.05 (d, J = 5.2
Hz, 2H), 7.79 (d, J = 8.5 Hz, 2H), 7.29 (t, J = 73.9 Hz, 1H), 7.22 (d, J
= 8.4 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ: 152.3, 151.7, 145.6,
141.6, 132.4, 131.5, 128.8, 128.3, 118.8, 116.2 (t, J = 257.9 Hz).
o
line. Reduced pressure means under rotary evaporation at 40 C from
900–50 mbar. Flash chromatography was performed on Davisil Grace
Davison 40–63 µm (230–400 mesh) silica gel or on a Biotage Isolera
One. Analytical thin layer chromatography was performed on Merck
Silica Gel 60 F254 precoated aluminium plates (0.2 mm) and
visualised with UV irradiation (254 nm) and potassium permanganate,
anisaldehyde or ninhydrin staining. High temperature reactions were
tert-Butyl
(E)-3-((2-(6-chloropyrazin-2-
yl)hydrazinylidene)methyl)pyrrolidine-1-carboxylate (2b). From
1-boc-3-pyrrolidine-carbaldehyde (200 mg, 1.00 mmol) and 1 (145
mg, 1.00 mmol); purified by automated flash chromatography on
silica (Biotage Isolera, 6–50% EtOAc in hexanes) to give 2b as an
6
ACS Paragon Plus Environment

Page 7 of 18
Journal of Medicinal Chemistry
off-white powder (215 mg, 66%); Rf 0.27 (25% EtOAc in hexanes);
product, which was purified by automated flash chromatography on
mp 124–128 oC; 1H NMR (400 MHz, CDCl3) δ: 8.47 (s, 1H), 8.45 (s,
1H), 7.98 (s, 1H), 7.16 (d, J = 4.3 Hz, 1H), 3.61 (dt, J = 20.6 & 9.8
Hz, 1H), 3.54–3.44 (m, 1H), 3.44–3.32 (m, 2H), 3.09 (s, 1H), 2.18–
2.06 (m, 1H), 2.06–1.90 (m, 1H), 1.46 (s, 9H); 13C NMR (101 MHz,
CDCl3) δ: 154.6, 152.0, 146.2, 144.9, 134.0, 129.1, 79.6, 48.9, 45.2,
41.4, 40.5, 28.6; m/z (ESI+) 348 ([M+Na]+, 100%); HRMS (ESI+)
found 348.1194 ([M+Na]+), C14H20ClN5O2Na+ requires 348.1198.
silica (Biotage Isolera, 12–100% EtOAc in hexanes) to give 3a as a
reddish-brown solid (4.91 g, 73%); Rf 0.42 (50% EtOAc in hexanes);
mp 130–133 oC; 1H NMR (300 MHz, CDCl3) δ: 9.34 (s, 1H), 7.88 (s,
1H), 7.65 (d, J = 7.6 Hz, 2H), 7.27 (d, J = 7.7 Hz, 2H), 6.64 (t, J =
73.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ: 153.2, 147.4, 147.3,
143.2, 133.2, 130.0, 123.8, 122.0, 118.8, 115.6 (t, J = 261.4 Hz).
1
2
3
4
5
6
7
8
9
tert-Butyl
3-(5-chloro-[1,2,4]triazolo[4,3-a]pyrazin-3-
yl)pyrrolidine-1-carboxylate (3b). From 2b (160 mg, 0.49 mmol) to
give the crude product, which was purified by automated flash
chromatography on silica (Biotage Isolera, 12–100% EtOAc in
hexanes) to give 3b as a pale yellow powder (101 mg, 63%); Rf 0.07
(50% EtOAc in hexanes); mp 157–160 oC; 1H NMR (400 MHz,
CDCl3) δ: 9.23 (s, 1H), 7.84 (s, 1H), 4.45–4.32 (m, 1H), 4.00–3.70
(m, 3H), 3.55 (dt, J = 10.7 & 7.3 Hz, 1H), 2.90–2.50 (m, 1H), 2.54–
2.35 (m, 1H), 1.46 (s, 9H); 13C NMR (101 MHz, CDCl3) δ: 154.4,
149.3, 147.7, 143.3, 129.5, 121.3, 79.8, 51.4 (d, J = 57.4 Hz), 45.6,
36.7 (d, J = 85.9 Hz), 31.2 (d, J = 63.2 Hz), 28.6; m/z (ESI+) 346
([M+Na]+, 100%); HRMS (ESI+) found 346.1040 ([M+Na]+),
C14H18ClN5O2Na+ requires 346.1041.
(E)-2-Chloro-6-(2-((tetrahydrofuran-3-
yl)methylene)hydrazinyl)pyrazine (2c). From tetrahydrofuran-3-
carbaldehyde (50% in H2O, 800 mg, 1.88 mmol) and 1 (578 mg, 1.88
mmol); purified by automated flash chromatography on silica
(Biotage Isolera, 6–50% EtOAc in hexanes) to give 2c as an off-white
powder (662 mg, 73%); Rf 0.28 (25% EtOAc in hexanes); mp 135–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
1
138 C; H NMR (400 MHz, CDCl3) δ: 8.46 (s, 1H), 8.40 (s, 1H),
7.98 (s, 1H), 7.14 (d, J = 5.9 Hz, 1H), 3.95 (ddd, J = 13.8, 8.4 & 6.6
Hz, 2H), 3.89–3.71 (m, 2H), 3.17 (dq, J = 13.8 & 6.3 Hz, 1H), 2.20
(dtd, J = 12.8, 7.8 & 5.4 Hz, 1H), 2.10–1.95 (m, 1H); 13C NMR (101
MHz, CDCl3) δ: 152.0, 146.2, 146.0, 134.0, 129.1, 70.9, 68.2, 42.0,
30.6; m/z (ESI+) 249 ([M+Na]+, 100%); HRMS (ESI+) found
227.0691 ([M+H]+), C9H11ClN4OH+ requires 227.0694.
5-Chloro-3-(tetrahydrofuran-3-yl)-[1,2,4]triazolo[4,3-
a]pyrazine (3c). From 2c (600 mg, 2.65 mmol) to give the crude
product, which was purified by automated flash chromatography on
silica (Biotage Isolera, 12–100% EtOAc in hexanes) to give 3c as a
pale yellow powder (457 mg, 77%); Rf 0.06 (50% EtOAc in hexanes);
tert-Butyl
(E)-4-((2-(6-chloropyrazin-2-
yl)hydrazinylidene)methyl)piperidine-1-carboxylate (2d). From 1-
boc-4-piperidine-carboxaldehyde (400 mg, 1.88 mmol) and 1 (271
mg, 1.88 mmol); filtered and washed with EtOH to give 2d as a light
brown powder (352 mg, 55%); Rf 0.52 (25% EtOAc in hexanes); mp
o
1
mp 99–102 C; H NMR (400 MHz, CDCl3) δ: 9.22 (s, 1H), 7.83 (s,
1H), 4.42 (dq, J = 8.7 & 6.3 Hz, 1H), 4.30 (dd, J = 8.5 & 7.3 Hz, 1H),
4.24–4.17 (m, 1H), 4.17–4.11 (m, 1H), 4.05 (td, J = 8.1 & 5.9 Hz,
1H), 2.77 (ddt, J = 12.2, 7.9 & 6.0 Hz, 1H), 2.48 (dddd, J = 12.5, 8.7,
7.8 & 6.5 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ: 149.8, 147.8,
143.4, 129.4, 121.3, 72.9, 68.6, 37.7, 32.4; m/z (ESI+) 247 ([M+Na]+,
100%); HRMS (ESI+) found 225.0538 ([M+H]+), C9H9ClN4OH+
requires 225.0538.
o
1
179–183 C; H NMR (400 MHz, CDCl3) δ: 8.47 (s, 1H), 8.15 (s,
1H), 7.99 (s, 1H), 7.09 (s, 1H), 4.06 (br s, 1H), 3.85 (br s, 1H), 2.93
(q, J = 12.2 Hz, 2H), 2.47 (dq, J = 9.3 & 4.5 Hz, 1H), 1.99 (dd, J =
9.2 & 3.8 Hz, 1H), 1.74 (dd, J = 8.6 & 3.9 Hz, 1H), 1.66–1.44 (m,
2H), 1.46 (s, 9H); 13C NMR (101 MHz, CDCl3) δ: 154.9, 152.1,
146.2, 145.1, 134.0, 129.2, 79.9, 38.7, 28.6, 28.5, 24.4; m/z (ESI+)
362 ([M+Na]+, 100%); HRMS (ESI+) found 362.1350 ([M+Na]+),
C15H22ClN5O2Na+ requires 362.1354.
tert-Butyl
4-(5-chloro-[1,2,4]triazolo[4,3-a]pyrazin-3-
yl)piperidine-1-carboxylate (3d). From 2d (300 mg, 0.88 mmol) to
give the crude product, which was purified by automated flash
chromatography on silica (Biotage Isolera, 12–100% EtOAc in
hexanes) to give 3d as a pale yellow powder (245 mg, 82%); Rf 0.14
(50% EtOAc in hexanes); mp 112–116 oC; 1H NMR (400 MHz,
CDCl3) δ: 9.21 (s, 1H), 7.82 (s, 1H), 4.50 (d, J = 12.5 Hz, 1H), 4.16
(s, 1H), 3.81 (t, J = 10.8 Hz, 1H), 3.28 (t, J = 11.8 Hz, 1H), 2.88 (s,
1H), 2.31 (d, J = 13.2 Hz, 1H), 2.12 (qd, J = 12.8 & 3.6 Hz, 1H), 1.91
(dq, J = 9.7 & 3.0 Hz, 1H), 1.64 (q, J = 11.6 & 10.8 Hz, 1H), 1.44 (s,
9H); 13C NMR (101 MHz, CDCl3) δ: 154.5, 150.2, 147.3, 143.3,
129.4, 121.6, 80.1, 35.6, 31.0, 28.6, 25.0; m/z (ESI+) 360 ([M+Na]+,
100%); HRMS (ESI+) found 360.1197 ([M+Na]+), C15H20ClN5O2Na+
requires 360.1198.
(E)-2-Chloro-6-(2-((tetrahydro-2H-pyran-4-
yl)methylene)hydrazinyl)pyrazine
(2e).
From
4-
formyltetrahydropyran (200 mg, 1.75 mmol) and 1 (253 mg, 1.75
mmol); filtered and washed with EtOH to give 2e as a brown powder
(284 mg, 67%); Rf 0.33 (25% EtOAc in hexanes); mp 160–168 oC; 1H
NMR (400 MHz, CDCl3) δ: 8.47 (s, 1H), 8.40 (s, 1H), 7.98 (s, 1H),
7.09 (d, J = 4.7 Hz, 1H), 4.01 (dt, J = 11.1 & 3.5 Hz, 2H), 3.47 (td, J
= 11.5 & 2.2 Hz, 2H), 2.72–2.40 (m, 1H), 1.85–1.73 (m, 2H), 1.73–
1.57 (m, 2H); 13C NMR (101 MHz, CDCl3) δ: 152.2, 148.3, 146.1,
133.8, 129.2, 67.3, 37.9, 30.1; m/z (ESI+) 263 ([M+Na]+, 100%);
HRMS (ESI+) found 241.0848 ([M+H]+), C10H13ClN4OH+ requires
241.0851.
(E)-2-Chloro-6-(2-(4-iodobenzylidene)hydrazinyl)pyrazine (2f).
From S13 (175 mg, 0.75 mmol) and 1 (109 mg, 0.75 mmol) to give 2f
as a light yellow powder (200 mg, 74%); carried forward without
further purification or characterisation; Rf 0.26 (10% EtOAc in
hexanes).
5-Chloro-3-(tetrahydro-2H-pyran-4-yl)-[1,2,4]triazolo[4,3-
a]pyrazine (3e). From 2e (230 mg, 0.96 mmol) to give the crude
product, which was purified by automated flash chromatography on
silica (Biotage Isolera, 12–100% EtOAc in hexanes) to give 3e as pale
yellow crystalline powder (145 mg, 63%); Rf 0.06 (50% EtOAc in
2-Chloro-6-(2-((E)-((2r,3R,4r,5S)-4-iodocuban-1-
o
1
hexanes); mp 185–193 C; H NMR (400 MHz, CDCl3) δ: 9.23 (s,
1H), 7.82 (s, 1H), 4.27–4.09 (m, 2H), 3.95 (tt, J = 11.3 & 3.7 Hz,
1H), 3.61 (td, J = 11.7 & 2.1 Hz, 2H), 2.45–2.21 (m, 2H), 2.17–2.03
(m, 2H); 13C NMR (101 MHz, CDCl3) δ: 151.9, 147.4, 143.4, 129.5,
121.4, 67.5, 34.3, 32.4; m/z (ESI+) 261 ([M+Na]+, 100%); HRMS
(ESI+) found 239.0693 ([M+H]+), C10H11ClN4OH+ requires 239.0694.
yl)methylene)hydrazinyl) pyrazine (2g). From S15 (60.0 mg, 0.23
mmol) and 1 (33.6 mg, 0.23 mmol) to give 2g as an off-white powder
(90.1 mg, >100%); carried forward without further purification or
characterisation; Rf 0.73 (2% MeOH in CH2Cl2).
General Procedure for the Synthesis of 3a–g. The product from
General Procedure 1 (1 equiv.) was stirred into CH2Cl2 (112 mM).
PhI(OAc)2 (1 equiv.) was added and the reaction stirred at rt until
completion as indication by TLC. The reaction was quenched with
sat. aq. NaHCO3, diluted with CH2Cl2 and the organic layer separated.
The aqueous layer was extracted with CH2Cl2 (3 ×) and the combined
organic layers were washed with brine, dried (MgSO4), filtered and
concentrated under reduced pressure to give the corresponding crude
material which was purified by flash chromatography on silica to give
the compounds 3a–g.
5-Chloro-3-(4-iodophenyl)-[1,2,4]triazolo[4,3-a]pyrazine (3f).
From 2f (200 mg, 0.56 mmol) to give the crude product, which was
purified by automated flash chromatography on silica (Biotage
Isolera, 12–100% EtOAc in hexanes) to give 3f as a light brown
powder (157 mg, 79%); carried forward without further
characterisation; Rf 0.42 (50% EtOAc in hexanes); mp decomposed
o
1
>200 C; H NMR (300 MHz, CDCl3) δ: 9.34 (s, 1H), 7.88 (dapp, J =
5.8 Hz, 3H), 7.37 (d, J = 7.9 Hz, 2H).
5-Chloro-3-(4-iodocuban-1-yl)-[1,2,4]triazolo[4,3-a]pyrazine
(3g). From 2g (80.0 mg, 0.21 mmol) to give the crude product, which
was purified by automated flash chromatography on silica (Biotage
5-Chloro-3-(4-(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-
a]pyrazine (3a). From 2a (6.70 g, 22.4 mmol) to give the crude
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 18
Isolera, 0–10% MeOH in CH2Cl2) to give 3g as a brown powder (62.0
125.2, 118.6, 115.7 (t, J = 261.0 Hz), 108.4, 70.1, 31.6, 29.9; 19F
mg, 78%); Rf 0.37 (2% MeOH in CH2Cl2); mp decomposed >150 oC;
1H NMR (300 MHz, CDCl3) δ: 9.20 (s, 1H), 7.87 (s, 1H), 4.95–4.56
(m, 3H), 4.54–4.12 (m, 3H); 13C NMR (75 MHz, CDCl3) δ: 147.7,
142.8, 129.1, 120.9, 54.7, 53.0, 51.7, 50.4, 34.5; m/z (ESI+) 405
([M+Na]+, 100%); HRMS (ESI+) found 404.9375 ([M+Na]+),
C13H8ClIN4Na+ requires 404.9374.
NMR (376 MHz, CDCl3) δ: -81.33; m/z (ESI+) 419 ([M+Na]+,
100%); HRMS (ESI+) found 397.1469 ([M+H]+), C21H18F2N4O2H+
requires 397.1471.
1
2
3
4
5
6
7
8
5-(2-Cyclopropylethoxy)-3-(4-(difluoromethoxy)phenyl)-
[1,2,4]triazolo[4,3-a]pyrazine (8). From 2-cyclopropylethanol (50.0
mg, 0.58 mmol) and 3a (172 mg, 0.58 mmol) to give the crude
product, which was purified by automated flash chromatography on
silica (Biotage Isolera, 25–100% EtOAc in hexanes) to give an orange
solid; repurifed by preparative TLC (75% EtOAc in hexanes) to give
8 as a yellow powder (42.3 mg, 21%); Rf 0.58 (100% EtOAc); mp
120–124 oC; 1H NMR (400 MHz, CDCl3) δ: 9.03 (s, 1H), 7.71 (d, J =
8.8 Hz, 2H), 7.32 (s, 1H), 7.23 (d, J = 8.7 Hz, 2H), 6.60 (t, J = 73.2
Hz, 1H), 4.25 (t, J = 6.3 Hz, 2H), 1.51 (q, J = 6.4 Hz, 2H), 0.54–0.19
(m, 3H), -0.01–-0.10 (m, 2H); 13C NMR (101 MHz, CDCl3) δ: 152.5,
148.0, 146.4, 144.4, 136.2, 132.6, 125.1, 118.8, 115.7 (t, J = 261.4
Hz), 108.3, 71.3, 33.5, 7.4, 4.3; 19F NMR (376 MHz, CDCl3) δ: -
81.43; m/z (ESI+) 369 ([M+Na]+, 100%), 715 ([2M+Na]+, 35%);
HRMS (ESI+) found 347.1313 ([M+H]+), C17H16F2N4O2H+ requires
347.1314.
General Procedure for the Synthesis of 4–31. Nucleophile (1.0
equiv.) was added to PhMe (168 mM) along with the product from
General Procedure 2 (1.0 equiv.), KOH (3.0 equiv.) and 18-crown-6
(0.1 equiv.). The reaction was stirred at rt until completion as
indicated by TLC. The reaction was diluted with H2O, then extracted
with EtOAc. The organic layers were washed with H2O until the
aqueous layer became neutral, followed by brine, dried (MgSO4),
filtered and concentrated under reduced pressure to give the crude
material, which was purified by flash chromatography on silica to give
compounds 4–31.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-(4-(Difluoromethoxy)phenyl)-5-phenoxy-[1,2,4]triazolo[4,3-
a]pyrazine (4). From phenol (31.7 mg, 0.34 mmol) and 3a (100 mg,
0.34 mmol); the solvent was removed and the residue directly purified
by automated flash chromatography on silica (Biotage Isolera, 25–
100% EtOAc in hexanes) to give 4 as an off-white powder (44.9 mg,
3-(4-(Difluoromethoxy)phenyl)-5-(2-(oxetan-3-yl)ethoxy)-
[1,2,4]triazolo[4,3-a]pyrazine (9). From S1 (35.0 mg, 0.34 mmol)
and 3a (102 mg, 0.34 mmol) to give the crude product, which was
purified by automated flash chromatography on silica (Biotage
Isolera, 1–10% MeOH in CH2Cl2) to give 9 as an orange powder
o
1
38%); Rf 0.41 (100% EtOAc); mp 113–117 C; H NMR (400 MHz,
CDCl3) δ: 9.12 (s, 1H), 7.72 (d, J = 8.7 Hz, 2H), 7.36 (t, J = 7.9 Hz,
2H), 7.25–7.19 (m, 2H), 7.15 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.0 Hz,
2H), 6.54 (t, J = 73.3 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ: 153.1,
152.6 (t, J = 2.8 Hz), 148.2, 146.5, 142.7, 138.4, 132.3, 130.6, 126.5,
124.4, 118.8, 115.7 (t, J = 261.0 Hz), 114.2; 19F NMR (376 MHz,
CDCl3) δ: -81.36; m/z (ESI+) 377 ([M+Na]+, 100%); HRMS (ESI+)
found 355.0998 ([M+H]+), C18H12F2N4O2H+ requires 355.1001.
o
1
(21.6 mg, 17%); Rf 0.06 (100% EtOAc); mp 110–114 C; H NMR
(400 MHz, CDCl3) δ: 9.04 (s, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.30 (s,
1H), 7.25 (d, J = 7.5 Hz, 2H), 6.62 (t, J = 73.1 Hz, 1H), 4.21–3.96
(m, 1H), 3.91–3.71 (m, 1H), 3.65 (qapp, J = 7.7 Hz, 1H), 3.48 (ddapp, J
= 9.0 & 7.0 Hz, 1H), 3.34 (dd, J = 9.1 & 4.7 Hz, 1H), 2.47 (hept, J =
7.1 Hz, 1H), 1.86 (dtd, J = 12.9, 8.1 & 5.2 Hz, 1H), 1.42 (td, J = 13.0
& 7.6 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ: 152.4, 147.8, 146.2,
144.0, 136.8, 132.4, 125.2, 119.0, 115.6 (t, J = 262.0 Hz), 108.4,
72.3, 69.8, 67.5, 38.3, 28.4; m/z (ESI+) 385 ([M+Na]+, 100%), 747
([2M+Na]+, 47%); HRMS (ESI+) found 385.1087 ([M+Na]+),
C17H16F2N4O3Na+ requires 385.1083.
5-(Benzyloxy)-3-(4-(difluoromethoxy)phenyl)-
[1,2,4]triazolo[4,3-a]pyrazine (5). From benzyl alcohol (70.0 µL,
0.67 mmol) and 3a (200 mg, 0.67 mmol) to give the crude product,
which was purified by automated flash chromatography on silica
(Biotage Isolera, 25–100% EtOAc in hexanes) to give 5 as a pale
yellow powder (135 mg, 54%); Rf 0.08 (50% EtOAc in hexanes); mp
152–156 oC; 1H NMR (300 MHz, CDCl3) δ: 9.02 (s, 1H), 7.58 (d, J =
8.5 Hz, 2H), 7.42 (s, 1H), 7.38–7.22 (m, 3H), 7.07 (d, J = 7.3 Hz,
2H), 6.95 (d, J = 8.4 Hz, 2H), 6.46 (t, J = 73.4 Hz, 1H), 5.19 (s, 2H);
13C NMR (75 MHz, CDCl3) δ: 152.4, 147.9, 146.5, 143.9, 136.7,
132.9, 132.5, 129.3, 128.8, 128.2, 124.5, 118.3, 115.7 (t, J = 260.4
Hz), 108.9, 72.9; m/z (ESI+) 391 ([M+Na]+, 100%), 759 ([2M+Na]+,
71%); HRMS (ESI+) 391.0975 ([M+Na]+), C19H14F2N4O2Na+
requires 391.0977.
6-(2-((3-(4-(Difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-
a]pyrazin-5-yl)oxy)ethyl)-2-oxa-6-azaspiro[3.3]heptane (10). From
2-[2-oxa-6-azaspiro[3.3]heptan-6-yl]ethan-1-ol (100 mg, 0.70 mmol)
and 3a (207 mg, 0.70 mmol) to give the crude product, which was
purified by automated flash chromatography on silica (Biotage
Isolera, 1–10% MeOH in CH2Cl2) to give 10 as a light brown powder
o
1
(145 mg, 52%); Rf 0.07 (5% MeOH in CH2Cl2); mp 136–141 C; H
NMR (400 MHz, CDCl3) δ: 9.03 (s, 1H), 7.75 (d, J = 8.8 Hz, 2H),
7.28 (s, 1H), 7.26 (d, J = 8.8 Hz, 2H), 6.64 (t, J = 73.0 Hz, 1H), 4.62
(s, 4H), 4.16 (t, J = 5.2 Hz, 2H), 3.09 (s, 4H), 2.58 (t, J = 5.2 Hz,
2H); 13C NMR (101 MHz, CDCl3) δ: 152.2, 147.9, 146.3, 144.0,
136.7, 132.7, 125.1, 119.0, 115.5 (t, J = 262.4 Hz), 108.6, 81.0, 70.0,
64.2, 56.4, 39.5; m/z (ESI+) 426 ([M+Na]+, 100%); HRMS (ESI+)
found 426.1353 ([M+Na]+), C19H19F2N4O3Na+ requires 426.1348.
3-(4-(Difluoromethoxy)phenyl)-5-phenethoxy-
[1,2,4]triazolo[4,3-a]pyrazine (6). From 2-phenylethanol (80.8 µL,
0.67 mmol) and 3a (200 mg, 0.67 mmol) to give the crude product,
which was purified by automated flash chromatography on silica
(Biotage Isolera, 25–100% EtOAc in hexanes) to give 6 as a pale
yellow powder (189 mg, 73%); Rf 0.10 (50% EtOAc in hexanes); mp
115–117 oC; 1H NMR (500 MHz, DMSO-d6) δ: 9.03 (s, 1H), 7.77 (d,
J = 8.6 Hz, 2H), 7.59 (s, 1H), 7.36 (t, J = 73.7 Hz, 1H), 7.30 (d, J =
8.6 Hz, 2H), 7.20 – 7.13 (m, 3H), 6.97 – 6.84 (m, 2H), 4.50 (t, J = 6.4
Hz, 2H), 2.88 (t, J = 6.4 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ:
152.0, 147.4, 145.5, 143.8, 137.3, 135.0, 132.6, 128.6, 128.2, 126.4,
124.7, 117.6, 116.1 (t, J = 258.2 Hz), 108.8, 71.2, 33.8; m/z (ESI+)
405 ([M+Na]+, 100%), 787 ([2M+Na]+, 71%); HRMS (ESI+)
405.1131 ([M+Na]+), C20H16F2N4O2Na+ requires 405.1133.
3-(4-(Difluoromethoxy)phenyl)-5-(2-(pyrrolidin-1-yl)ethoxy)-
[1,2,4]triazolo[4,3-a]pyrazine
(11).
From
1-(2-
hydroxyethyl)pyrrolidine (59.1 µL, 0.51 mmol) and 3a (150 mg, 0.51
mmol); the solvent was removed and the residue directly purified by
automated flash chromatography on silica (Biotage Isolera, 1–10%
MeOH in CH2Cl2) to give 11 as a brown powder (70.2 mg, 37%); Rf
o
1
0.12 (5% MeOH in CH2Cl2); mp 96–102 C; H NMR (400 MHz,
CD3OD) δ: 8.98 (s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.54 (s, 1H), 7.33
(d, J = 8.8 Hz, 2H), 7.00 (t, J = 73.5 Hz, 1H), 4.43 (t, J = 5.2 Hz,
2H), 2.77 (t, J = 5.2 Hz, 2H), 2.32 (t, J = 6.5 Hz, 4H), 1.87–1.59 (m,
4H); 13C NMR (101 MHz, CD3OD) δ: 151.9, 149.5, 149.1, 146.0,
136.3, 134.0, 125.9, 119.5, 117.4 (t, J = 259.5 Hz), 110.1, 71.3, 55.1,
54.6, 24.2; m/z (ESI+) 398 ([M+Na]+, 100%); HRMS (ESI+) found
376.1582 ([M+H]+), C18H19F2N5O2H+ requires 376.1580.
3-(4-(Difluoromethoxy)phenyl)-5-(3-phenylpropoxy)-
[1,2,4]triazolo[4,3-a]pyrazine (7). From 3-phenylpropanol (45.9 µL,
0.34 mmol) and 3a (100 mg, 0.34 mmol); the solvent was removed
and the residue directly purified by automated flash chromatography
on silica (Biotage Isolera, 25–100% EtOAc in hexanes) to give 7 as a
dark yellow crystalline powder (111 mg, 83%); Rf 0.42 (100%
EtOAc); mp 153–157 oC; 1H NMR (400 MHz, CDCl3) δ: 9.03 (s, 1H),
7.75 (d, J = 8.8 Hz, 2H), 7.32–7.16 (m, 6H), 6.96 (d, J = 7.0 Hz, 2H),
6.47 (t, J = 73.3 Hz, 1H), 4.19 (t, J = 5.9 Hz, 2H), 2.35 (t, J = 7.6 Hz,
2H), 1.96 (dt, J = 8.4 & 6.4 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ:
152.7, 147.9, 146.3, 144.2, 140.0, 136.5, 132.6, 128.8, 128.3, 126.6,
3-(4-(Difluoromethoxy)phenyl)-5-(2-(piperidin-1-yl)ethoxy)-
[1,2,4]triazolo[4,3-a]pyrazine
(12).
From
1-(2-
hydroxyethyl)piperidine (67.1 µL, 0.51 mmol) and 3a (150 mg, 0.51
mmol); the solvent was removed and the residue directly purified by
automated flash chromatography on silica (Biotage Isolera, 1–10%
8
ACS Paragon Plus Environment

Page 9 of 18
Journal of Medicinal Chemistry
MeOH in CH2Cl2) to give 12 as a brown powder (70.2 mg, 36%); Rf
1H(min)), 5.90 (dd, J = 5.8 & 3.2 Hz, 1H(min)), 5.74 (dd, J = 5.7 & 2.8
Hz, 1H(maj)), 4.09 (ddd, J = 140.2, 8.8 & 6.2 Hz, 2H(min)), 3.88 (dt, J =
90.9 & 9.1 Hz, 2H(maj)), 2.89–2.65 (m, 2H(comb)), 2.43–2.20 (m,
3H(comb)), 1.71 (ddd, J = 12.4, 9.1 & 3.7 Hz, 1H(maj)), 1.64 (dq, J = 9.5
& 4.8 Hz, 1H(min)), 1.47–1.35 (m, 1H(comb)), 1.34–1.23 (m, 1H(comb)),
1.21–1.12 (m, 3H(comb)), 1.10–0.99 (m, 1H(min)), 0.47 (ddd, J = 11.7,
4.1 & 2.6 Hz, 1H(maj)); 13C NMR (101 MHz, CDCl3, present as a
mixture of endo and exo isomers) δ: 152.4, 152.3, 147.9, 146.3, 146.3,
144.24, 144.22, 138.6, 137.3, 136.3, 136.1, 135.7, 132.6, 132.5,
131.5, 129.1, 128.7, 125.3, 125.1, 119.1, 118.7, 115.7 (t, J = 261.6
Hz), 115.6 (t, J = 261.9 Hz), 108.2, 75.0, 74.4, 49.4, 44.8, 43.7, 43.4,
42.3, 41.6, 38.1, 37.9, 29.4, 28.9; m/z (ESI+) 407 ([M+Na]+, 100%);
HRMS (ESI+) found 407.1294 ([M+Na]+), C20H18F2N4O2Na+ requires
407.1290.
o
1
0.21 (5% MeOH in CH2Cl2); mp 123–129 C; H NMR (400 MHz,
CD3OD) δ: 8.97 (s, 1H), 7.79 (d, J = 8.7 Hz, 2H), 7.53 (s, 1H), 7.33
(d, J = 8.7 Hz, 2H), 7.01 (t, J = 73.5 Hz, 1H), 4.43 (t, J = 5.1 Hz,
2H), 2.63 (t, J = 5.1 Hz, 2H), 2.21 (br sapp, 4H), 1.73–1.35 (m, 4H),
1.41–1.33 (m, 2H); 13C NMR (101 MHz, CD3OD) δ: 154.3, 149.0,
147.8, 146.0, 136.2, 133.9, 125.9, 119.4, 117.4 (t, J = 259.4 Hz),
110.1, 70.3, 57.7, 55.4, 26.5, 24.7; m/z (ESI+) 412 ([M+Na]+, 100%);
HRMS (ESI+) found 390.1737 ([M+H]+), C19H21F2N5O2H+ requires
390.1736.
1
2
3
4
5
6
7
8
4-(2-((3-(4-(Difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-
9
a]pyrazin-5-yl)oxy)ethyl)morpholine
(13).
From
4-(2-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
hydroxyethyl)morpholine (61.2 µL, 0.51 mmol) and 3a (150 mg, 0.51
mmol); the solvent was removed and the residue directly purified by
automated flash chromatography on silica (Biotage Isolera, 1–10%
MeOH in CH2Cl2) to give 13 as a light brown powder (141 mg, 71%);
Rf 0.26 (5% MeOH in CH2Cl2); mp 160–164 oC; 1H NMR (400 MHz,
CD3OD) δ: 8.97 (s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.53 (s, 1H), 7.33
(d, J = 8.7 Hz, 2H), 7.00 (t, J = 73.5 Hz, 1H), 4.42 (t, J = 5.1 Hz,
2H), 3.62–3.42 (m, 4H), 2.61 (t, J = 5.0 Hz, 2H), 2.37–2.04 (m, 4H);
13C NMR (101 MHz, CD3OD) δ: 154.3, 150.1, 149.0, 146.1, 136.2,
134.0, 125.9, 119.4, 117.5 (t, J = 259.3 Hz), 110.1, 70.2, 67.7, 57.5,
54.5; m/z (ESI+) 414 ([M+Na]+, 100%); HRMS (ESI+) found
392.1530 ([M+H]+), C18H19F2N4O3H+ requires 392.1529.
5-(((3r,5r,7r)-Adamantan-1-yl)methoxy)-3-(4-
(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine
(17).
From 1-adamantanemethanol (100 mg, 0.60 mmol) and 3a (178 mg,
0.60 mmol) to give the crude product, which was purified by
automated flash chromatography on silica (Biotage Isolera, 25–100%
EtOAc in hexanes) to give 17 as a yellow powder (140 mg, 55%); Rf
0.53 (100% EtOAc); mp 210–219 oC; 1H NMR (400 MHz, CDCl3) δ:
9.01 (s, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.29 (s, 1H), 7.26 (d, J = 8.7
Hz, 2H), 6.59 (t, J = 73.2 Hz, 1H), 3.71 (s, 2H), 1.88 (br s, 3H), 1.67
(d, J = 12.3 Hz, 3H), 1.48 (d, J = 11.6 Hz, 3H), 1.26 (dapp, J = 2.5 Hz,
6H); 13C NMR (101 MHz, CDCl3) δ: 152.6, 147.9, 146.0, 144.9,
136.2, 132.4, 125.5, 118.9, 115.7 (t, J = 261.2 Hz), 108.7, 81.6, 38.8,
36.6, 33.6, 27.8; m/z (ESI+) 449 ([M+Na]+, 100%), 875 ([2M+Na]+,
44%); HRMS (ESI+) found 449.1761 ([M+Na]+), C23H24F2N4O2Na+
requires 449.1760.
tert-Butyl
4-(2-((3-(4-(difluoromethoxy)phenyl)-
[1,2,4]triazolo[4,3-a]pyrazin-5-yl)oxy)ethyl)piperazine-1-
carboxylate (14). From S2 (200 mg, 0.87 mmol) and 3a (258 mg,
0.87 mmol) to give the crude product, which was purified by
automated flash chromatography on silica (Biotage Isolera, 2–20%
MeOH in CH2Cl2) to give 14 as a yellow powder with trace amounts
of inseparable S2 (205 mg, 48%); Rf 0.01 (100% EtOAc); mp 134–
5-(2-((1s,3s)-Adamantan-1-yl)ethoxy)-3-(4-
(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine
(18).
o
1
139 C; H NMR (400 MHz, CDCl3) δ: 9.04 (s, 1H), 7.74 (d, J = 8.8
Hz, 2H), 7.32 (s, 1H), 7.24 (d, J = 8.8 Hz, 2H), 6.62 (t, J = 73.1 Hz,
1H), 4.30 (t, J = 5.2 Hz, 2H), 3.33–3.18 (m, 4H), 2.59 (t, J = 5.2 Hz,
2H), 2.25–2.05 (m, 4H), 1.45 (s, 9H); 13C NMR (101 MHz, CDCl3) δ:
154.7, 152.4, 147.9, 146.4, 144.0, 136.7, 132.6, 125.1, 118.9, 115.6 (t,
J = 262.0 Hz), 108.6, 80.0, 69.0, 56.1, 53.1, 28.5; 19F NMR (376
MHz, CDCl3) δ: -81.50; m/z (ESI+) 513 ([M+Na]+, 100%); HRMS
(ESI+) found 491.2213 ([M+H]+), C23H28F2N6O4H+ requires
491.2213.
From 1-adamantaneethanol (250 mg, 1.39 mmol) and 3a (411 mg,
1.39 mmol) to give the crude product, which was purified by
automated flash chromatography on silica (Biotage Isolera, 25–100%
EtOAc in hexanes) to give 18 as a light brown powder (363 mg,
o
1
59%); Rf 0.63 (100% EtOAc); mp 182–185 C; H NMR (400 MHz,
CDCl3) δ: 9.00 (s, 1H), 7.73–7.64 (m, 2H), 7.29 (s, 1H), 7.25–7.20
(m, 2H), 6.61 (t, J = 73.2 Hz, 1H), 4.24 (t, J = 6.7 Hz, 2H), 1.87 (s,
3H), 1.66 (d, J = 12.3 Hz, 3H), 1.52 (d, J = 11.4 Hz, 3H), 1.39 (t, J =
6.7 Hz, 2H), 1.29 (d, J = 2.4 Hz, 6H); 13C NMR (101 MHz, CDCl3) δ:
152.5, 147.9, 146.4, 144.2, 136.2, 132.7, 125.1, 118.7, 115.6 (t, J =
261.6 Hz), 108.3, 67.5, 42.3, 41.9, 36.8, 31.7, 28.5; m/z (ESI+) 463
([M+Na]+, 100%); HRMS (ESI+) found 463.1916 ([M+Na]+),
C24H26F2N4O2Na+ requires 463.1916.
3-(4-(Difluoromethoxy)phenyl)-5-(2-((1R,5S)-6,6-
dimethylbicyclo[3.1.1]hept-2-en-2-yl)ethoxy)-[1,2,4]triazolo[4,3-
a]pyrazine (15). From (1R)-(–)-nopol (57.6 µL, 0.34 mmol) and 3a
(100 mg, 0.34 mmol); the solvent was removed and the residue
directly purified by automated flash chromatography on silica
(Biotage Isolera, 25–100% EtOAc in hexanes) to give 15 as a light
5-(2-(Cuban-1-yl)ethoxy)-3-(4-(difluoromethoxy)phenyl)-
[1,2,4]triazolo[4,3-a]pyrazine (19). From 2-cubylethan-1-ol[57] (19.0
mg, 0.12 mmol) and 3a (37.0 mg, 0.12 mmol) to give the crude
product, which was purified by automated flash chromatography on
silica (Biotage Isolera, 18–100% EtOAc in hexanes) to give 19 as a
pale yellow powder (28.0 mg, 55%); Rf 0.35 (75% EtOAc in
o
red powder (103 mg, 71%); Rf 0.49 (100% EtOAc); mp 134–137 C;
1H NMR (400 MHz, CDCl3) δ: 9.01 (s, 1H), 7.71 (d, J = 8.8 Hz, 2H),
7.29 (s, 1H), 7.24 (d, J = 8.8 Hz, 2H), 6.61 (t, J = 73.3 Hz, 1H), 4.97
(dt, J = 2.9 & 1.3 Hz, 1H), 4.29–4.11 (m, 2H), 2.39–2.22 (m, 3H),
2.13 (br s, 1H), 2.07 (br s, 1H), 2.05–1.98 (m, 1H), 1.83 (td, J = 5.7 &
1.5 Hz, 1H), 1.20 (s, 3H), 0.98 (dapp, J = 8.6 Hz, 1H), 0.63 (s, 3H);
13C NMR (101 MHz, CDCl3) δ: 152.5, 148.0, 146.4, 144.1, 142.4,
136.4, 132.6, 125.1, 120.0, 118.7, 115.7 (t, J = 261.3 Hz), 108.4,
69.3, 45.5, 40.7, 38.1, 35.5, 31.7, 31.4, 26.3, 21.1; m/z (ESI+) 449
([M+Na]+, 100%), 875 ([2M+Na]+, 33%); HRMS (ESI+) found
449.1760 ([M+Na]+), C23H24F2N4O2Na+ requires 449.1760.
o
1
hexanes); mp 146–149 C; H NMR (300 MHz, CDCl3) δ: 9.05 (s,
1H), 7.73 (d, J = 8.7 Hz, 2H), 7.33 (s, 1H), 7.24 (d, J = 8.6 Hz, 2H),
6.61 (t, J = 73.2 Hz, 1H), 4.26 (t, J = 6.5 Hz, 2H), 3.98 (ddq, J = 7.2,
4.6 & 2.2 Hz, 1H), 3.78 (q, J = 5.0 Hz, 3H), 3.54 (ddd, J = 5.7, 4.4 &
2.3 Hz, 3H), 1.95 (t, J = 6.5 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ:
152.5, 147.9, 146.4, 144.2, 136.3, 132.7, 124.9, 119.1, 118.6, 115.7,
112.2, 108.4, 68.2, 55.7, 48.6, 48.3, 44.3, 31.8; m/z (ESI+) 431
([M+Na]+, 100%), 839 ([2M+Na]+, 23%); HRMS (ESI+) found
431.1291 ([M+Na]+), C22H18F2N4O2Na+ requires 431.1290.
5-(((1S,2S,4S)-Bicyclo[2.2.1]hept-5-en-2-yl)methoxy)-3-(4-
(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine
(16).
From 5-norbornene-2-methanol (40.8 µL, 0.34 mmol) and 3a (100
mg, 0.34 mmol); the solvent was removed and the residue directly
purified by automated flash chromatography on silica (Biotage
Isolera, 25–100% EtOAc in hexanes) to give 16 (present as a mixture
of endo and exo isomers) as a yellow powder (80.0 mg, 62%); Rf 0.63
(100% EtOAc); mp 122–128 oC; 1H NMR (400 MHz, CDCl3, present
as a mixture of endo and exo isomers, ∼1:0.78 maj/min) δ: 9.01 (s,
1H(min) ), 9.00 (s, 1H(maj)), 7.72 (d, J = 8.7 Hz, 4H(comb)), 7.37–7.14 (m,
6H(comb)), 6.61 (t, J = 73.2 Hz, 1H(min)), 6.59 (t, J = 73.2 Hz, 1H(maj)),
6.15 (dd, J = 5.7 & 3.0 Hz, 1H(maj)), 6.06 (dd, J = 5.7 & 3.0 Hz,
5-(3-(Cuban-1-yl)propoxy)-3-(4-(difluoromethoxy)phenyl)-
[1,2,4]triazolo[4,3-a]pyrazine (20). From S3 (20.0 mg, 0.12 mmol)
and 3a (37.0 mg, 0.12 mmol) to give the crude product, which was
purified by automated flash chromatography on silica (Biotage
Isolera, 18–100% EtOAc in hexanes) to give 20 as a light brown
powder (24.0 mg, 47%); Rf 0.33 (75% EtOAc in hexanes); mp 128–
132 C; H NMR (300 MHz, CDCl3) δ: 9.06 (s, 1H), 7.72 (d, J = 8.7
Hz, 2H), 7.32 (s, 1H), 7.23 (d, J = 8.5 Hz, 2H), 6.59 (t, J = 73.2 Hz,
1H), 4.22 (t, J = 6.2 Hz, 2H), 4.04 (qt, J = 4.5 & 2.2 Hz, 1H), 3.86 (q,
J = 5.1 Hz, 3H), 3.63 (dt, J = 5.4 & 2.3 Hz, 3H), 1.75 (s, 4H); 13C
o
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 18
NMR (75 MHz, CDCl3) δ: 152.6, 147.9, 146.3, 144.3, 136.3, 132.5,
solvent was removed and the residue directly purified by automated
125.1, 119.2, 118.6, 115.7, 112.2, 108.4, 71.4, 58.2, 48.7, 48.2, 44.1,
29.1, 23.6; m/z (ESI+) 445 ([M+Na]+, 100%), 867 ([2M+Na]+, 13%);
HRMS (ESI+) found 445.1448 ([M+Na]+), C23H20F2N4O2Na+ requires
445.1446.
flash chromatography on silica (Biotage Isolera, 25–100% EtOAc in
hexanes) to give 25 as a light brown powder (16.2 mg, 34%); Rf 0.43
(100% EtOAc); mp 137–143 oC; 1H NMR (500 MHz, CDCl3) δ: 9.05
(s, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.25 (d, J = 8.8 Hz, 1H), 7.23 (s,
1H), 6.63 (t, J = 73.1 Hz, 1H), 3.98 (t, J = 6.6 Hz, 2H), 1.91 (t, J =
6.6 Hz, 2H), 2.86–1.05 (m, 11H); 13C NMR (126 MHz, CDCl3) δ:
152.6, 147.9, 146.4, 143.3, 137.2, 132.5, 124.9, 119.0, 115.7 (t, J =
261.7 Hz), 108.6, 70.8, 68.6, 59.2, 36.4; m/z (ESI+) 449 ([M+H]+,
1
2
3
4
5
6
7
8
3-(4-(Difluoromethoxy)phenyl)-5-(2-(octahydro-1H-2,4,1-
(epiethane[1,1,2]triyl)cyclobuta[cd]pentalen-7-yl)ethoxy)-
[1,2,4]triazolo[4,3-a]pyrazine (21). From S7 (20.0 mg, 0.11 mmol)
and 3a (31.2 mg, 0.11 mmol); the solvent was removed and the
residue directly purified by automated flash chromatography on silica
(Biotage Isolera, 25–100% EtOAc in hexanes) to give 21 as a light
brown powder (14.5 mg, 31%); Rf 0.35 (100% EtOAc); mp 136–142
100%);
C16H22B10F2N4O2H+ requires 449.2801.
tert-Butyl 3-(5-phenethoxy-[1,2,4]triazolo[4,3-a]pyrazin-3-
HRMS
(ESI+)
found
449.2806
([M+H]+),
9
1
oC; H NMR (400 MHz, CDCl3) δ: 9.02 (s, 1H), 7.70 (d, J = 8.8 Hz,
yl)pyrrolidine-1-carboxylate (26). From 2-phenylethanol (25.9 µL,
0.22 mmol) and 3b (70.0 mg, 0.22 mmol); the solvent was removed
and the residue directly purified by automated flash chromatography
on silica (Biotage Isolera, 25–100% EtOAc in hexanes) to give 26 as
a pale yellow powder (38.7 mg, 44%); Rf 0.14 (100% EtOAc); mp
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2H), 7.29 (s, 1H), 7.23 (d, J = 8.7 Hz, 2H), 6.61 (t, J = 73.2 Hz, 1H),
4.18 (tq, J = 5.8 & 2.5 Hz, 2H), 2.60 (q, J = 6.5 Hz, 1H), 2.48 (dq, J
= 27.4 & 6.8 Hz, 2H), 2.30 (dt, J = 7.8 & 4.0 Hz, 1H), 2.25–2.19 (m,
1H), 2.19–2.13 (m, 1H), 2.01–1.97 (m, 1H), 1.91 (q, J = 6.5 Hz, 2H),
1.68–1.49 (m, 4H), 1.18–1.05 (m, 2H); 13C NMR (101 MHz, CDCl3)
δ: 148.0, 146.4, 145.8, 144.3, 136.3, 132.5, 125.1, 118.6, 115.7,
108.4, 71.2, 47.4, 47.0, 44.5, 42.3, 41.8, 41.5, 38.8, 38.4, 36.3, 34.2,
28.7, 28.6; m/z (ESI+) 473 ([M+Na]+, 100%); HRMS (ESI+) found
451.1941 ([M+H]+), C25H24F2N4O2H+ requires 451.1940.
o
1
123–126 C; H NMR (400 MHz, CDCl3) δ: 8.91 (s, 1H), 7.47–7.04
(m, 6H), 4.60 (dapp, J = 17.1 Hz, 2H), 3.79 (dapp, J = 7.0 Hz, 2H), 3.69
(s, 1H), 3.40 (br s, 1H), 3.27 (t, J = 7.0 Hz, 2H), 2.67 (br s, 1H), 2.38
(br s, 1H), 2.16 (br s, 1H), 1.49 (s, 9H); 13C NMR (126 MHz, CDCl3)
δ: 154.6, 154.4, 148.1, 147.9, 147.8, 144.1, 136.8, 136.7, 136.2,
136.1, 129.2, 129.1, 128.6, 128.5, 127.6, 127.5, 108.0, 107.9, 79.7,
71.0, 51.5, 50.9, 45.7, 45.4, 37.0, 36.2, 35.12, 35.07, 30.9, 30.3, 28.7
(mixture of amide rotamers); m/z (ESI+) 432 ([M+Na]+, 100%);
HRMS (ESI+) found 432.2000 ([M+Na]+), C22H27N5O3Na+ requires
432.2006.
3-(4-(Difluoromethoxy)phenyl)-5-((3-
(difluoromethyl)bicyclo[1.1.1]pentan-1-yl)methoxy)-
[1,2,4]triazolo[4,3-a]pyrazine (22). From S8 (15.0 mg, 101 µmol)
and 3a (30.0 mg, 101 µmol); the solvent was removed and the residue
directly purified by automated flash chromatography on silica
(Biotage Isolera, 25–100% EtOAc in hexanes) to give 22 as a yellow
5-Phenethoxy-3-(tetrahydrofuran-3-yl)-[1,2,4]triazolo[4,3-
a]pyrazine (27). From 2-phenylethanol (107 µL, 0.89 mmol) and 3c
(200 mg, 0.89 mmol); the solvent was removed and the residue
directly purified by automated flash chromatography on silica
(Biotage Isolera, 25–100% EtOAc in hexanes) to give 27 as a yellow
o
1
powder (11.1 mg, 27%); Rf 0.40 (100% EtOAc); mp 129–134 C; H
NMR (500 MHz, CDCl3) δ: 9.05 (s, 1H), 7.69 (d, J = 8.8 Hz, 2H),
7.29 (d, J = 8.7 Hz, 2H), 7.28 (s, 1H), 6.61 (t, J = 73.0 Hz, 1H), 5.62
(t, J = 56.3 Hz, 1H), 4.20 (s, 2H), 1.63 (s, 6H); 13C NMR (126 MHz,
CDCl3) δ: 152.2, 147.8, 146.1, 144.2, 136.8, 132.4, 125.5, 119.5,
115.6, 112.6, 108.5, 70.4, 47.6 (t, J = 3.9 Hz), 39.0, 37.2; m/z (ESI+)
431 ([M+Na]+, 100%); HRMS (ESI+) found 409.1281 ([M+H]+),
C19H16F4N4O2H+ requires 409.1282.
o
1
powder (205 mg, 74%); Rf 0.11 (100% EtOAc); mp 127–133 C; H
NMR (400 MHz, CDCl3) δ: 8.91 (s, 1H), 7.41–7.33 (m, 2H), 7.30 (td,
J = 8.3, 7.8 & 4.0 Hz, 3H), 7.25 (s, 1H), 4.89–4.48 (m, 2H), 4.37–
3.73 (m, 5H), 3.27 (t, J = 6.6 Hz, 2H), 2.86–2.47 (m, 1H), 2.31–2.10
(m, 1H); 13C NMR (101 MHz, CDCl3) δ: 148.5, 147.9, 144.1, 136.8,
136.2, 129.2, 128.7, 127.6, 107.8, 72.7, 71.1, 68.5, 37.6, 35.1, 31.8;
m/z (ESI+) 333 ([M+Na]+, 100%); HRMS (ESI+) found 311.1499
([M+H]+), C17H18N4O2H+ requires 311.1503.
5-((1,2-Dicarba-closo-decaborane-1-yl)ethoxy)-3-(4-
(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine
(23).
From S9 (50.0 mg, 0.27 mmol) and 3a (78.8 mg, 0.27 mmol) to give
the crude product, which was purified by automated flash
chromatography on silica (Biotage Isolera, 25–100% EtOAc in
hexanes) to give 23 as a light brown powder (43.6 mg, 37%); Rf 0.17
(100% EtOAc); mp 179–184 oC; 1H NMR (400 MHz, CDCl3) δ: 9.10
(s, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.33 (s, 1H), 7.29 (d, J = 8.7 Hz,
2H), 6.66 (t, J = 72.7 Hz, 1H), 4.32 (t, J = 5.6 Hz, 2H), 2.55 (t, J =
5.6 Hz, 2H), 3.11–1.14 (m, 11H); 13C NMR (101 MHz, CDCl3) δ:
152.6, 147.9, 145.8, 142.9, 137.8, 132.5, 124.8, 119.3, 115.4 (t, J =
263.3 Hz), 108.7, 71.0, 68.5, 60.1, 36.4; m/z (ESI+) 449 ([M+H]+,
20%), 471 ([M+Na]+, 100%); HRMS (ESI+) found 471.2610
([M+Na]+), C16H22B10F2N4O2Na+ requires 471.2614.
tert-Butyl
4-(5-phenethoxy-[1,2,4]triazolo[4,3-a]pyrazin-3-
yl)piperidine-1-carboxylate (28). From 2-phenylethanol (70.9 µL,
0.59 mmol) and 3d (200 mg, 0.59 mmol); the solvent was removed
and the residue directly purified by automated flash chromatography
on silica (Biotage Isolera, 25–100% EtOAc in hexanes) to give 28 as
a pale yellow powder (174 mg, 69%); Rf 0.26 (100% EtOAc); mp
o
1
145–149 C; H NMR (400 MHz, CDCl3) δ: 8.89 (s, 1H), 7.56–7.10
(m, 6H), 4.80–4.41 (m, 2H), 4.26–4.04 (m, 1H), 3.56–3.40 (m, 1H),
3.37–3.15 (m, 2H), 2.87 (t, J = 12.2 Hz, 2H), 2.13 (dapp, J = 10.7 Hz,
2H), 1.86 (dq, J = 9.3 & 2.8 Hz, 1H), 1.59–1.39 (m, 2H), 1.46 (s,
9H); 13C NMR (101 MHz, CDCl3) δ: 154.7, 149.0, 147.5, 144.3,
136.5, 129.0, 128.9, 127.3, 107.8, 79.9, 71.4, 48.7, 44.7, 35.5, 34.7,
28.6, 25.2; m/z (ESI+) 446 ([M+Na]+, 100%); HRMS (ESI+) found
424.2339 ([M+H]+), C23H29N5O3H+ requires 424.2343.
5-((1,7-Dicarba-closo-decaborane-1-yl)ethoxy)-3-(4-
(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine
(24).
From S10 (20.0 mg, 106 µmol) and 3a (31.5 mg, 106 µmol) the
solvent was removed and the residue directly purified by automated
flash chromatography on silica (Biotage Isolera, 25–100% EtOAc in
hexanes) to give a yellow powder (11.1 mg, 23%); repurified by
automated reversed-phase flash chromatography on silica (Biotage
Isolera, 5–100% MeOH in H2O) to give 24 as a light yellow powder
5-Phenethoxy-3-(tetrahydro-2H-pyran-4-yl)-[1,2,4]triazolo[4,3-
a]pyrazine (29). From 2-phenylethanol (50.2 µL, 0.42 mmol) and 3e
(100 mg, 0.42 mmol); the solvent was removed and the residue
directly purified by automated flash chromatography on silica
(Biotage Isolera, 25–100% EtOAc in hexanes) to give 29 as a pale
yellow powder (95.2 mg, 70%); Rf 0.10 (100% EtOAc); mp 146–149
o
1
(8.3 mg, 17%); Rf 0.36 (100% EtOAc); mp 158–162 C; H NMR
(400 MHz, CDCl3) δ: 9.07 (s, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.31–
7.24 (m, 3H), 6.63 (t, J = 73.1 Hz, 1H), 4.16 (t, J = 6.5 Hz, 2H), 2.25
(t, J = 6.5 Hz, 2H), 3.28–0.98 (m, 11H); 13C NMR (101 MHz, CDCl3)
δ: 152.6, 147.9, 146.3, 143.3, 137.4, 132.5, 125.0, 119.2, 115.6 (t, J =
262.0 Hz), 108.7, 71.2, 68.9, 55.5, 34.8; 19F NMR (376 MHz, CDCl3)
δ: -81.47; m/z (ESI+) 471 ([M+Na]+, 100%); HRMS (ESI+) found
449.2788 ([M+H]+), C16H22B10F2N4O2H+ requires 499.2787.
1
oC; H NMR (400 MHz, CDCl3) δ: 8.92 (s, 1H), 7.42–7.35 (m, 2H),
7.33–7.26 (m, 4H), 4.62 (t, J = 6.7 Hz, 2H), 4.37–3.87 (m, 2H), 3.53
(tt, J = 11.4 & 3.8 Hz, 1H), 3.37 (td, J = 11.7 & 2.1 Hz, 2H), 3.29 (t,
J = 6.6 Hz, 2H), 2.24–2.06 (m, 2H), 1.95–1.78 (m, 2H); 13C NMR
(101 MHz, CDCl3) δ: 150.9, 147.6, 144.2, 136.9, 136.0, 129.2, 128.6,
127.6, 107.8, 70.6, 67.5, 34.8, 34.2, 32.0; m/z (ESI+) 347 ([M+Na]+,
100%); HRMS (ESI+) found 325.1656 ([M+H]+), C18H20N4O2H+
requires 325.1659.
5-((1,12-Dicarba-closo-decaborane-1-yl)ethoxy)-3-(4-
(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine
(25).
From S12 (20.0 mg, 106 µmol) and 3a (31.5 mg, 106 µmol); the
10
ACS Paragon Plus Environment

Page 11 of 18
Journal of Medicinal Chemistry
5-(3,4-Difluorophenethoxy)-3-(4-iodophenyl)-
[1,2,4]triazolo[4,3-a]pyrazine (30). From
(400 MHz, CDCl3) δ: 8.90 (s, 1H), 7.48–6.67 (m, 6H), 4.77–4.40 (m,
2-(3,4-
2H), 4.04 (dt, J = 8.1 & 4.4 Hz, 1H), 3.68–3.51 (m, 2H), 3.39–3.23
(m, 3H), 3.09 (ddd, J = 12.5, 9.0 & 4.1 Hz, 1H), 2.33–1.90 (m, 1H),
1.90–1.64 (m, 3H) (amine NH signal not seen); 13C NMR (101 MHz,
CDCl3) δ: 147.5, 147.4, 143.9, 136.4, 136.0, 129.1, 128.7, 127.4,
108.5, 71.2, 46.9, 44.1, 34.5, 32.0, 29.2, 21.1; m/z (ESI+) 324
([M+H]+, 100%); HRMS (ESI+) found 324.1813 ([M+H]+),
C18H21N5OH+ requires 324.1819.
1
2
3
4
5
6
7
8
difluorophenyl)ethanol (44.4 mg, 0.28 mmol) and 3f (100 mg, 0.28
mmol) to give the crude product, which was purified by automated
flash chromatography on silica (Biotage Isolera, 25–100% EtOAc in
hexanes) to give 30 as a pale yellow powder (81.1 mg, 61%); Rf 0.50
(100% EtOAc); mp 155–157 oC; 1H NMR (300 MHz, CDCl3) δ: 9.02
(s, 1H), 7.83 (d, J = 7.7 Hz, 2H), 7.40 (d, J = 7.6 Hz, 2H), 7.31 (s,
1H), 7.00 (q, J = 8.7 Hz, 1H), 6.54 (t, J = 9.2 Hz, 2H), 4.42 (t, J = 5.9
Hz, 3H), 2.92 (t, J = 5.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ:
150.3 (dd, J = 249.0 & 12.7 Hz), 149.5 (dd, J = 248.0 & 12.5 Hz),
147.9, 146.3, 143.8, 137.1, 136.8, 133.3 (dd, J = 5.6 & 4.0 Hz),
132.3, 127.4, 124.5 (dd, J = 6.1 & 3.6 Hz), 117.5 (d, J = 17.4 Hz),
117.4 (d, J = 17.3 Hz), 108.5, 96.7, 70.9, 33.9; m/z (ESI+) 501
([M+Na]+, 100%); HRMS (ESI+) found 500.9986 ([M+Na]+),
C19H13F2IN4ONa+ requires 500.9994.
5-(((1R,2R,4S,5S,6S)-3-Oxatricyclo[3.2.1.02,4]octan-6-yl)methoxy)-
3-(4-(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine
(35).
1
From the late-stage biofunctionalization of 16; H NMR (600 MHz,
DMSO-d6) δ: 9.05 (s, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.57 (s, 1H), 7.35
(t, J = 73.1 Hz, 1H), 7.34 (d, J = 8.7 Hz, 2H), 4.31–4.13 (m, 2H), 3.13
(d, J = 3.5 Hz, 1H), 3.09 (d, J = 2.7 Hz, 1H), 2.35 (br d, J = 2.5 Hz,
1H), 2.16–2.09 (m, 1H), 1.89–1.78 (m, 1H), 1.46 (ddd, J = 12.6, 10.2,
4.3 Hz, 1H), 1.09–1.02 (m, 1H), 0.73 (ddd, J = 12.6, 4.8, 2.5 Hz, 1H),
0.62 (d, J = 9.5 Hz, 1H); HRMS (ESI+) found 401.1416 ([M+H]+),
C20H18F2N4O3H+ requires 401.1420.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-(3,4-Difluorophenethoxy)-3-(4-iodocuban-1-yl)-
[1,2,4]triazolo[4,3-a]pyrazine
(31).
From
2-(3,4-
dilfuorophenyl)ethanol (20.7 mg, 0.13 mmol) and 3g (50.0 mg, 0.13
mmol) to give the crude product, which was purified by automated
flash chromatography on silica (Biotage Isolera, 25–100% EtOAc in
hexanes) to give 31 as a brown powder (19.9 mg, 30%); Rf 0.23
(100% EtOAc); mp 147–153 oC; 1H NMR (300 MHz, CDCl3) δ: 8.86
(s, 1H), 7.47–6.73 (m, 4H), 4.64–4.35 (m, 5H), 4.26 (s, 3H), 3.16 (t, J
= 7.5 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ: 148.3, 146.3, 143.3,
136.7, 132.2, 125.0, 118.3, 118.0, 117.8, 108.1, 74.1, 71.4, 54.9, 52.2,
35.4, 34.2, 29.8; m/z (ESI+) 527 ([M+Na]+, 100%); HRMS (ESI+)
found 527.0150 ([M+Na]+), C21H15F2IN4ONa+ requires 527.0151.
5-(((1R,2S,4R,5S,6S)-3-Oxatricyclo[3.2.1.02,4]octan-6-yl)methoxy)-
3-(4-(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine
(36).
1
From the late-stage biofunctionalization of 16; H NMR (600 MHz,
DMSO-d6) δ: 9.04 (s, 1H), 7.81 (d, J = 8.7 Hz, 2H), 7.52 (s, 1H), 7.37
(d, J = 8.6 Hz, 2H), 7.37 (t, J = 73.6 Hz, 1H), 4.19–3.96 (m, 2H), 3.08
(d, J = 3.8 Hz, 1H), 2.84 (d, J = 3.5 Hz, 1H), 2.34–2.26 (m, 1H),
1.91–1.78 (m, 1H), 1.72–1.60 (m, 1H), 1.25 (ddd, J = 11.1, 8.5, 2.3
Hz, 1H), 0.98 (dt, J = 12.7, 4.2 Hz, 1H), 0.94 (d, J = 10.1 Hz, 1H),
0.71 (d, J = 10.1 Hz, 1H); HRMS (ESI+) found 401.1417 ([M+H]+),
C20H18F2N4O3H+ requires 401.1420.
3-(4-(Difluoromethoxy)phenyl)-5-(2-(piperazin-1-yl)ethoxy)-
[1,2,4]triazolo[4,3-a]pyrazine (32). Compound 14 (150 mg, 0.31
mmol, 1.00 equiv.) was dissolved in CH2Cl2 (0.88 mL). TFA (0.26
mL, 3.42 mmol, 11.2 equiv.) was added and the reaction stirred at rt
overnight. The solvent was removed and the residue directly purified
by automated flash chromatography on silica (Biotage Isolera, 2–15%
MeOH in CH2Cl2) to give 32 as a light yellow powder (106 mg,
89%); mp 140–148 oC; 1H NMR (400 MHz, CD3OD) δ: 8.99 (s, 1H),
7.81 (d, J = 8.8 Hz, 2H), 7.53 (s, 1H), 7.35 (d, J = 8.7 Hz, 2H), 7.03
(t, J = 73.5 Hz, 1H), 4.42 (t, J = 5.0 Hz, 2H), 3.16–2.94 (m, 4H), 2.70
(t, J = 5.0 Hz, 2H), 2.58–2.35 (m, 4H) (amine NH signal not seen);
13C NMR (101 MHz, CD3OD) δ: 154.2, 149.0, 147.8, 146.0, 136.3,
134.0, 126.0, 119.4, 117.4 (t, J = 259.5 Hz), 110.1, 70.1, 56.6, 50.6,
44.8; m/z (ESI+) 413 ([M+Na]+, 100%); HRMS (ESI+) found
319.1687 ([M+H]+), C18H20F2N6O2H+ requires 391.1689.
3-(2-((3-(4-(Difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-
a]pyrazin-5-yl)oxy)ethyl)cuban-1-ol (37). From the late-stage
biofunctionalization of 19; 1H NMR (600 MHz, DMSO-d6) δ: 9.05
(s, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.58 (s, 1H), 7.36 (t, J = 73.7 Hz,
1H), 7.31 (d, J = 8.5 Hz, 2H), 6.02 (s, 1H), 4.30 (t, J = 6.2 Hz, 2H),
3.81 (dq, J = 5.5, 2.7 Hz, 1H), 3.41 (dt, J = 5.1, 2.5 Hz, 2H), 3.37 (q, J
= 5.1 Hz, 2H), 1.85 (t, J = 6.2 Hz, 2H). One cubane C–H at δ 3.33
obscured by solvent peak; HRMS (ESI+) found 425.1400 ([M+H]+),
C22H18F2N4O3H+ requires 425.1420.
4-(2-((3-(4-(Difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-
a]pyrazin-5-yl)oxy)ethyl)cuban-1-ol (38). From the late-stage
1
biofunctionalization of 19; H NMR (600 MHz, DMSO-d6) δ: 9.04 (s,
1H), 7.82 (d, J = 8.6 Hz, 2H), 7.58 (s, 1H), 7.37 (t, J = 73.7 Hz, 1H),
7.31 (d, J = 8.6 Hz, 2H), 6.15 (s, 1H), 4.32 (t, J = 6.0 Hz, 2H), 3.58 (t,
J = 5.2 Hz, 3H), 3.13 (t, J = 5.2 Hz, 3H), 1.89 (t, J = 6.0 Hz, 2H);
HRMS (ESI+) found 425.1402 ([M+H]+), C22H18F2N4O3H+ requires
425.1420.
5-Phenethoxy-3-(pyrrolidin-3-yl)-[1,2,4]triazolo[4,3-a]pyrazine
(33). Compound 26 (20.0 mg, 48.8 µmol, 1.00 equiv.) was dissolved
in CH2Cl2 (0.13 mL). TFA (42.1 µL, 0.55 mmol, 11.2 equiv.) was
added and the reaction stirred at rt overnight. The solvent was
removed and the residue directly purified by automated flash
chromatography on silica (Biotage Isolera, 2–10% MeOH in CH2Cl2)
to give a clear oil; repurified by automated reversed-phase flash
chromatography on silica (Biotage Isolera, 5–100% MeOH in H2O) to
give 33 as a white powder (9.1 mg, 60%); mp 78–85 C; H NMR
(400 MHz, CDCl3) δ: 8.88 (s, 1H), 7.40–7.33 (m, 2H), 7.33–7.26 (m,
3H), 7.23 (s, 1H), 4.59 (t, J = 6.6 Hz, 2H), 3.86 (ddt, J = 8.7, 7.5 &
3.6 Hz, 1H), 3.27 (t, J = 6.6 Hz, 2H), 3.30–3.17 (m, 2H), 3.00 (dq, J
= 41.7 & 7.5 Hz, 2H), 2.45 (br s, 1H), 2.34–2.18 (m, 1H), 2.16–1.94
(m, 1H); 13C NMR (101 MHz, CDCl3) δ: 150.7, 147.8, 144.3, 136.7,
136.3, 129.1, 128.7, 127.5, 107.7, 70.9, 53.6, 47.4, 37.9, 35.1, 32.6;
m/z (ESI+) 310 ([M+H]+, 100%); HRMS (ESI+) found 310.1659
([M+H]+), C17H19N5OH+ requires 310.1662.
5-((7,8-Dicarba-nido-undecaborane-7-yl)ethoxy)-3-(4-
(difluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine
(39).
Adapted from the procedure of Yoo et al.[58]: Compound 23 (20.0 mg,
44.6 µmol, 1 equiv.) and CsF (20.3 mg, 134 µmol, 3 equiv.) were
dissolved in EtOH (1 mL) and heated at reflux for 25 h. The solvent
was removed. Acetone (2 mL) was added, the solution filtered
through a fritted glass funnel, washed several times with acetone and
the solvent removed to give the crude product as an off-white solid;
purified by automated flash chromatography on silica (Biotage
Isolera, 2–10% MeOH in CH2Cl2) to give 39 as a yellow powder
(16.4 mg, 65%); Rf 0.13 (10% MeOH in CH2Cl2); mp 181–190 oC; 1H
NMR (500 MHz, CDCl3) δ: 8.94 (s, 1H), 7.76 (d, J = 8.9 Hz, 2H),
7.50 (s, 1H), 7.28 (d, J = 8.9 Hz, 2H), 6.91 (t, J = 73.8 Hz, 1H), 4.56
(s, 2H), 4.46–4.29 (m, 2H), 1.94–1.65 (m, 2H) (carborane BH signals
not seen); 13C NMR (126 MHz, CD3OD) δ: 154.6, 149.0, 148.0,
146.3, 135.6, 133.9, 125.6, 119.2, 117.7 (t, J = 257.9 Hz), 109.9,
74.2, 38.8 (carborane C signals not seen); m/z (ESI+) 484 ([M+2Na]+,
o
1
5-Phenethoxy-3-(piperidin-4-yl)-[1,2,4]triazolo[4,3-a]pyrazine
(34). Compound 28 (100 mg, 0.24 mmol, 1.00 equiv.) was dissolved
in CH2Cl2 (0.62 mL). TFA (0.2 mL, 2.64 mmol, 11.2 equiv.) was
added and the reaction stirred at rt overnight. The solvent was
removed and the residue directly purified by automated flash
chromatography on silica (Biotage Isolera, 210% MeOH in CH2Cl2)
to give a white solid; repurified by automated reversed-phase flash
chromatography on silica (Biotage Isolera, 5–100% MeOH in H2O) to
give 34 as a white powder (61.7 mg, 81%); mp 175–180 oC; 1H NMR
100%);
HRMS
(ESI+)
found
484.2381
([M+2Na]+),
C16H22B9CsF2N4O2Na2+ requires 484.2375.
2-(Oxetan-3-yl)ethan-1-ol (S1). Lithium 2-(oxetan-3-yl)acetate
(150 mg) was converted to the free acid by addition of conc. HCl and
extraction with EtOAc to give a colorless oil (125 mg). The free acid
(100 mg, 0.86 mmol, 1 equiv.) was dissolved in anhydrous THF (2
o
mL) and cooled to 0 C. LiAlH4 (1 M in THF, 0.55 mL, 0.55 mmol,
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 18
0.64 equiv.) was added dropwise and the reaction mixture stirred for
10 min at 0 C, then at rt. The reaction was cooled in an ice bath and
Compound S4 (120 mg, 0.42 mmol, 1.0 equiv.) was suspended in
o
EtOH (8 mL) and NaBH4 (17.4 mg, 0.42 mmol, 1.1 equiv.) was added
1
2
3
4
5
6
7
8
o
the LAH quenched with EtOAc dropwise. A sat. aq. solution of
Rochelle’s salt was added and the mixture stirred at 0 oC, then at rt for
1.5 h. The organic layer was separated and the aqueous layer
extracted with EtOAc (2 ×). The combined organic layers were dried
(MgSO4), filtered and concentrated under reduced pressure to give S1
as a yellow oil (41.4 mg, 47%); carried forward without further
purification or characterisation.
portionwise keeping the temperature below 30 C. The reaction was
stirred for 1 h then filtered. The filtrate was reduced to half the
volume and poured onto a H2O (8 mL) and AcOH (0.13 mL) mixture
and left to stand for 2 h. The solid was filtered, washed with H2O and
dried in vacuo to give the crude product as white crystals (91.8 mg,
76%); carried forward without further purification; the remaining
crude material was purified by recrystallisation from 50% aq. acetone
o
o
to give S5 as white plates; mp 138–143 C (lit.[61] 136–137 C); IR
νmax (film) /cm–1 1742; 1H NMR (400 MHz, CDCl3) δ: 3.91 (d, J = 9.9
Hz, 1H), 3.05–2.73 (m, 2H), 2.59 (qd, J = 12.2, 10.8 & 4.8 Hz, 3H),
2.47 (d, J = 8.8 Hz, 1H), 2.24 (br d, J = 17.9 Hz, 2H), 1.92 (dt, J =
9.9 & 3.1 Hz, 1H), 1.81 (d, J = 13.2 Hz, 1H), 1.73 (d, J = 8.1 Hz,
6H), 1.70 (sapp, 1H), 1.30–1.08 (m, 2H); 13C NMR (101 MHz, CDCl3)
δ: 166.0, 165.8, 105.4, 47.4, 46.4, 46.3, 44.3, 42.5, 42.1, 41.7, 41.5,
38.4, 36.4, 34.1, 29.2, 28.8, 28.3. Spectroscopic data matched those in
the literature.[61]
tert-Butyl 4-(2-hydroxyethyl)piperazine-1-carboxylate (S2)
Following the procedure of del Prado et al.[59]: A solution of Boc2O
(2.18 mL, 9.45 mmol, 1.23 equiv.) in CH2Cl2 (7 mL) was added
dropwise to a solution of 1-(2-hydroxyethyl)piperazine (1.00 g, 7.68
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
mmol, 1.00 equiv.) in CH2Cl2 (10 mL) at 0 C. The reaction was
warmed to rt and stirred for 2 h. Sat. aq. NH4Cl (6 mL) was added and
the organic layer separated. The aqueous layer was extracted with
CH2Cl2 (2 × 8 mL) and the combined organic layers were dried
(Na2SO4), filtered and concentrated under reduced pressure to give the
crude product; purified by DCVC (25–100% EtOAc in hexanes) to
give S2 as a clear oil that crystallised on standing (674 mg, 38%); Rf
0.05 (100% EtOAc, KMnO4 stain); mp 45–52 oC; IR νmax (film) /cm–1
2-(Octahydro-1H-2,4,1-
(epiethane[1,1,2]triyl)cyclobuta[cd]pentalen-7-yl)acetic acid (S6).
Following the procedure of Aleksandrov et al.[61]: Compound S5
(65.0 mg, 0.23 mmol) was dissolved in a mixture of AcOH (0.5 mL)
and conc. HCl (0.1 mL) and heated at reflux for 2.5 h. The reaction
was cooled to rt and poured onto H2O (3.75 mL), cooled in ice,
filtered and washed with H2O to give the crude product as an off-
white powder (32.5 mg, 70%); carried forward without further
purification; the remaining crude material was purified by
1
3426, 2974, 2933, 2868, 2812, 1695; H NMR (300 MHz, CDCl3) δ:
3.61 (t, J = 5.4 Hz, 2H), 3.42 (t, J = 5.1 Hz, 4H), 2.54 (t, J = 5.4 Hz,
2H), 2.44 (t, J = 5.1 Hz, 4H), 1.45 (s, 9H) (alcohol OH signal not
seen); 13C NMR (75 MHz, CDCl3) δ: 154.8, 79.8, 59.5, 57.9, 52.9,
28.6 (1 obscured signal). Spectroscopic data matched those in the
literature.[59]
1
recrystallisation from hexane to give S6 as white crystals; H NMR
3-Cubylpropan-1-ol (S3). Adapted from the procedure of Priefer
et al.[60]: 3-Cubylacetic acid (58.0 mg, 0.36 mmol) was suspended in
anhydrous THF (5 mL) under Ar. Borane dimethylsulfide complex
(2.0 M in Et2O, 0.51 mL, 1.02 mmol) was added and the solution was
left to stir for 16 h. Sat. aq. NH4Cl (1 mL) was cautiously added and
the mixture was left to stir for 20 min. The THF was removed in
vacuo, the residue was then diluted with H2O (10 mL) and extracted
with CH2Cl2 (3 × 10 mL). The combined organic layers were dried
(MgSO4), filtered and concentrated under reduced pressure. The crude
product was purified by flash chromatography on silica (50% EtOAc
in petroleum ether) to give S3 as a yellow oil (55.0 mg, 94%); IR νmax
(300 MHz, CDCl3) δ: 2.82–2.49 (m, 6H), 2.30 (br s, 1H), 2.27–2.14
(m, 4H), 1.93 (tt, J = 6.8 & 2.8 Hz, 1H), 1.69 (s, 1H), 1.68–1.59 (m,
1H), 1.17 (d, J = 10.7 Hz, 1H), 1.03 (dt, J = 12.7 & 3.5 Hz, 1H).
Spectroscopic data matched those in the literature.[61]
2-(Octahydro-1H-2,4,1-
(epiethane[1,1,2]triyl)cyclobuta[cd]pentalen-7-yl)ethan-1ol (S7).
Compound S6 (25.0 mg, 0.12 mmol, 1 equiv.) was dissolved in
anhydrous THF (1 mL) and cooled to 0 oC. LiAlH4 (1 M in THF, 78.3
µL, 78.3 µmol, 0.64 equiv.) was added dropwise and the reaction
mixture stirred for 10 min at 0 oC, then at rt. The reaction was cooled
in an ice bath and the LAH quenched with EtOAc dropwise. A sat. aq.
1
(film) /cm–1 3307, 2968, 1444, 1214, 1054, 881, 841; H NMR (400
o
MHz, CDCl3) δ: 4.07–4.01 (m, 1H), 3.87–3.84 (m, 3H), 3.74–3.71
(m, 3H), 3.65 (t, J = 6.6 Hz, 2H), 1.65–1.54 (m, 4H), 1.36 (br s, 1H);
13C NMR (101 MHz, CDCl3) δ: 63.5, 59.0, 48.7, 48.5, 44.2, 29.4,
27.7; HRMS (ESI+) found 163.1115 ([M+H]+), C11H14OH+ requires
163.1117.
solution of Rochelle’s salt was added and the mixture stirred at 0 C,
then at rt for 1.5 h. The organic layer was separated and the aqueous
layer extracted with EtOAc (2 ×). The combined organic layers were
dried (MgSO4), filtered and concentrated under reduced pressure to
give the crude product as a clear oil (25.6 mg, >100%); carried
forward without further purification or characterisation.
2,2-Dimethyl-5-(octahydro-1H-2,4,1-
(epiethane[1,1,2]triyl)cyclobuta[cd]pentalen-7ylidene)-1,3-
dioxane-4,6-dione (S4). Following the procedure of Aleksandrov et
(3-(Difluoromethyl)bicyclo[1.1.1]pentan-1-yl)methanol (S8). 3-
(Difluoromethyl)bicyclo[1.1.1]pentane-1-carboxylic acid (25.8 mg,
159 µmol, 1.0 equiv.) was dissolved in THF (72.3 µL, 2.2 M) and
al.[61]
:
Octahydro-1H-2,4,1-
o
(epiethane[1,1,2]triyl)cyclobuta[cd]pentalen-7-one (150 mg, 0.94
mmol, 1.0 equiv.) was dissolved in pyridine (1 mL) and
isopropylidene malonate (162 mg, 1.12 mmol, 1.2 equiv.) was added.
The mixture was agitated several times within the first hour to
dissolve the acid, then left to stand for 5 days. The reaction mixture
was poured over H2O (20 mL) and the precipitate filtered to give the
crude product as a white powder (153 mg, 57%); carried forward
without further purification; the remaining crude material was purified
by recrystallisation from 50% aq. acetone to give S4 as white plates;
cooled to 0 C. LiAlH4 (1 M in THF, 239 µL, 239 µmol, 1.5 equiv.)
was added and the reaction allowed to warm to rt. The reaction was
quenched with H2O (18 µL) and anhydrous Na2SO4 (103 mg) and
stirred for 20 min. The mixture was filtered, washed with THF and the
filtrate concentrated under reduced pressure to give S8 as a pale
yellow oil (19.6 mg, 83%); carried forward without further
purification or characterisation; Rf 0.34 (25% EtOAc in hexanes,
1
KMnO4 stain); H NMR (200 MHz, CDCl3) δ: 5.70 (t, J = 56.5 Hz,
1H), 3.66 (s, 2H), 1.81 (s, 6H) (alcohol OH signal not seen).
o
o
mp 160–164 C (lit.[61] 157–158 C); IR νmax (film) /cm–1 1726, 1606;
1H NMR (400 MHz, CDCl3) δ: 4.24 (ddd, J = 8.4, 5.9 & 2.4 Hz, 1H),
4.16–3.98 (m, 1H), 3.18 (td, J = 8.6 & 3.5 Hz, 1H), 2.94 (d, J = 10.1
Hz, 1H), 2.79 (q, J = 6.4 & 6.0 Hz, 1H), 2.66 (q, J = 7.0 & 6.4 Hz,
1H), 2.58–2.45 (m, 2H), 1.92 (d, J = 10.9 Hz, 1H), 1.71 (d, J = 5.3
Hz, 6H), 1.52 (d, J = 10.9 Hz, 1H), 1.40–1.15 (m, 2H); 13C NMR
(101 MHz, CDCl3) δ: 194.6, 161.2, 161.0, 111.5, 103.9, 50.8, 50.6,
47.8, 47.5, 44.2, 43.0, 42.8, 39.8, 36.9, 30.9, 27.7, 27.1. Spectroscopic
data matched those in the literature.[61]
1,2-Dicarba-closo-dodecaborane-1-ethanol (S9). Following the
procedure of Li et al.[62]: To a solution of closo-1,2-carborane (100
mg, 0.69 mmol, 1 equiv.) in anhydrous THF (2.25 mL) was added n-
o
BuLi (1.6 M in hexanes, 0.43 mL, 0.69 mmol, 1 equiv.) at –78 C
under N2. Stirring was continued for 30 min, then ethylene oxide (2.5
M in THF, 0.42 mL, 1.04 mmol, 1.5 equiv.) was added dropwise.
Stirring was continued for 1 h at 0 oC, then the reaction was quenched
with sat. aq. NH4Cl (1 mL). The aqueous phase was extracted with
EtOAc (3 × 1.5 mL) and the combined organic layers dried (Na2SO4),
filtered and concentrated under reduced pressure to give the crude
product, which was purified by automated flash chromatography on
silica (Biotage Isolera, 5–40% EtOAc in hexanes) to give S9 as a
2,2-Dimethyl-5-(octahydro-1H-2,4,1-
(epiethane[1,1,2]triyl)cyclobuta[cd]pentalen-7yl)-1,3-dioxane-4,6-
dione (S5). Following the procedure of Aleksandrov et al.[61]
:
12
ACS Paragon Plus Environment

Page 13 of 18
Journal of Medicinal Chemistry
white solid (110 mg, 66%); 1H NMR (400 MHz, CDCl3) δ: 3.98 (br s,
1-Iodo-4-(hydroxymethyl)cubane (S14). Following the procedure
of Griffiths et al.[69]: 4-Iodocubanecarboxylic acid[65] (200 mg, 0.73
mmol) was dissolved in anhydrous THF (7 mL) under Ar and cooled
1H), 3.79 (t, J = 5.9 Hz, 2H), 2.49 (t, J = 5.9 Hz, 2H), 2.80–1.42 (m,
11H); 13C NMR (101 MHz, CDCl3) δ: 73.2, 60.8, 60.7, 39.9; m/z
(ESI–) 189 ([M-H]–, 100%); HRMS (ESI–) found 189.2058 ([M-H]–),
C4H15B10O– requires 189.2059. Spectroscopic data matched those in
the literature.[63]
1
2
3
4
5
6
7
8
o
to 0 C. Borane dimethylsulfide complex (0.23 mL, 1.16 mmol) was
o
added and the reaction stirred at 0 C for 20 min, then at rt for 4 h.
The solution was quenched with H2O and stirred overnight. After
adding EtOAc, the solution was washed with H2O, brine, dried
(MgSO4), filtered and concentrated under reduced pressure to give
S14 as a white solid (162 mg, 85%); carried forward without further
1,7-Dicarba-closo-dodecaborane-1-ethanol (S10). Adapted from
the procedure of Li et al.[62]: To a solution of closo-1,7-carborane (100
mg, 0.69 mmol, 1 equiv.) in anhydrous THF (2.25 mL) was added n-
1
purification; Rf 0.80 (100% EtOAc); H NMR (300 MHz, CDCl3) δ:
o
BuLi (1.6 M in hexanes, 0.43 mL, 0.69 mmol, 1 equiv.) at –78 C
4.21 (dd, J = 5.8 & 4.4 Hz, 3H), 4.04 (dd, J = 5.8 & 4.2 Hz, 3H), 3.77
(s, 2H) (alcohol OH signal not seen); 13C NMR (75 MHz, CDCl3) δ:
63.2, 59.1, 54.8, 48.0, 39.0. Spectroscopic data matched those in the
literature.[69]
under N2. Stirring was continued for 30 min, then ethylene oxide (2.5
M in THF, 0.42 mL, 1.04 mmol, 1.5 equiv.) was added dropwise.
Stirring was continued for 1 h at 0 oC, then the reaction was quenched
with sat. aq. NH4Cl (1 mL). The aqueous phase was extracted with
EtOAc (3 × 1.5 mL) and the combined organic layers dried (Na2SO4),
filtered and concentrated under reduced pressure to give the crude
product, which was purified by automated flash chromatography on
silica (Biotage Isolera, 6–50% EtOAc in hexanes) to give S10 as a
white solid (30.7 mg, 23%); Rf 0.47 (25% EtOAc in hexanes, KMnO4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1-Iodocubane-4-carboxaldehyde (S15). Following the procedure
of Griffiths et al.[69]: A stirring solution of oxalyl chloride (63.8 µL,
0.75 mmol) in anhydrous CH2Cl2 (1 mL) was prepared under Ar at –
78 oC. Anhydrous DMSO (0.11 mL, 1.54 mmol) in anhydrous CH2Cl2
(1 mL) was added dropwise. After 20 min at –78 oC, a solution of S14
(162 mg, 0.62 mmol) in anhydrous CH2Cl2 (4.25 mL) was added
1
stain); H NMR (200 MHz, CDCl3) δ: 3.88–3.32 (m, 2H), 2.94 (s,
o
1H), 2.51–2.00 (m, 2H), 3.29–0.71 (m, 11H); m/z (APCI+) 191
([M+H]+, 100%); HRMS (ESI–) found 189.2058 ([M-H]–),
C4H15B10O– requires 189.2059. Spectroscopic data matched those in
the literature.[64]
dropwise under Ar. The mixture was maintained at –78 C for 1.5 h,
then anhydrous Et3N (0.39 mL, 2.80 mmol) was added. The mixture
was warmed to rt and quenched with H2O (4 mL). The aqueous layer
was extracted with CH2Cl2 (2 × 4 mL) and the combined organic
layers washed with H2O (4 mL), brine (4 mL), dried (MgSO4), filtered
and concentrated under reduced pressure to give the crude product,
which was purified by automated flash chromatography on silica
(Biotage Isolera, 12–100% EtOAc in hexanes) to give S15 as a white
solid (130 mg, 81%); Rf 0.84 (50% EtOAc in hexanes); mp 124–130
1-(2-Benzyloxyethyl)-1,12-dicarba-closo-dodecaborane (S11).
Following the procedure of Fujii et al.[65]: To a solution of closo-1,12-
carborane (120 mg, 0.83 mmol, 1.0 equiv.) in anhydrous Et2O (2.88
mL) was added n-BuLi (1.6 M in hexanes, 572 µL, 0.92 mmol, 1.1
o
equiv.) at 0 C under Ar. The reaction was stirred at rt for 1 h, then
o
1
oC (lit.[69] 106–109 C); H NMR (300 MHz, CDCl3) δ: 9.74 (s, 1H),
4.56–4.47 (m, 3H), 4.33–4.25 (m, 3H); 13C NMR (75 MHz, CDCl3) δ:
197.1, 62.8, 54.8, 49.0, 35.6. Spectroscopic data matched those in the
literature.[69]
benzyl 2-bromoethyl ether (163 µL, 1.00 mmol, 1.2 equiv.) was added
o
and the reaction stirred for 16 h. The reaction was cooled to 0 C,
quenched with H2O and diluted with EtOAc. The organic layer was
washed with H2O, brine, dried (Na2SO4), filtered and concentrated
under reduced pressure to give the crude product, which was purified
by automated flash chromatography on silica (Biotage Isolera, 6–50%
EtOAc in hexanes) to give S11 as a clear colorless oil (108 mg, 47%);
In Vitro Antiplasmodial Activity (Drug Discovery Unit,
University of Dundee).[70] Cultures of the widely-used malaria
reference strain of chloroquine-sensitive P. falciparum strain 3D7
were maintained in a 1.25% or 5% suspension of human red blood
cells cultured in RPMI 1640 medium supplemented with 0.5%
Albumax II (available from Gibco Life Technologies, San Diego, CA,
cat. no. 11021-037), 12 mM sodium bicarbonate, 0.2 mM
hypoxanthine, (pH 7.3), and 20 mg/L gentamicin at 37 oC, in a
humified atmosphere of 1% O2, 3% CO2 with a gas balance of
nitrogen. Growth inhibition of the P. falciparum cultures was
quantified in a 10-point dose response curve with a 1 in 3 dilution
series. This 384 well plate based fluorescence assay utilises the
binding of SYBRgreen I (Thermo Fisher Scientific/Invitrogen cat. no.
S7585) to double stranded DNA, which greatly increases the
fluorescent signal at 528 nm after excitation at 485 nm. Mefloquine
1
Rf 0.83 (10% EtOAc in hexanes, KMnO4 stain); H NMR (200 MHz,
CDCl3) δ: 7.63–6.97 (m, 5H), 4.40 (s, 2H), 3.26 (t, J = 7.2 Hz, 2H),
2.64 (br s, 1H), 1.95 (t, J = 7.1 Hz, 2H), 3.72–0.63 (m, 10H).
Spectroscopic data matched those in the literature.[65]
1,12-Dicarba-closo-dodecaborane-1-ethanol (S12) Adapted from
the procedure of Mandal et al.[66]: Compound S11 (90 mg, 0.32 mmol,
1.00 equiv.) and 5% Pd/C (34.4 mg, 0.02 mmol, 0.05 equiv.) were
suspended in EtOH (2 mL). The reaction purged with Ar and Et3SiH
(0.52 mL, 3.23 mmol, 10.0 equiv.) was added dropwise and the
reaction stirred at rt. The mixture was filtered through a pad of celite
and concentrated under reduced pressure to give S12 as a pale yellow
solid (40.7 mg, 67%); carried forward without further purification; Rf
푍′
1
was used as a drug control to monitor the quality of the assay ( = 0.6
0.26 (10% EtOAc in hexanes, KMnO4 stain); H NMR (400 MHz,
푍′
to 0.8, where
positive and negative controls on a screen plate). Dose-response
curves were determined from minimum of independent
is a measure of the discrimination between the
CDCl3) δ: 3.45 (t, J = 7.0 Hz, 2H), 2.66 (br s, 1H), 2.91–1.26 (m,
11H), 1.90 (t, J = 7.0 Hz, 2H). Spectroscopic data matched those in
the literature.[63]
a
3
experiments. Compound bioactivity was expressed as IC50, the
concentration of compound causing 50% inhibition. IC50 values were
determined from a minimum of 3 independent experiments. All data
was processed using IDBS ActivityBase® raw data was converted
into per cent inhibition through linear regression by setting the high
inhibition control as 100% and the no inhibition control as 0%.
4-Iodobenzaldehyde (S13). Following the procedure of Yamashita
et al.[67]: 1,3-Dimethyl-2-imidazolidinone (15 mL) was added to 4-
bromobenzaldehyde (1.00 g, 5.40 mmol, 1.0 equiv.), KI (8.07 g, 48.6
mmol 9.0 equiv.) and CuI (3.19 g, 17.8 mmol, 3.1 equiv.). The
mixture was purged with N2 and heated with vigorous stirring at 200
oC for 23 h. After cooling to rt, brine and ice were added and the
reaction was placed in an ice bath for 3 h. The precipitated inorganic
salts were removed by filtration and the filtrate extracted with Et2O.
The combined organic layers were washed with brine, dried
(Na2SO4), filtered and concentrated under reduced pressure to give the
crude product, which was purified by automated flash
chromatography on silica (Biotage Isolera, 12–75% EtOAc in
hexanes) to give S13 as a yellow powder (263 mg, 21%); Rf 0.87
푍′
Quality control criteria for passing plates were as follows: >0.5,
S:B >3, %CV(no inhibition control) <15. The formula used to
)
푍′ = 1 ― 3 × (푆푡퐷푒푣ℎ푖푔ℎ + 푆푡퐷푒푣푙표푤
calculate
) . All IC50 Curve fitting was
퐴퐵푆(푀푒푎푛ℎ푖푔ℎ ― 푀푒푎푛푙표푤
undertaken using XLFit version 4.2 using Model 205 with the
퐵 ― 퐴
푦 = 퐴 +
퐴
= %
following 4 parametric equation:
, where
퐷
퐶
1 + (
)
푥
o
o
1
(50% EtOAc in hexanes); mp 72–79 C (lit.[68] 77–78 C); H NMR
(400 MHz, CDCl3) δ: 9.96 (s, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.59 (d, J
= 8.5 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ: 191.5, 138.6, 135.7,
130.9, 103.0. Spectroscopic data matched those in the literature.[67]
퐵
퐶
퐷
푥
inhibition at bottom, = % inhibition at top, = IC50, = slope,
=
푦
inhibitor concentration and = % inhibition. If curve did not reach
100% of inhibition, B was fixed to 100 only when at least 50% of
inhibition was reached.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 18
Cytotoxicity Studies (Drug Discovery Unit, University of
similarly, except that the MeCN composition was raised to 70%
Dundee).[70] In vitro cytotoxicity studies were carried out using
HepG2 (Human Caucasian hepatocyte carcinoma, ECACC cat. no.
85011430) used as indicators for general mammalian cell toxicity.
HepG2 in vitro cytotoxicity can be assessed using the assay procedure
as described.[71]
instead of 55%.
1
2
3
4
ASSOCIATED CONTENT
Supporting Information
5
6
7
8
Kinetic Solubility Estimation using Nephelometry (Centre for Drug
Candidate Optimization, Monash Institute of Pharmaceutical
Sciences). Compound in DMSO was spiked into either pH 6.5
phosphate buffer or 0.01 M HCl (approx. pH 2.0) at 7 concentrations
with the final DMSO concentration of each being 1%. After 30
minutes had elapsed, samples were then analyzed for precipitation via
Nephelometry to determine a solubility range.[72]
The Supporting Information is available free of charge on the
ACS Publications website.
Table of molecular formula strings, in vitro potency values and
calculated properties for target compounds (CSV)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1H and 13C NMR spectra for synthesized compounds (PDF)
Graphical table of in vitro potencies for target compounds (PDF)
Distribution Coefficient Estimation using Chromatography (Centre
for Drug Candidate Optimization, Monash Institute of Pharmaceutical
Sciences). Partition coefficient values (LogD) of the test compounds
were estimated at pH 7.4 by correlation of their chromatographic
retention properties (mean of 2 injections) against the characteristics
of a series of standard compounds with known partition coefficient
values. The method employed is a gradient HPLC based derivation of
the method developed by Lombardo.[73]
AUTHOR INFORMATION
Corresponding Author
* matthew.todd@ucl.ac.uk
Author Contributions
In Vitro Metabolic Stability (Centre for Drug Candidate
Optimization, Monash Institute of Pharmaceutical Sciences). The
metabolic stability assay was performed by incubating each test
compound with liver microsomes at 37 oC and a protein concentration
of 0.4 mg/mL. The metabolic reaction was initiated by the addition of
an NADPH-regenerating system and quenched at 5 time points over a
60 minute incubation period by the addition of acetonitrile containing
diazepam as internal standard. Control samples (containing no
NADPH) were included (and quenched at 2, 30 and 60 minutes) to
monitor for potential degradation in the absence of cofactor. All liver
microsomes used in these experiments were purchased from
XenoTech. Microsomal incubations were performed at a substrate
concentration of 1 µM. The half-life and intrinsic clearance were
determined by exponential regression of the concentration vs time
data.
E.G.T. led the synthetic chemistry (laboratory-based and data
interpretation), supervised by M.H.T., S.D.H., C.M.W., G.P.S.
and L.M.R. provided reagents, protocols and experimental advice.
I. H. developed the Pf assay, manually prepared and analyzed the
compounds, collated all data and contributed to the paper
(methods section and data table). M. A. carried out the routine Pf
assay and analysis. R.S., G.S.W. and R.S.O. performed the
biofunctionalization experiments and contributed data
interpretation. E.G.T. and M.H.T. wrote the paper with inputs
from all authors. M.H.T. conceived the study, secured the funding
and founded Open Source Malaria where much of the work took
place.
Funding Sources
We thank the Australian Research Council and the Medicines for
Malaria Venture for support (LP150101226).
Late-Stage Biofunctionalization of Compounds 16 and 19
(Obach Lab, Pfizer). Compound 19 (40 µM) was incubated with dog
liver microsomes (1 mg/mL) in a total volume of 40 mL potassium
phosphate buffer (0.1 M; pH 7.4) containing MgCl2 (3.3 mM) and
NADPH (1.3 mM). The incubation was done in a 500 mL Erlenmeyer
Conflict of Interest
CSIRO (GPS) and UQ (CMW) have a formal relationship with
Boron Molecular Pty Ltd. who supply cubane intermediates.
RSO, GSW, and RS are employed by and shareholders in Pfizer
Inc.
o
flask in a shaking water bath maintained at 37 C open to the air.
After 1 h, the incubation was terminated by addition of MeCN (40
mL) and the precipitated protein was removed by spinning at 1700 ×
g for 5 min. The supernatant was partially evaporated in a vacuum
centrifuge for 1.5 h and to the remaining liquid was added formic acid
(0.5 mL), MeCN (0.5 mL) and H2O to a final volume of 50 mL. This
mixture was spun in a centrifuge at 40000 × g for 30 min. The
clarified supernatant was applied to a Varian Polaris C18 column (4.6
× 250 mm; 5 µm) at 0.8 mL/min through a Jasco HPLC pump. After
the entire solution was applied and another 5 mL of 0.1% formic acid
in H2O was washed through the system, the column was moved to an
HPLC-UV-MS (Thermo Velos LTQ mass spectrometer, equipped
with a Waters Acquity HPLC-UV system) in line with a fraction
collector (Leap Analytics). A mobile phase gradient was applied at
0.8 mL/min beginning with 2% MeCN in 0.1% aqueous formic acid,
raised to 20% MeCN at 1 min, held until 5 min, then increased
linearly to 55% MeCN at 80 min, followed by a 10 min wash at 95%
MeCN and 10 min re-equilibration to initial conditions. Fractions
were collected every 20 sec. Fractions predicted to contain products
of interest eluted around 39.5 and 42.0 min and were individually
analysed on a Thermo Orbitrap Elite UHPLC-UV-HRMS system to
ascertain identity and purity for pooling and qNMR analysis. Pooled
fractions were evaporated in a vacuum centrifuge and the residue was
taken up in DMSO-d6 (0.05 mL). Compound 16 was subjected to a
similar procedure except that rabbit liver microsomes were used as
the source of enzyme, the substrate concentration was 25 µM, and the
incubation time was 45 min. The fractionation was carried out
ACKNOWLEDGMENT
Synthetic precursor 3-(difluoromethyl)bicyclo[1.1.1]pentane-1-
carboxylic acid was provided to Prof. M. H. Todd by Pfizer
through its Compound Transfer Program. We thank the Kassiou
group (The University of Sydney) for the precursor to compound
S4. We thank the Centre for Drug Candidate Optimisation,
Monash University for conducting the kinetic solubility,
chromatographic LogD and microsomal stability studies. We
thank contributors to Open Source Malaria, including Chris
Swain, Mark Coster and Anthony Sama, for useful discussions.
ABBREVIATIONS
APCI, atmospheric-pressure chemical ionization; BCP,
bicyclo[1.1.1]pentane; BNCT, boron neutron capture therapy;
CCDC, Cambridge Crystallographic Data Centre; CNS, central
nervous system; DCVC, dry column vacuum chromatography;
hERG, human ether-à-go-go related gene; HLM, human liver
microsomes; LpPLA2, lipoprotein-associated phospholipase A2;
MLM, mouse liver microsomes; MMV, Medicines for Malaria
Venture; OSM, Open Source Malaria; RCH, rat cryopreserved
hepatocytes; UCSF, University of California San Francisco.
14
ACS Paragon Plus Environment

Page 15 of 18
Journal of Medicinal Chemistry
[16] Stockdale, T. P.; Williams, C. M. Pharmaceuticals that
Contain Polycyclic Hydrocarbon Scaffolds. Chem. Soc. Rev. 2015,
44, 7737-7763.
[17] Locke, G. M.; Bernhard, S. S. R.; Senge, M. O.
Nonconjugated Hydrocarbonds as Rigid-Linear Motifs: Isosteres for
Material Sciences and Bioorganic and Medicinal Chemistry. Chem.
Eur. J. 2018, 25, 4590-4647.
[18] Wlochal, J.; Davies, R. D. M.; Burton, J. Cubanes in
Medicinal Chemistry: Synthesis of Functionalized Building Blocks.
Org. Lett. 2014, 16, 4094-4097.
[19] Biegawiewicz, K. F.; Griffiths, J. R.; Savage, G. P.;
Tsanaktsidis, J.; Priefer, R. Cubane: 50 Years Later. Chem. Rev. 2015,
115, 6719-6745.
[20] Reekie, T. A.; Williams, C. M.; Rendina, L. M.; Kassiou, M.
Cubanes in Medicinal Chemistry. J. Med. Chem. 2019, 62, 1078-
1095.
REFERENCES
1
2
3
4
5
6
7
8
[1] Ritchie, T. J.; Macdonald, S. J. F. The Impact of Aromatic Ring
Count on Compound Developability – Are too many Aromatic Rings
a Liability in Drug Design? Drug Discov. Today 2009, 14, 1011-
1020.
[2] Ritchie, T. J.; Macdonald, S. J. F. Physicochemical Descriptors
of Aromatic Character and Their Use in Drug Discovery. J. Med.
Chem. 2014, 57, 7206-7215.
[3] Ishikawa, M.; Hashimoto, Y. Improvement in Aqueous
Solubility in Small Molecule Drug Discovery Programs by Disruption
of Molecular Planarity and Symmetry. J. Med. Chem. 2011, 54, 1539-
1554.
[4] Walker, M. A. Novel Tactics for Designing Water-Soluble
Molecules in Drug Discovery. Expert Opin. Drug Discovery 2014, 9,
1421-1433.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[5] Ritchie, T. J.; Macdonald, S. J. F. Heterocyclic Replacements
for Benzene: Maximising ADME Benefits by Considering Individual
Ring Isomers. Eur. J. Med. Chem. 2016, 124, 1057-1068.
[21] Chalmers, B. A.; Xing, H.; Houston, S.; Clark, C.;
Ghassabian, S.; Kuo, A.; Cao, B.; Reitsma, A.; Murray, C.-E. P.;
Stok, J. E.; Boyle, G. M.; Pierce, C. J.; Littler, S. W.; Winkler, D. A.;
Bernhardt, P. V.; Pasay, C.; De Voss, J. J.; McCarthy, J.; Parsons, P.
G.; Walter, G. H.; Smith, M. T.; Cooper, H. M.; Nilsson, S. K.;
Tsanaktsidis, J.; Savage, G. P.; Williams, C. M. Cubane as a Benzene
Bioisostere. Angew. Chem Int. Ed. 2016, 55, 3580-3585; Angew.
Chem. 2016, 128, 3644-3649.
[22] Houston, S. D.; Fahrenhorst-Jones, T.; Xing, H.; Chalmers, B.
A.; Sykes, M. L.; Stok, J. E.; Soto, C. F.; Burns, J. M.; Bernhardt, P.
V.; De Voss, J. J.; Boyle, G. M.; Smith, M. T.; Tsanaktsidis, J.;
Savage, G. P.; Avery, V. M.; Williams, C. M. The Cubane Paradigm
in Bioactive Molecule Discovery: Further Scope, Limitations and the
Cyclooctatetraene Complement. Org. Biomol. Chem. 2019, 17, 6790-
6798.
[23] Caputo, D. F. J.; Arroniz, C.; Dürr, A. B.; Mousseau, J. J.;
Stepan, A. F.; Mansfield, S. J.; Anderson, E. A. Synthesis and
Applications of Highly Functionalized 1-Halo-3-substituted
Bicyclo[1.1.1]pentanes. Chem. Sci. 2018, 9, 5295-5300.
[24] Stepan, A. F.; Subramanyam, C.; Efremov, I. V.; Dutra, J. K.;
O’Sullivan, T. J.; DiRico, K. J.; McDonald, W. S.; Won, A.; Dorff, P.
H.; Nolan, C. E.; Becker, S. L.; Pustilnik, L. R.; Riddell, D. R.;
Kauffman, G. W.; Kormos, B. L.; Zhang, L.; Lu, Y.; Capetta, S. H.;
Green, M. E.; Karki, K.; Sibley, E.; Atchison, K. P.; Hallgren, A. J.;
Oborski, C. E.; Robshaw, A. E.; Sneed, B.; O’Donnell, C. J.
Application of the Bicyclo[1.1.1]pentane Motif as a Nonclassical
Phenyl Ring Bioisostere in the Design of a Potent and Orally Active
γ-Secretase Inhibitor. J. Med. Chem. 2012, 55, 3414-3424.
[25] Measom, N. D.; Down, K. D.; Hirst, D. J.; Jamieson, C.;
Manas, E. S.; Patel, V. K.; Somers, D. O. Investigation of a
Bicyclo[1.1.1]pentane as a Phenyl Replacement within an LpPLA2
Inhibitor. ACS Med. Chem. Lett. 2017, 8, 43-48.
[26] Ma, X.; Pham, L. N. Selected Topics in the Syntheses of
Bicyclo[1.1.1]pentane (BCP) Analogues. Asian J. Org. Chem. 2019,
9, 8-22.
[27] Nugent, J.; Arroniz, C.; Shire, B. R.; Sterling, A. J.; Pickford,
H. D.; Wong, M. L. J.; Mansfield, S. J.; Caputo, D. F. J.; Owen, B.;
Mousseau, J. J.; Duarte, F.; Anderson, E. A. A General Route to
Bicyclo[1.1.1]pentanes through Photoredox Catalysis. ACS Catal.
2019, 9, 9568-9574.
[28] Issa, F.; Kassiou, M.; Rendina, L. M. Boron in Drug
Discovery: Carboranes as Unique Pharmacophores in Biologically
Active Compounds. Chem. Rev. 2011, 111, 5701-5722.
[29] Beer, M. L.; Lemon, J.; Valliant, J. F. Preparation and
Evaluation of Carborane Analogues of Tamoxifen. J. Med. Chem.
2010, 53, 8012-8020.
[30] Scholz, M.; Bensdorf, K.; Gust, R.; Hey-Hawkins, E. Asborin:
The Carbaborane Analogue of Aspirin. ChemMedChem 2009, 4, 746-
748.
[6]
WHO
World
Malaria
Report
2018.
https://www.who.int/malaria/publications/world-malaria-report-
2018/en/ (accessed Apr 4, 2018).
[7] Fight Against Malaria Stalling and Could Reverse, Warns 2017
World Malaria Report. https://www.devex.com/news/fight-against-
malaria-stalling-and-could-reverse-warns-2017-world-malaria-report-
91636 (accessed Mar 24, 2018).
[8] Global Malaria Report Reveals Africa’s Hits and Missed:
Here’s What to Do. https://www.devex.com/news/fight-against-
malaria-stalling-andcould-reverse-warns-2017-world-malaria-report-
91636 (accessed Mar 24, 2018).
[9] a) Williamson, A. E.; Ylioja, P. M.; Robertson, M. N.;
Antonova-Koch, Y.; Avery, V.; Baell, J. B.; Batchu, H.; Batra, S.;
Burrows, J. N.; Bhattacharyya, S.; Calderon, F.; Charman, S. A.;
Clark, J.; Crespo, B.; Dean, M.; Debbert, S. L.; Delves, M.; Dennis,
A. S. M.; Deroose, F.; Duffy, S.; Fletcher, S.; Giaever, G.;
Hallyburton, I.; Gamo, F.-J.; Gebbia, M.; Guy, R. K.; Hungerford, Z.;
Kirk, K.; Lafuente-Monasterio, M. J.; Lee, A.; Meister, S.; Nislow,
C.; Overington, J. P.; Papadatos, G.; Patiny, L.; Pham, J.; Ralph, S.
A.; Ruecker, A.; Ryan, E.; Southan, C.; Srivastava, K.; Swain, C.;
Tarnowski, M. J.; Thompson, P.; Turner, P.; Wallace, I. M.; Wells, T.
N. C.; White, K.; White, L.; Willis, P.; Winzeler, E. A.; Wittlin, S.;
Todd, M. H. Open Source Drug Discovery: Highly Potent
Antimalarial Compounds Derived from the Tres Cantos Arylpyrroles.
ACS Cent. Sci. 2016, 2, 687-701. b) Electronic Laboratory Notebook
available at https://ses.library.usyd.edu.au/handle/2123/21118 (DOI:
10.25910/5d7af492b2c63).
[10] Potential new class of antimalarials now open source.
https://www.mmv.org/newsroom/news/potential-new-class-
antimalarials-now-open-source (accessed Nov 8, 2018).
[11] a) OSM Series
4
Wiki In Vivo Efficacy.
https://github.com/OpenSourceMalaria/Series4/wiki/In-Vivo-Efficacy
(accessed Nov 14, 2019). b) Compound 6, containing a phenyl ring,
was chosen as the comparison compound over either of the known in
vivo actives because the latter contain difluorophenyl motifs; the
comparison is valid because the potencies, solubilities and in vitro
clearance values for the three compounds are similar (manuscript in
preparation).
[12] Friedman, H. L. in First Symposium on Chemical-Biological
Correlation, May 26-27, 1950, NAS-NRS: Washington, DC, 1951,
295-358.
[13] Meanwell, N. A. Synopsis of some Recent Tactical
Application of Bioisosteres in Drug Design. J. Med. Chem. 2011, 54,
2529-2591.
[14] Wanka, L.; Iqbal, K.; Schreiner, P. R. The Lipophilic Bullet
Hits the Targets: Medicinal Chemistry of Adamantane Derivatives.
Chem Rev. 2013, 113, 3516-3604.
[31] Scholz, M.; Steinhagen, M.; Heiker, J. T.; Beck-Sickinger, A.
G.; Hey-Hawkins, E. Asborin Inhibits Aldo/Keto Reductase 1A1.
ChemMedChem 2011, 6, 89-93.
[32] Wilkinson, S. M.; Gunosewoyo, H.; Barron, M. L.; Boucher,
A.; McDonnell, M.; Turner, P.; Morrison, D. E.; Bennett, M. R.;
[15] Carreira, E. M.; Fessard, T. C. Four-Membered Ring-
Containing Spirocycles: Synthetic Strategies and Opportunities.
Chem. Rev. 2014, 114, 8257-8322.
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 18
McGregor, I. S.; Rendina, L. M.; Kassiou, M. The First CNS-Active
Carborane: A Novel P2X7 Receptor Antagonist with Antidepressant
Activity. ACS Chem Neurosci. 2014, 5, 335-339.
[51] Bandak, D.; Babii, O.; Vasiuta, R.; Komarov, I. V.;
Mykhailiuk, P. K. Design and Synthesis of Novel 19F-Amino Acid: A
Promising 19F NMR Label for Peptide Studies. Org. Lett. 2015, 17,
226-229.
1
2
3
4
5
6
7
8
[33] Nicolaou, K. C.; Vourloumis, D.; Totokotsopoulos, S.;
Papakyriakou, A.; Karsunky, H.; Fernando, H.; Gavrilyuk, J.; Webb,
D.; Stepan, A. F. Synthesis and Biopharmaceutical Evaluation of
Imatinib Analogues Featuring Unusual Structural Motifs.
ChemMedChem 2016, 11, 31-37.
[34] Auberson, Y. P.; Brocklehurst, C.; Furegati, M.; Fessard, T.
C.; Koch, G.; Decker, A.; La Vecchia, L.; Briard, E. Improving
Nonspecific Binding and Solubility: Bicycloalkyl Groups and
Cubanes as para-Phenyl Bioisosteres. ChemMedChem 2017, 12, 590-
598.
[52] Lamoureux, G.; Artavia, G. Use of the Adamantane Structure
in Medicinal Chemistry. Curr. Med. Chem. 2010, 17, 2967-2978.
[53] Xing, H.; Houston, S. D.; Chen, X.; Ghassabian, S.;
Fahrenhorst-Jones, T.; Kuo, A.; Murray, C.-E. P.; Conn, K.-A.;
Jaeschke, K. N.; Jin, D.-Y.; Pasay, C.; Bernhardt, P. V.; Burns, J. M.;
Tsanaktsidis, J.; Savage, G. P.; Boyle, G. M.; De Voss, G. J.;
McCarthy, J.; Walter, G. H.; Burne, T. H. J.; Smith, M. T.; Tie, J.-K.;
Williams, C. M. Cyclooctatetraene: A Bioactive Cubane Paradigm
Complement. Chem. Eur. J. 2019, 25, 2729-2734.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[35] Shultz, M. D. Two Decades under the Influence of the Rule of
Five and the Changing Properties of Approved Oral Drugs. J. Med.
Chem. 2019, 62, 1701-1714.
[36] OSM Series 4 Wiki Mechanism of Action: Possible PfATP4
Activity Deduced from Parasite Ion Regulation Assays.
https://github.com/OpenSourceMalaria/Series4/wiki/Mechanism-of-
Action%3A-Possible-PfATP4-Activity-Deduced-from-Parasite-Ion-
Regulation-Assays (accessed Apr 19, 2020).
[37] Lehane, A. M.; Ridgway, M. C.; Baker, E.; Kirk, K. Diverse
Chemotypes Disrupt Ion Homeostasis in the Malaria Parasite. Mol.
Microbiol. 2014, 94, 327-339.
[38] Scholz, M.; Hey-Hawkins, E. Carbaboranes as
Pharmacophores: Properties, Synthesis, and Application Strategies.
Chem. Rev. 2011, 111, 7035-7062.
[39] Lesnikowski, Z. J. Boron Units as Pharmacophores – New
Applications and Opportunities of Boron Cluster Chemistry. Collect.
Czech. Chem. Commun. 2007, 72, 1646-1658.
[40] Valliant, J. F.; Schaffer, P.; Stephenson, K. A.; Britten, J. F.
Synthesis of Boroxifen, A Nido-Carborane Analogue of Tamoxifen. J.
Org. Chem. 2002, 67, 383-387.
[41] Reynolds, R. C.; Campbell, S. R.; Fairchild, R. G.; Kisliuk, R.
L.; Micca, P. L.; Queener, S. F.; Riordan, J. M.; Sedwick, W. D.;
Waud, W. R.; Leung, A. K. W.; Dixon, R. W.; Suling, W. J.; Borhani,
D. W. Novel Boron-Containing, Nonclassical Antifolates: Synthesis
and Preliminary Biological and Structural Evaluation. J. Med. Chem.
2007, 50, 3283-3289.
[42] Eaton, P. E. Cubanes: Starting Materials for the Chemistry of
the 1990s and the New Centure. Angew. Chem. Int. Ed. 1992, 31,
1421-1436.
[54] Morel-Desrosiers, N.; Morel, J.-P. Standard Molar Enthalpies,
Volumes, and Heat Capacities of Adamantane in Cyclohexane, n-
Hexane, and Carbon Tetrachloride. Interpretation using the Scaled-
Particle Theory. J. Solution Chem. 1979, 8, 579-592.
[55] Bohn, R. K.; Bohn, M. D. The Molecular Structures of 1,2-,
1,7-, and 1,12-dicarboclosododecarborane (12), B10C2H12. Inorg.
Chem. 1971, 10, 350-355.
[56] Bradac, J.; Furek, Z.; Janezic, D.; Molan, S.; Smerkolj, I.;
Stanovnik, B.; Tisler, M.; Vercek, B. Heterocycles. 167.
Telesubstitution and other Transformations of Imidazo[1,2-a]- and s-
Triazolo[4,3-a]pyrazines. J. Org. Chem. 1977, 42, 4197-4201.
[57] Houston, S. D.; Xing, H.; Bernhardt, P. V.; Vanden Berg, T.
J.; Tsanaktsidis, J.; Savage, G. P.; Williams, C. M. Cyclooctatetraenes
through Valence Isomerization of Cubanes: Scope and Limitations.
Chem. Eur. J. 2019, 25, 2735-2739.
[58] Yoo, J.; Hwang, J.-W.; Do, Y. Facile and Mild Deboronation
of o-Carboranes Using Cesium Fluoride. Inorg. Chem. 2001, 40, 568-
570.
[59] del Prado, A.; Navarro, R.; Levkin, P.; Gallardo, A.; Elvira,
C.; Reinecke, H. Dual Stimuli-Responsive Polyamines Derived from
Modified N-Vinylpyrrolidones through CuAAC Click Chemistry. J.
Polym. Sci., Part A: Polym. Chem. 2016, 54, 1098-1108.
[60] Priefer, R.; Farrell, P. G.; Harpp, D. N. Effective Synthetic
Routes to Cubylcarbinol Derivatives. Synthesis 2002, 18, 2671-2673.
[61] Aleksandrov, A. M.; Pehk, T. J.; Petrenko, A. E.; Watson, W.
H. Accessible Route to 4-Substituted “Bird-Cage” Hydrocarbon
Derivatives. J. Org. Chem. 1994, 59, 3709-3711.
[62] Li, K.; Wang, Y.; Yang, G.; Byun, S.; Rao, G.; Shoen, C.;
Yang, H.; Gulati, A.; Crick, D. C.; Cynamon, M.; Huang, G.;
Docampo, R.; No, J. H.; Oldfield, E. Oxa, Thia, Heterocycle, and
Carborane Analogues of SQ109: Bacterial and Protozoal Cell Growth
Inhibitors. ACS Infect. Dis. 2015, 1, 215-221.
[63] Nekvinda, J.; Grüner, B.; Gabel, D.; Nau, W. M.; Assaf, K. I.
Host-Guest Chemistry of Carboranes: Synthesis of Carboxylate
Derivatives and their Binding to Cyclodextrins. Chem. Eur. J. 2018,
24, 12970-12975.
[64] Agarwal, H. K.; Buszek, B.; Ricks, K. G.; Tjarks, W.
Synthesis of Closo-1,7-Carboranyl Alkyl Amines. Tetrahedron Lett.
2011, 52, 5664-5667.
[65] Fujii, S.; Kano, A.; Songkram, C.; Masuno, H.; Taoda, Y.;
Kawachi, E.; Hirano, T.; Tanatani, A.; Kagechika, H. Synthesis and
Structure–Activity Relationship of p-Carborane-Based Non-
Secosteroidal Vitamin D Analogs. Bioorg. Med. Chem. 2014, 22,
1227-1235.
[66] Mandal, P. K.; McMurray, J. S. Pd–C-Induced Catalytic
Transfer Hydrogenation with Triethylsilane. J. Org. Chem. 2007, 72,
6599-6601.
[67] Yamashita, K.-I.; Tsuboi, M.; Asano, M. S.; Sugiura, K.-I.
Facile Aromatic Finkelstein Iodination (AFI) Reaction in 1,3-
Dimethyl-2-imidazolidinone (DMI). Synth. Commun. 2011, 42, 170-
175.
[68] Yang, H.; Li, Y.; Jiang, M.; Wang, J.; Fu, H. Ceneral Copper-
Catalyzed Transformations of Functional Groups from Arylboronic
Acids in Water. Chem. Eur. J. 2011, 17, 5652-5660.
[43] Bashir-Hashemi, A. Cubanes: Super explosives and potential
pharmaceutical intermediates. A. NASA Conf. Pub. 1994, 127-130.
[44] Uetrecht, J. P.; Trager, W. Drug Metabolism: Chemical and
Enzymatic Aspects, CRC Press, 2007, pp 71-75.
[45] Feng, Y.; Liu, L.; Wang, J.-T.; Zhao, S.-W.; Guo, Q.-X.
Homolytic C–H and N–H Bond Dissociation Energies of Strained
Organic Compounds. J. Org. Chem. 2004, 69, 3129-3138.
[46] Jin, Y.; Lipscomb, J. D. Probing the Mechanism of C–H
Activation: Oxidation of Methylcubane by Soluble Methane
Monooxygenase
from
Methylosinus
trichosporium
OB3b.
Biochemistry 1999, 38, 6178-6186.
[47] Sarkar, M. R.; Houston, S. D.; Savage, G. P.; Williams, C. M.;
Krenske, E. H.; Bell, S. G.; De Voss, J. J. Rearrangement-Free
Hydroxylation of Methylcubanes by a Cytochrome P450: The Case
for Dynamical Coupling of C–H Abstraction and Rebound. J. Am.
Chem. Soc. 2019, 141, 19688-19699.
[48] Eaton, P. E.; Yip, Y. C. The Preparation and Fate of
Cubylcarbinyl Radicals. J. Am. Chem. Soc. 1991, 113, 7692-7697.
[49] Annese, C.; D’Accolti, L.; Fusco, C.; Gandolfi, R.; Eaton, P.
E.; Curci, R. Oxyfunctionalization of Non-Natural Targets by
Dioxiranes. 6. On the Selective Hydroxylation of Cubane. Org. Lett.
2009, 11, 3574-3577.
[50] Collin, D. E.; Folgueiras-Amador, A. A.; Pletcher, D.; Light,
M. E.; Linclau, B.; Brown, R. C. D. Cubane Electrochemistry: Direct
Conversion of Cubane Carboxylic Acids to Alkoxy Cubanes Using
the Hofer-Moest Reaction under Flow Conditions. Chem. – Eur. J.
2019, 26, 374-378.
[69] Griffiths, J. R.; Tsanaktsidis, J.; Savage, G. P.; Priefer, R.
Thermochemical Properties of Iodinated Cubane Derivatives.
Thermochim. Acta 2010, 499, 15-20.
16
ACS Paragon Plus Environment

Page 17 of 18
Journal of Medicinal Chemistry
[70] Baragaña, B.; Forte, B.; Choi, R.; Hewitt, S. N.; Bueren-
C.; Gilbert, I. H. Lysyl-tRNA Synthetase as a Drug Target in Malaria
and Cryptosporidiosis. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 7015-
7020.
[71] Mersch-Sundermann, V.; Knasmüller, S.; Wu, X.-J.; Darroudi,
F.; Kassie, F. Use of a Human-Derive Liver Cell Line for the
Detection of Cytoprotective, Antigenotoxic and Cogenotoxic Agents.
Toxicology 2004, 198, 329-340.
[72] Bevan, C. D.; Lloyd, R. S. A High-Throughput Screening
Method for the Determination of Aqueous Drug Solubility Using
Laser Nephelometry in Microtiter Plates. Anal. Chem. 2000, 72,
1781-1787.
[73] Lombardo, F.; Shalaeva, M. Y.; Tupper, K. A.; Gao, F.
ElogDoct: A Tool for Lipophilicity Determination in Drug Discovery.
2. Basic and Neutral Compounds. J. Med. Chem. 2001, 44, 2490-
2497.
Calabuig, J. A.; Pisco, J. P.; Peet, C.; Dranow, D. M.; Robinson, D.
A.; Jansen, C.; Norcross, N. R.; Vinayak, S.; Anderson, M.; Brooks,
C. F.; Cooper, C. A.; Damerow, S.; Delves, M.; Dowers, K.; Duffy,
J.; Edwards, T. E.; Hallyburton, I.; Horst, B. G.; Hulverson, M. A.;
Ferguson, L.; Jiménez-Díaz, M. B.; Jumani, R. S.; Lorimer, D. D.;
Love, M. S.; Maher, S.; Matthews, H.; McNamara, C. W.; Miller, P.;
O’Neill, S.; Ojo, K. K.; Osuna-Cabello, M.; Pinto, E.; Post, J.; Riley,
J.; Rottmann, M.; Sanz, L. M.; Scullion, P.; Sharma, A.; Shepherd, S.
M.; Shishikura, Y.; Simeons, F. R. C.; Stebbins, E. E.; Stojanovski,
L.; Straschil, U.; Tamaki, F. K.; Tamjar, J.; Torrie, L. S.; Vantaux, A.;
Witkowski, B.; Wittlin, S.; Yogavel, M.; Zuccotto, F.; Angulo-
Barturen, I.; Sinden, R.; Baum, J.; Gamo, F.-J.; Mäser, P.; Kyle, D.
E.; Winzeler, E. A.; Myler, P. J.; Wyatt, P. G.; Floyd, D.; Matthews,
D.; Sharma, A.; Striepen, B.; Huston, C. D.; Gray, D. W.; Fairlamb,
A. H.; Pisliakov, A. V.; Walpole, C.; Read, K. D.; Van Voorhis, W.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 18
SYNOPSIS TOC (Word Style “SN_Synopsis_TOC”). If you are submitting your paper to a journal that requires a synopsis
graphic and/or synopsis paragraph, see the Instructions for Authors on the journal’s homepage for a description of what
needs to be provided and for the size requirements of the artwork.
1
2
3
Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the manuscript title,
should give the reader a representative idea of one of the following: A key structure, reaction, equation, concept, or theorem,
etc., that is discussed in the manuscript. Consult the journal’s Instructions for Authors for TOC graphic specifications.
4
5
6
7
8
Insert Table of Contents artwork here
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18
ACS Paragon Plus Environment