Design, synthesis and evaluation of photoactivatable derivatives of microtubule (MT)-active [1,2,4]triazolo[1,5-a]pyrimidines

Article abstract of DOI:10.1016/j.bmcl.2018.05.010

The [1,2,4]triazolo[1,5-a]pyrimidines comprise a promising class of non-naturally occurring microtubule (MT)-active compounds. Prior studies revealed that different triazolopyrimidine substitutions can yield molecules that either promote MT stabilization or disrupt MT integrity. These differences can have important ramifications in the therapeutic applications of triazolopyrimidines and suggest that different analogues may exhibit different binding modes within the same site or possibly interact with tubulin/MTs at alternative binding sites. To help discern these possibilities, a series of photoactivatable triazolopyrimidine congeners was designed, synthesized and evaluated in cellular assays with the goal of identifying candidate probes for photoaffinity labeling experiments. These studies led to the identification of different derivatives that incorporate a diazirine ring in the amine substituent at position 7 of the triazolopyrimidine heterocycle, resulting in molecules that either promote stabilization of MTs or disrupt MT integrity. These photoactivatable candidate probes hold promise to investigate the mode of action of MT-active triazolopyrimidines.

Full text of DOI:10.1016/j.bmcl.2018.05.010

Accepted Manuscript
Design, synthesis and evaluation of photoactivatable derivatives of microtubule
(MT)-active [1,2,4]triazolo[1,5-a]pyrimidines
Killian Oukoloff, Jane Kovalevich, Anne-Sophie Cornec, Yuemang Yao,
Zachary A. Owyang, Michael James, John Q. Trojanowski, Virginia M.-Y. Lee,
Amos B. Smith III, Kurt R. Brunden, Carlo Ballatore
PII:
S0960-894X(18)30400-1
https://doi.org/10.1016/j.bmcl.2018.05.010
BMCL 25827
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 March 2018
18 April 2018
5 May 2018
Please cite this article as: Oukoloff, K., Kovalevich, J., Cornec, A-S., Yao, Y., Owyang, Z.A., James, M.,
Trojanowski, J.Q., Lee, V.M., Smith, A.B. III, Brunden, K.R., Ballatore, C., Design, synthesis and evaluation of
photoactivatable derivatives of microtubule (MT)-active [1,2,4]triazolo[1,5-a]pyrimidines, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.010
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Design, synthesis and evaluation of
photoactivatable derivatives of microtubule
(MT)-active [1,2,4]triazolo[1,5-
a]pyrimidines.
Killian Oukoloffa, Jane Kovalevichb , Anne-Sophie Cornecc, Yuemang Yaob, Zachary A. Owyanga, Michael
Jamesb, John Q. Trojanowskib, Virginia M.-Y. Leeb, Amos B. Smith IIIc, Kurt R. Brundenb*, and Carlo
Ballatorea*.

Bioorganic & Medicinal Chemistry Letters
journal hom epage: www.els evier.com
Design, synthesis and evaluation of photoactivatable derivatives of microtubule
(MT)-active [1,2,4]triazolo[1,5-a]pyrimidines.
Killian Oukoloffa, Jane Kovalevichb , Anne-Sophie Cornecc, Yuemang Yaob, Zachary A. Owyanga,
Michael Jamesb, John Q. Trojanowskib, Virginia M.-Y. Leeb, Amos B. Smith IIIc, Kurt R. Brundenb*, and
Carlo Ballatorea*.
a Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093
b Center for Neurodegenerative Disease Research, Institute on Aging, University of Pennsylvania, 3600 Spruce Street, Philadelphia, PA 19104-6323
c Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, 231 South 34th St., Philadelphia, PA 19104-6323
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The [1,2,4]triazolo[1,5-a]pyrimidines comprise a promising class of non-naturally occurring
microtubule (MT)-active compounds. Prior studies revealed that different triazolopyrimidine
substitutions can yield molecules that either promote MT stabilization or disrupt MT integrity.
These differences can have important ramifications in the therapeutic applications of
triazolopyrimidines and suggest that different analogs may exhibit different binding modes
within the same site or possibly interact with tubulin/MTs at alternative binding sites. To help
discern these possibilities, a series of photoactivatable triazolopyrimidine congeners was
designed, synthesized and evaluated in cellular assays with the goal of identifying candidate
probes for photoaffinity labeling experiments. These studies led to the identification of different
derivatives that incorporate a diazirine ring in the amine substituent at position 7 of the
triazolopyrimidine heterocycle, resulting in molecules that either promote stabilization of MTs
or disrupt MT integrity. These photoactivatable candidate probes hold promise to investigate the
mode of action of MT-active triazolopyrimidines.
Keywords:
Triazolopyrimidine
Microtubule stabilization
Photoaffinity labeling
CNS drug discovery
*Corresponding authors:
*C.B.: e-mail, cballatore@ucsd.edu
*K.R.B.: e-mail, kbrunden@upenn.edu
2017 Elsevier Ltd. All rights reserved
.
Originally reported as anti-fungal agents, microtubule (MT)-
active [1,2,4]triazolo[1,5-a]pyrimidines and related heterocyclic
molecules1-5 have since attracted attention as potential candidates
for a variety of applications including cancer chemotherapy,6 as
well as neurodegenerative disease treatment.7-10 The mechanism
of action of this class of compounds has been the focus of several
studies and appears to be distinct from that of other MT-targeting
agents. Competition binding experiments first revealed that
that 2 binds exclusively to MTs and not to unpolymerized tubulin
heterodimers.11
cevipabulin6 (1, Figure 1),
a typifying example of the
triazolopyrimidine class and a potent anti-cancer compound,
competes with vincristine but not taxol or colchicine for binding
to MTs, suggesting that compounds from this class may interact
with MTs at the binding site of vinca alkaloids. This hypothesis
has recently been confirmed by X-ray crystallography and mode-
of-action studies11 in which triazolopyrimidine congener 2
(Figure 1) was found to bind at the interface between two
consecutive longitudinally-associated tubulin heterodimeric
subunits, in a region that largely overlaps with the vinca binding
site. However, opposite to the activity of vincristine/vinblastine,
the binding of 2 to MTs was found to promote MT-assembly and
stabilization. Interestingly, the same studies also demonstrated
Figure 1. Structures of selected MT-active triazolo-
pyrimidines; dotted lines highlight sites chosen for insertion of
photoactivatable moieties.

However, prior investigations with 1 clearly suggested that
this compound, like vincristine, can in fact interact with tubulin
heterodimers and interfere with the rate of exchange of the
guanosine triphosphate (GTP).12 Furthermore, side-by-side
evaluation of the MT-stabilizing properties of different
triazolopyrimidines in cell-based assays highlighted important
differences in activity depending on whether or not these
molecules contained an alkoxide side chain at the 4 position of
the fluorinated phenyl ring.8 Indeed, whereas triazolopyrimidine
lacking the alkoxy side chain, such as 3 and 4 (Figure 1), were
found to stabilize MTs and preserve MT integrity, other
examples with the side chain, including 1 and 5 (Figure 1), were
found to affect markers of stable MTs with a bell-shaped dose-
response relationship, and to produce a proteasomal-dependent
degradation of tubulin, thereby leading to an overall disruption of
MT integrity.8 Consistent with this structure-activity relationship
(SAR), we confirmed that compound 2, which compared to 1
features a relatively bulky bicyclic amine substituent at position 7
but lacks the alkoxy side chain, produces MT-stabilization in
HEK-293 cells, as evidenced by an elevation in acetylated α-
tubulin, a known marker of stable MTs,13 without reducing total
tubulin levels (see Table 1). Taken together, these observations
suggest that different triazolopyrimidine congeners may interact
with tubulin/MTs in different manners depending on the
particular substitution pattern, thereby producing different effects
on MT structure and function. Such differences are likely to have
important implications in terms of therapeutic applications of
triazolopyrimidines as, depending on the particular clinical
indication, only one of these effects (i.e., preservation or
disruption of MT integrity) may be desirable.8 Whereas the X-ray
crystallography studies with 2 provide a detailed insight on how
MT-stabilizing triazolopyrimidines interact with and stabilize
MTs, further investigations are likely needed to fully elucidate
the mode of action of other MT-binding triazolopyrimidine
congeners that can lead to altered MT morphology and tubulin
degradation, such as 1.
Photoaffinity labeling (PAL) experiments are commonly
utilized to identify biological target(s) and/or investigate the
mode of action of small molecules, and can be useful to
complement other techniques such as NMR or X-ray
crystallography.14 To enable PAL experiments, a series of
triazolopyrimidine derivatives bearing known photoreactive
moieties were designed, synthesized and evaluated with the goal
of identifying candidate probes. In particular, we explored
derivatives in which arylazides or diazirines were
appended/incorporated at the end of the alkoxy side chain, or at
positions 7 or 5 of the triazolopyrimidine core (see Figure 1).
These studies led to the identification of selected
photoactivatable derivatives that retain the characteristic
biological activity of MT-active triazolopyrimidines that are
either lacking or bearing the alkoxy side chain.
The synthesis of test compounds was largely based on
strategies that had been previously described by Wyeth6 for the
synthesis of triazolopyrimidine 1 and related derivatives (Scheme
1). Thus, azidated derivatives 6, 7,7 and 8, in which the
photoactivatable group is at C-5 (7 and 8) or C-7 (6), were
accessed respectively from triazolopyrimidine 9, 10 and 1 upon
reaction with sodium azide. Triazolopyrimidine derivative 11,
which comprises an aryl diazirine fragment appended at the
terminal amine of the alkoxy side chain, was readily accessed via
N-acylation of known compound 126 with commercially
available diazirine 13. Finally, triazolopyrimidine 14–16, which
incorporate a diazirine ring into the amine fragment linked at C-
5, were accessed from dichloride 9 upon reaction with the
appropriate diazirine containing amine (17–19) that were
prepared from N-Boc protected amino acids (see Supporting
Information). Further reaction of 14–16 with the appropriate
alkoxy side chain resulted in derivatives 20–22.
CF3
CF3
H
N
NH2
H
N
F
F
F
O
F
F
or
X
NH
R
Cl
HO
NaN3
R
N
N
N
N
N
N
N
N
N
DMF, 16h, rt
F
NaH, DMSO/THF
60°C, 16h
F
F
N
Y
N
Y
N
Cl
4-(3-(trifluoromethyl)-3H-
diazirin-3-yl)benzoic acid (13)
(9)
Y = Cl; R = H (12)
X = N3; Y = Cl (6)
CDI, CH2Cl2,
0˚C - rt, 40h
O
CF3
R1
R1
Y = Cl; R =
X =
X =
; Y = Cl (10)
; Y = N3 (7)
(11)
CF3
Boc
OH
NH
CF3
NH
-Cl+H3N
N
H
NaN3
N
N
4 steps
O
N
N
R
R
R
1 = (S)-CH3
1 = (R)-CH3
1 = H
Y =Cl; R = CH3 (1)
Y =N3; R = CH3 (8)
R
R
R
1 = (S)-CH3 (17)
1 = (R)-CH3 (18)
1 = H (19)
NaN3
N
N
N
N
R1
F
O
N
R1 NH
R
F
F
R1 NH
HO
N
-Cl+H3N
R
N
N
N
N
N
N
(9)
NaH, DMSO/THF
60°C, 16h
F
N
N
F
Cl
DMF, TEA, 1h, rt
N
N
Cl
R
1 = (S)-CH3; R = CH3 (20)
1 = (R)-CH3; R = CH3 (21)
1 = H; R = CH3 (22)
R
R
R
1 = (S)-CH3 (14)
1 = (R)-CH3 (15)
1 = H (16)
R
R
R
H2CO, NaBH4
1 = H; R = H (23)
Scheme 1. Synthesis of triazolopyrimidine derivatives.

After synthesis, each of the candidate probes was evaluated in
a cell-based MT assay8 in which acetylated α-tubulin and total α-
tubulin levels were determined by ELISA in cell lysates after 4 h
of incubation with the test compound at either 1 or 10 µM. As
summarized in Table 1, azidated derivatives 6 and 7 were found
to be essentially inactive independent of the position of the azide
in the triazolopyrimidine heterocycle. Addition of the alkoxy side
chain to the fluorinated ring of derivative 7 resulted in a
biologically active derivative (8) that was considerably less
potent than either 1 or 5, but which at 10 µM compound
concentration was able to produce a significant increase in
acetylated α-tubulin levels as well as a concurrent reduction in
total α-tubulin. These results further confirmed that the addition
of the side chain may be critical to impart MT-disrupting activity
to triazolopyrimidines, although it is interesting to note that
derivative 11, which carries a modified alkoxide side chain, did
not produce a significant reduction in total α-tubulin levels at
compound concentrations (10 µM) that are active in the
acetylated α-tubulin assay. Among other diazirine containing
derivatives, incorporation of the photoactivatable moiety into the
amine substituent at C-7 resulted in active derivatives (14–16)
that were similar to 2–4 in producing an elevation of acetylated
α-tubulin without a significant loss of total α-tubulin, suggesting
that these analogs are MT-stabilizing and do not interfere with
MT integrity.
Compound 16 appeared to be especially promising as this analog
produced a statistically significant increase in acetylated α-
tubulin at both 1 and 10 µM compound concentration. Further
introduction of the alkoxy side chain at the 4 position of the
fluorinated phenyl of this triazolopyrimidine, as well as 14 and
15, caused the anticipated shift in mode of action from MT-
stabilizing (14–16) to analogs (20–22) that cause a reduction in
total α-tubulin levels in a manner that appears to be comparable
to 1 and 5 (cf., Table 1). Among these derivatives, 21 and 22
were found to be most active, with both compounds exhibiting a
bell-shaped dose-response and inducing a profound loss of total
tubulin in a dose-dependent manner, as previously noted for 1
and 5 (see Figure 2). Given these properties, 22 holds promise as
a candidate PAL probe to investigate at the molecular level the
mode of action of triazolopyrimidines that, like 1, disrupt MT
integrity. In addition, the presence of the alkoxy side chain
provides convenient access to radiolabeled derivatives of 22 (e.g.,
by methylating the terminal amine of 23 via reductive amination
in the presence of tritiated NaBH4, Scheme 1) that may facilitate
the detection of covalently modified tubulin fragments after the
PAL experiments.
In summary, these studies reveal that incorporation of
photoactivatable diazirine rings in the C-7 substituent of MT-
active [1,2,4]triazolo[1,5-a]pyrimidines can result in derivatives
that retain significant biological activity. Furthermore, depending
on whether or not these derivatives are modified further with an
appropriate alkoxy side chain, the mode of action of these
compounds can be switched from MT-stabilizing agents that
preserve MT integrity, to MT-disrupting compounds that cause a
reduction in tubulin levels. As a result, these derivatives hold
promise as candidate probes to investigate the mode of action of
MT-active triazolopyrimidines.
Table 1. Relative changes in acetylated α-tubulin and total α-
tubulin levels in HEK-293 cells after 4 h incubation with test
compounds at either 1 or 10 µM.
Compound
Acetylated α-Tubulin
Total α-Tubulin
1 µM
7.07**
±0.36
10 µM
0.1**
±0.01
1 µM
0.51**
±0.08
0.29**
±0.03
1.12
±0.08
1.18 ±
0.09
1.10
±0.17
ND
0.74**
±0.14
ND
10 µM
0.14**
±0.03
0.14**
±0.01
1.38**
±0.19
1.22 ±
0.18
0.90 ±
0.16
Cevipabulin (1)
5
2
3
4
5.83** ±
0.35
3.65** ±
0.15
2.78** ±
0.17
2.14** ±
0.15
Inactive
0.97 ±
0.06
Inactive
1.30±
0.28
0.30** ±
0.15
7.43** ±
0.93
5.04** ±
0.45
2.59** ±
0.15
Inactive
4.99** ±
0.27
Inactive
3.65** ± 1.31**±
6
8
ND
0.46**
±0.06
ND
0.88 ±
0.05
7
11
0.74
1.85** ±
0.08
0.28* ±
0.03
0.13
ND
14
20
15
ND
0.94 ±
0.02
Figure 2. Relative changes of acetylated α-tubulin (A) and total
α-tubulin (B) caused by 22 in HEK-293 cells after 4 h of
incubation. **, p<0.01 as determined by one-way ANOVA and
Dunnett’s post-hoc analysis.
0.78**
± 0.02
6.7** ±
0.45
1.42 ±
0.08
0.69**±
0.05
0.92*†
± 0.01
0.73**
± 0.02
0.89**†
4.28** ±
0.37
± 0.04
21
16
22
Acknowledgments
21.7** ±
0.93 ±
0.03
3.42** ±
0.06
0.29** ±
0.03
1.01 ±
0.05
1.03 ±
0.04
0.19**
± 0.02
0.4
1.56** ±
0.1
10.5** ±
0.36
1.08 ±
0.04
0.36**
± 0.04
Financial support for this work has been provided by NIH/NIA
Grant AG044332 and the National Foundation for Cancer
Research (ABS).
Values represent the average and SD of assays run in triplicate. ND = not
determined. *, p<0.05; **, p<0.01 relative to vehicle-treated control cells as
determined by one-way ANOVA with Dunnett’s post-hoc analysis. Assay-to-
assay variability suggests that changes of <15% in α-Tubulin are not
meaningful.
References and notes
1.
Crowley, P. J.; Lamberth, C.; Müller, U.; Wendeborn, S.;
Nebel, K.; Williams, J.; Sageot, O.-A.; Carter, N.; Mathie, T.;
Kempf, H.-J.; Godwin, J.; Schneiter, P.; Dobler, M. R. Synthesis and
fungicidal activity of tubulin polymerisation promoters. Part 1:
pyrido[2,3-b]pyrazines. Pest. Manag. Sci. 2010, 66, 178-185.

2.
Lamberth, C.; Trah, S.; Wendeborn, S.; Dumeunier, R.;
9.
Cornec, A. S.; James, M. J.; Kovalevich, J.; Trojanowski,
Courbot, M.; Godwin, J.; Schneiter, P. Synthesis and fungicidal
activity of tubulin polymerisation promoters. Part 2: Pyridazines.
Bioorg. Med. Chem. 2012, 20, 2803-2810.
J. Q.; Lee, V. M.; Smith, A. B., III; Ballatore, C.; Brunden, K. R.
Pharmacokinetic, pharmacodynamic and metabolic characterization
of a brain retentive microtubule (MT)-stabilizing triazolopyrimidine.
Bioorg. Med. Chem. Lett. 2015, 25, 4980-2.
3.
Lamberth, C.; Dumeunier, R.; Trah, S.; Wendeborn, S.;
Godwin, J.; Schneiter, P.; Corran, A. Synthesis and fungicidal
activity of tubulin polymerisation promoters. Part 3: imidazoles.
Bioorg. Med. Chem. 2013, 21, 127-134.
10.
M.; Smith, A. B., III. Non-Naturally Occurring Small Molecule
Microtubule-Stabilizing Agents: Potential Tactic for CNS-
Directed Therapies. ACS Chem. Neurosci. 2017, 8, 5-7.
11. Sáez-Calvo, G.; Sharma, A.; Balaguer, F. d. A.; Barasoain,
I.; Rodríguez-Salarichs, J.; Olieric, N.; Muñoz-Hernández, H.;
Berbís, M. Á.; Wendeborn, S.; Peñalva, M. A.; Matesanz, R.;
Canales, Á.; Prota, A. E.; Jímenez-Barbero, J.; Andreu, J. M.;
Lamberth, C.; Steinmetz, M. O.; Díaz, J. F. Triazolopyrimidines Are
Microtubule-Stabilizing Agents that Bind the Vinca Inhibitor Site of
Tubulin. Cell Chem. Biol. 2017, 24, 737-750 e6.
Ballatore, C.; Brunden, K. R.; Trojanowski, J. Q.; Lee, V.
A
4.
Zhang, N.; Ayral-Kaloustian, S.; Nguyen, T.; Hernandez,
R.; Beyer, C. 2-cyanoaminopyrimidines as a class of antitumor
agents that promote tubulin polymerization. Bioorg. Med. Chem.
Lett. 2007, 17, 3003-3005.
5.
Zhang, N.; Ayral-Kaloustian, S.; Nguyen, T.; Hernandez,
R.; Lucas, J.; Discafani, C.; Beyer, C. Synthesis and SAR of 6-
chloro-4-fluoroalkylamino-2-heteroaryl-5-
(substituted)phenylpyrimidines as anti-cancer agents. Bioorg. Med.
Chem. 2009, 17, 111-118.
12.
Beyer, C. F.; Zhang, N.; Hernandez, R.; Vitale, D.;
6.
Zhang, N.; Ayral-Kaloustian, S.; Nguyen, T.; Afragola, J.;
Nguyen, T.; Ayral-Kaloustian, S.; Gibbons, J. J. The microtubule-
active antitumor compound TTI-237 has both paclitaxel-like and
vincristine-like properties. Cancer Chemother. Pharmacol. 2009, 64,
681-689.
Hernandez, R.; Lucas, J.; Gibbons, J.; Beyer, C. Synthesis and SAR
of [1,2,4]triazolo[1,5-a]pyrimidines, a class of anticancer agents with
a unique mechanism of tubulin inhibition. J. Med. Chem. 2007, 50,
319-327.
13.
Fukushima, N.; Furuta, D.; Hidaka, Y.; Moriyama, R.;
7.
Lou, K.; Yao, Y.; Hoye, A. T.; James, M. J.; Cornec, A. S.;
Tsujiuchi, T. Post-translational modifications of tubulin in the
Hyde, E.; Gay, B.; Lee, V. M.; Trojanowski, J. Q.; Smith, A. B., III;
Brunden, K. R.; Ballatore, C. Brain-penetrant, orally bioavailable
microtubule-stabilizing small molecules are potential candidate
therapeutics for Alzheimer's disease and related tauopathies. J. Med.
Chem. 2014, 57, 6116-6127.
nervous system. J. Neurochem. 2009, 109, 683-693.
14.
Robinette, D.; Neamati, N.; Tomer, K. B.; Borchers, C. H.
Photoaffinity labeling combined with mass spectrometric approaches
as a tool for structural proteomics. Expert Rev. Proteomics 2006, 3,
399-408.
8.
Kovalevich, J.; Cornec, A. S.; Yao, Y.; James, M.; Crowe,
A.; Lee, V. M.; Trojanowski, J. Q.; Smith, A. B., III; Ballatore, C.;
Brunden, K. R. Characterization of brain-penetrant pyrimidine-
containing molecules with differential microtubule-stabilizing
activities developed as potential therapeutic agents for Alzheimer's
disease and related tauopathies. J. Pharmacol. Exp. Ther. 2016, 357,
432-450.

Highlights
1) Microtubule (MT)-active triazolopyrimidines
can preserve or disrupt MT integrity.
2) A series of photoactivatable derivatives was
designed, synthesized and evaluated.
3) Selected photoactivatable candidate probes
have been identified.
4) Such probes may be used in photoaffinity
labeling studies.