Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.09.007

Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.

Full text of DOI:10.1016/j.bmc.2016.09.007

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Scaffold-hopping from xanthines to tricyclic guanines: A case study
of dipeptidyl peptidase 4 (DPP4) inhibitors
a
a
a
a
Dmitri A. Pissarnitski a, , Zhiqiang Zhao , David Cole , Wen-Lian Wu , Martin Domalski ,
John W. Clader a, Giovanna Scapin b, Johannes Voigt b,y, Aileen Soriano c, Theresa Kelly c,
Mary Ann Powles d, Zuliang Yao d, Duane A. Burnett a,à
⇑
a Department of Medicinal Chemistry, Merck Research Laboratories, 2000 Galloping Hill Rd., Kenilworth, NJ 07033, United States
b Department of Structural Chemistry, Merck Research Laboratories, 2000 Galloping Hill Rd., Kenilworth, NJ 07033, United States
c In Vitro Pharmacology, Merck Research Laboratories, 2000 Galloping Hill Rd., Kenilworth, NJ 07033, United States
d In Vivo Pharmacology, Merck Research Laboratories, 2000 Galloping Hill Rd., Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective
bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray
crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding
mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship
between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine
replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhi-
bitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose
tolerance test.
Received 29 July 2016
Revised 31 August 2016
Accepted 2 September 2016
Available online xxxx
Keywords:
Bioisosteres
Tricyclic guanine
DPP4 inhibitor
FAP inhibitor
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
and costly high-throughput screening campaign. While in silico
approaches for scaffold-hopping have flourished in the past
Practitioners of medicinal chemistry define scaffold as the cen-
tral, invariable core of a chemical series, which bears one or more
points for the attachment of variable substituents. The term ‘scaf-
fold-hopping’ was coined by Schneider in 1999,1 and refers to
identification of a novel chemical series by re-engineering of the
central core of a known active series. The motivation behind scaf-
fold-hopping stems from the role the scaffold plays in defining the
overall properties of the molecular series. The scaffold does not
merely hold its substituents in the correct positions of space,
but also usually provides binding interactions with the biological
target. Alteration of the scaffold may have an effect on the affinity
of the series to the target of interest, selectivity versus anti-tar-
gets, physico-chemical properties (e.g., chemical stability, solubil-
ity, lipophilicity), and cellular permeation. In the pharmaceutical
industry, scaffold-hopping can provide a clinical candidate and
generate novel intellectual property as an alternative to a lengthy
decade2,3 resulting in the development of commercial soft-
ware,4–8 human creativity remains essential for design of previ-
ously unsynthesized structures. Among the approaches fruitfully
exercised by chemists on the intuitive level are heterocyclic
replacements, ring opening and closure, peptidomimetics, and
shape-based designs.9
Bioisosteric replacements of the xanthine scaffold I (Fig. 1A) are
interesting because of the wide biological activity of this substruc-
ture; search of CAS database reveals >19,000 molecules reported in
biological studies. A significant body of research on tricyclic ana-
logs of nucleobases has been accumulated since 70s starting with
Nelson Leonard’s etheno-bridged and lin-benzo-adenosines,10,11
and these designs continue to inspire modern-day medicinal
chemists.12,13
In the present work we discovered that tricyclic guanines II and
III can serve as isosteric replacements of xanthine scaffold I.
Scaffold III with alkyl substitution at N5 atom has been used pre-
viously in medicinal chemistry programs in the fields of PDE inhi-
bitors14–17 and P2X7 antagonists,18,19 whereas scaffold II with a
substitution at N6 has not been hitherto synthesized. Molecules
based on III have favorable drug-like properties, such as solubility
and pharmacokinetics. Solubility is governed by the basic nitrogen
⇑
Corresponding author.
y
Present address: Structural Chemistry, Gilead Sciences Inc., Foster City, CA 94404,
United States.
à
Present address: Forum Pharmaceuticals, 225 Second Avenue, Waltham, MA
02451, United States.
http://dx.doi.org/10.1016/j.bmc.2016.09.007
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Pissarnitski, D. A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.007

2
D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
A
O
R2
N
R2
3
O
4
R2
N
O
N
R1
N
5
R1
O
4
3
5
N
N
N
N
R3
R3
R3
2
R1
6
2
N
6
N
N
1
N
N
N
1
(CH2)n
(CH2)n
II
I
III
Xanthine
n=1: IMIDAZO[2,1-B]PURIN-4(5H)-ONE
n=2: PYRIMIDO[2,1-B]PURIN-4(3H)-ONE
B.
S1
S1
S1'
NC
S1'
S2'
Figure 2. Overlay of molecular models for scaffolds II and III with R1 and R2
substituents corresponding to linagliptin.
O
N
O
NH2
N
NH2
N
N
N
N
N
N
O
N
O
N
we describe bioisosteres of xanthines prepared on the basis of II
and III, and prove that SAR and binding mode of DPP4 inhibition
follows that of linagliptin series, rather than that of non-xanthine
inhibitors shown in Figure 1B.
S2
S2
1
2
Linagliptin
Alogliptin
S1
S1
N
F
S1
2. Results and discussion
2.1. Chemistry
F
F
NC
NC
N
O
NH2
O
NH2
NH
O
The known dichloropurine 628 was selectively mono-hydro-
lyzed in the basic conditions, yielding purinone 7 (Scheme 1),
which was then alkylated at the nitrogen with 4-methoxybenzyl
chloride using potassium carbonate as the base and potassium
iodide as the catalyst. Next, chlorine in 8 was displaced with
3-aminopropanol in the presence of Hunig’s base. This reaction
was complete in 30 min using a microwave reactor at 150 °C.
NMP was the preferred solvent for this reaction, and use of DMF
partially led to the incorporation of dimethylamino group into
the product, as a consequence of solvent decomposition with
release of dimethylamine. The primary alcohol in 9 was converted
to a leaving group using methanesulfonyl chloride, and formation
of cyclic guanine 10 was accomplished on heating of the interme-
diate mesylate in dioxane in the presence of Hünig’s base. Bromi-
nation of 10 at C2 position was smoothly accomplished using
DCM as the solvent and NBS as the brominating agent, and there
was no appreciable incorporation of bromine into the PMB group
under these conditions. The SEM group in 11 was more acid-labile
than PMB, allowing for a selective cleavage using TFA in DCM. The
two nitrogen atoms in the imidazole moiety of resulting 12 had
differential nucleophilicities, and the N3 was alkylated preferen-
tially, leading to intermediate 13.
Next, the aminopiperidine was installed at the C2 position of
the right hand side cyclic guanine by nucleophilic displacement
of the bromine in 13. Cleavage of the PMB group in 15 was accom-
plished by concentrated TFA on heating, generating 16 which was
used as a common intermediate for synthesis of 19 and 20.
Alkylation of 16 predominantly led to 17 (scaffold II), while small
amounts of regioisomeric 18 (scaffold III) were also isolated. Final
compounds 19 were obtained following the cleavage of phthalim-
ide protective group from molecules 17.
N
OH
S2-extensive
F3C
S2
N
OH
S2
N
S2
N
4
5
3
Saxagliptin
Sitagliptin
Vildagliptin
Figure 1. (A) Cyclic guanine scaffolds II and III are proposed as bioisosteric
replacement of xanthines I. (B) Structure and binding mode of xanthine-based DPP4
inhibitor linagliptin and other inhibitors approved in the US for treatment of type 2
diabetes.
(N6), which allows formulation as an HCl salt and the aliphatic
bridge connecting N6 to the bicyclic system enhances chemical
and metabolic stability with respect to N6-desamination, while
increasing fraction of sp3-hybridized carbons (Fsp3).20,21 Despite
the previous applications of III,22,23 it has not been noted whether
or not its SAR matches that of I,24 which would indicate a bioisos-
teric relationship between these two scaffolds. Crystal structures of
enzyme-bound III have not been reported, and so a rigorous con-
clusion about the equivalency of binding modes for I and III to
the biological targets could not be drawn. To bridge this gap, we
report here SAR and X-ray binding modes of II and III in the context
of DPP4 inhibition,25 a clinically validated method of glycemic con-
trol for type 2 diabetes mellitus, and compare them to the data for
the recently approved xanthine-based DPP4 inhibitor linagliptin.
To date, five DPP4 inhibitors have been approved for the treat-
ment of this disease in the USA (Fig. 1B), and their binding modes
are known.26 While all inhibitors occupy S1 and S2 pockets of
DPP4, the optimal groups binding at these positions vary (aromatic
for sitagliptin and alogliptin, heteroaliphatic for vildagliptin and
saxagliptin, and alkyne for linagliptin). Vildagliptin and
saxagliptin27 engage the nitrile in the formation of a covalent com-
plex at S1. Alogliptin and linagliptin, in addition to S1 and S2 sites,
also bind at the S10 site, and sitagliptin at the extension of S2-
pocket. A distinctive feature of xanthine-based linagliptin is bind-
ing at S20 site, which accommodates the characteristic
methylquinazoline group of this drug.
The route shown in Scheme 1 afforded a few representatives of
scaffold III: compound 20b was obtained from 15, compounds 20c,
d were prepared from 18c,d, and compound 20f from 16. A more
general approach to scaffold III is shown in Scheme 2. A sterically
hindered methylquinazoline analog 23a was prepared in a two-
step process including alkylation of 2129 with bromoacetonitrile,
followed by condensation of the nitrile with 2-aminoacetophenone
under acidic catalysis. Molecules 23n–p were obtained by alkyla-
tion of chloropurinone 21 with benzyl bromides. To construct
Comparison of molecular models of structures of type II and III
shows that R1 substituent for both of them occupies the same
region of space (Fig. 2), suggesting potential bioisosterism. Herein
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D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
O
Cl
O
N
O
N
O
N
O
N
O
N
H
N
PMB
Cl
PMB
HN
PMB
N
PMB
N
PMB
N
N
N
N
N
N
N
N
N
N
N
N
N
a
b
N
O
c
d
e
f
g
N
HN
N
N
N
N
Br
Br
N
N
8
Cl
N
6
Cl
SEM
SEM
SEM
SEM
SEM
SEM
7
OH
9
10
11
12
O
O
O
O
N
O
O
O
O
N
N
N
14,
N
i
j
h
O
O
O
O
PMB
N
PMB
R1
N
N
N
N
N
N
N
N
N
+
N
N
HN
N
N
N
N
(Major)
N
Br
N
R1
N
16
N
18c,d
N
N
N
N
N
N
N
13
15
17a-e
(Minor)
k
k
(for 20f)
(for 20b)
O
O
N
R1 groups
Protective groups:
O
NH2
NH2
R1
N
k
N
N
N
N
H3C
SEM
N
N
SiMe3
N
O
R1
MeO
N
H
f
N
N
N
N
PMB
20b-d, f
Scaffold III
19a-e
a
d
e
c
b
OMe
Scaffold II
Scheme 1. Initial route to scaffolds II and III. Reagents and conditions: (a) NaOH (0.2 M), 95 °C, 76%; (b) K2CO3, PMBCl, KI, DMF, 56%; (c) NH2(CH2)3OH, i-Pr2NEt, NMP,
microwave, 150 °C, 30 min, 85%; (d) (i) MsCl, Et3N, DCM; (ii) Dioxane, i-Pr2NEt, 80 °C, 52% over two steps; (e) NBS, DCM, 99%; (f) TFA, DCM, 79%; (g) 1-bromobut-2-yne,
i-Pr2NEt, DMF, 99%; (h) 3-(R)-piperidinyl phthalimide (14), i-Pr2NEt, NMP, 100 °C, 18 h, 76%; (i) TFA, 60 °C, 70%; (j) NaH, R1Cl or R1Br, DMF, followed by chromatography; (k)
H2N–NH2, DCM, MeOH, 34–62% (two steps) for 19, 0–15% (two steps) for 20c–d, 78% for 20b, 67% for 20f.
O
N
O
N
O
N
O
N
R2
N
NHBoc
O
N
O
N
O
N
PMB
N
PMB
N
R2
PMB
N
PMB
N
H
N
R1
N
R1
R1
R1
N
g
R1
N
R1
N
N
N
f
c
d
a
e
N
N
N
HN
N
N
N
N
Br
N
N
N
N
N
N
HN
Cl
Cl
22, R1=CH2CN
OH
21
n
n
n
n
n
b
32a
, n=2
33a-p, n=1
30a, n=2
31a-p
26a, n=2
28a
24a
25n-p
, n=2
29n-p, n=1
, n=2
, n=1
23a
23n-p
27n-p
, n=1
, n=1
R2 groups
R1 groups
NC
O
N
R2
NH2
R1
F F
H3C
N
CN
n
CN
N
N
h
CH3
b
N
NC
N
N
N
a-m
o
p
a, n-p
c
d
e
f
g
F
Scaffold III
n
Protective groups:
O
F
F
F
20a
, n=2
34a-p, n=1
NC
Boc
O
S
O
h
PMB
i
j
k
l
m
OMe
Scheme 2. Optimized route to scaffold III. Reagents and conditions: (a) R1Br, NaH, 55–72%; (b) 2-aminoacetophenone, HCl, dioxane, 58%; (c) NH2–CH2–(CH2)nOH, i-Pr2NEt,
NMP, microwave, 150 °C, 30 min, 49–68%; (d) MsCl, Et3N, DCM, 42–63%; (e) TFA, 70 °C, 53–61%; (f) (i) NBS, DMF, 0 °C; (ii) R2Br, i-Pr2NEt, DMF, 35–83%; (g) (R)-tert-butyl-
piperidin-3-yl-carbamate, i-Pr2NEt, NMP, 100 °C, 58–64%; (h) TFA/DCM, 83–99%.
tricyclic guanidines 26/27, the chlorine in 23 was displaced with
aminoalcohol, and the hydroxyl group of the resulting 24/25 was
converted to mesylate. This resulted in a spontaneous ring closure
in the same pot with displacement of mesylate. After removal of
PMB group in 26/27 by treatment with TFA, the resulting tricyclics
28/29 were brominated at C2 with NBS. The R2 group was then
installed at the N3 position of the cyclic guanine by alkylation in
the basic conditions, and aminopiperidine group was introduced
into the molecule by nucleophilic displacement of bromine. In
the final step, removal of Boc from the aminopiperidine moiety
was accomplished by TFA in DCM to afford 20a/34a–p.
2.2. X-ray analysis and SAR
X-ray analysis indicates that our inhibitors mimic the binding
mode of linagliptin and occupy the S1, S2, S10 and S20 subsites of
the DPP4 binding site. As illustrated by 34a (Fig. 3), the cyclic gua-
nine core is stacked against the side chain of Tyr547 in the S10 site,
the aminopiperidine nitrogen forms salt bridges with the con-
served acidic residues (Glu205 and Glu206) and is hydrogen
Figure 3. Binding of 34a to DPP4. Hydrogen bonds are shown as dotted lines. The
DPP4 subsites (S1, S2, S10 and S20) are labeled.
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4
D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
bonded to Tyr662 and one ordered water molecule. The butynyl
group extends into the S1 subsite, formed by the side chains of
Tyr631, Val656, Tyr662, Tyr666, Asn710, and the catalytic Ser630.
As was predicted by molecular modeling of unbound ligands,
X-ray analysis of enzyme-bound inhibitors indicated that S20 site
of DPP4 can be reached from both N5 of scaffold III and N6 of scaf-
fold II. However, there are important differences in the binding of
the scaffolds (Fig. 4).
weaker inhibition potency of scaffold II compared to scaffold III
(Table 1).
SAR of R2 group revealed that potency was highly dependent on
the size. For small substituents (methyl, cyanomethyl), only a mod-
est ꢀ0.5
lM potency was observed (34b,c).
A significant gain of potency was achieved for a larger cyclo-
propylmethyl in 34d, however further increase in size (34e, 34f)
led to a reduction in potency relative to 34d. On the other hand, flat-
For 34a (scaffold III), the phenyl of the methylquinazoline is
tening of the structure and addition of p-character as in alkene 34g
parallel to and forms
p
–
p
interactions with the side chain of
and ketone 34h resulted in compounds with single-digit nanomolar
potency, and 2-butynyl was the optimal substituent (34a).
Trp629 in the S20 subsite, and the methylene connecting the tri-
cyclic system to the methylquinazoline is within hydrophobic
interactions with the side chain of Tyr547. Corresponding analog
19a of scaffold II has crystallized in two conformations, shown in
Figure 4A as magenta and blue.
In contrast to alogliptin and sitagliptin, replacement of alkyne
at S1 site with a phenyl ring resulted in a moderate loss of potency
for 34i. Nevertheless, the potency could be slightly improved by
the introduction of a single fluorine atom into the phenyl group
(34j), whereas bis-fluorination (34k) or a cyano group (34l)
decreased the potency, and fluorinated thiophenyl group (34m)
had potency comparable to unsubstituted phenyl (34i).
The methylquinazoline is positioned farther away from the side
chain of Trp629 and it is no longer parallel to the indole of Trp629
(Fig. 4A). The methylene linker connecting the tricyclic system
with methylquinazoline in 19a has been moved by about 2 Å with
respect to the position observed in 34a, and the hydrophobic inter-
action with chain of Tyr547 is no longer possible (Fig. 4B). These
subtle changes in the binding of R1 group are responsible for the
Exploration of SAR at the R1 position of scaffold III indicated
that 20b with methoxy-substituted phenyl ring lost ꢀ30-fold of
potency compared to methylquinazoline 20a (Table 1). This result
could be due to the change of electron density on the aromatic
Figure 4. Overlay of 34a (yellow) and 19a (crystallized in two conformations, magenta and blue) in the DPP4 binding site. (A) Interactions with TRP-629. (B) Interactions with
TYR-547. Only one conformer of 19a (magenta) shown for clarity.
Table 1
In vitro data for representative inhibitors of scaffolds II and III
Scaffold
n
#
R1
R2
DPP4 IC50 (nM)
FAP IC50 (nM)
Ratio IC50, FAP/DPP4
II
II
II
II
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
19a
19b
19c
19d
19e
20a
20b
20c
20d
20f
34a
34b
34c
34d
34e
34f
34g
34h
34i
34j
34k
34l
34m
34n
34o
34p
(4-Methyl-2-quinazolinyl)methyl
4-MeO-benzyl
1-Naphthalenylmethyl
3-Phenylpropyl
2-Oxo-2-phenylethyl
(4-Methyl-2-quinazolinyl)methyl
4-MeO-benzyl
1-Naphthalenylmethyl
3-Phenylpropyl
H
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
(4-Methyl-2-quinazolinyl)methyl
2-Cyanobenzyl
2-Butynyl
2-Butynyl
2-Butynyl
2-Butynyl
2-Butynyl
2-Butynyl
2-Butynyl
2-Butynyl
2-Butynyl
2-Butynyl
2-Butynyl
Methyl
Cyanomethyl
Cyclopropylmethyl
2-Di-F-cyclopropylmethyl
Cyclobutylmethyl
3-Methyl-2-butenyl
2-Oxo-2-cyclopropylethyl
Benzyl
4376
790
483
>10,000
421
1.28
38.6
44.3
>10,000
6113
0.42
539
II
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
II
268
209
>50,000
22,406
1295
509
367
874
538
8.15
14.1
29.2
1.61
4.41
32.0
19.3
43.5
82.2
28.7
9.31
11.2
10.0
3122
383
14,740
3052
1045
105
755
469
107
4640
24.3
145
3-F-benzyl
4620
239
2,5-Di-F-phenyl
2-Cyanobenzyl
3-F-2-thiophenylmethyl
2-Butynyl
2-Butynyl
2-Butynyl
9920
228
15,288
11,446
>100,000
78,508
88,335
186
399
>1074
7010
8834
III
III
III
III
3-Cyanobenzyl
4-Cyanobenzyl
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D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 5. Binding of 34a (green), 34n (magenta) and 34p (orange) in the S20 subsite of DPP4 binding site (X-ray analysis). (A) The waters associated with each complex are in
the same color. Hydrogen bonds between 34a and the water molecules are shown as green dotted lines. (B) Surface representation of the binding site. Lys554 and Trp629 are
labeled.
OGTT Blood Glucose
3. Conclusion
Veh + Water
Veh + Dextrose, 5.0 g/kg
34a, 0.3 mg/kg
34a, 1.0 mg/kg
34a, 3.0 mg/kg
34a, 10.0 mg/kg
X-ray analysis of enzyme-bound representatives of scaffolds II
and III revealed similarities with the binding mode of linagliptin,
confirming the hypothesis of isosteric relationship of these scaf-
folds to xanthine. For scaffold III, the SAR of the R2 substituent with
respect to DPP4 inhibition matched that of linagliptin. Although
DPP4 inhibition potency similar to linagliptin was achieved in scaf-
fold III (34a), representatives of scaffold II were significantly less
active, as explained by suboptimal alignment of this scaffold with
the enzyme, shown in Figure 4. A limited exploration of SAR of R1
substituent (bound within S20 pocket) revealed that this position is
responsible for FAP inhibition activity. FAP inhibition was
mitigated by introduction of cyanobenzyl groups (34n–p), while
preserving DPP4 activity at 9–11 nM level.
400
300
200
100
-60
0
25
50
75
100
Time (min)
Figure 6. Effect of 34a on glucose plasma levels in oGTT mouse study. Fasted lean
C57BL/6 mice (n = 8 per group) were orally treated at t = ꢂ60 min with vehicle
(control group) or drug, and then at t = 0 min with water (control group) or dextrose
(5 g/kg).
4. Experimental
substituent or the loss of the polar nitrogens present on the quina-
zoline. To re-coop the potency, we introduced cyano groups on the
phenyl ring, both reducing electron density on the aromatic ring
and adding polarity. The resulting compounds 34n–p demon-
strated a substantially improved potency (ꢀ10 nM). The cyano
groups replaced some of the ordered water molecules present in
the S20 binding site, thus becoming part of the extended hydro-
gen-bonding network (Fig. 5).
Although selectivities more than 10,000-fold were observed for
compounds of scaffold III with respect to other isozymes of DPP
(data not shown), similarly to linagliptin, they co-inhibited fibrob-
last activation protein (FAP).30 This finding is in line with the bioisos-
teric relationship between the xanthine core of linagliptin and our
tricyclic guanines. Whereas R2 substituent did not show a significant
influence on FAP selectivity, R1 group had a large impact on FAP, and
compounds 34n–p had a notable reduction in FAP activity.
4.1. Synthesis of compounds
Reagents and anhydrous solvents were purchased from Sigma–
Aldrich and used without further purification. All reactions were
carried out under nitrogen atmosphere, unless otherwise noted.
Flow-injection MS analyses were conducted on Waters ZQ-4000
single quadrupole mass detector. LCMS analyses were conducted
on an Agilent-1200 LCMS system equipped with a single quadru-
pole mass detector; column: Zobrax SB-C18, 3 ꢁ 50 mm, 1.8
lM
column, column temperature: 50 °C, flow 1.0 mL/min, gradient
5–95% of solvent B in 2.5 min followed by hold. Flash chromatog-
raphy was carried out on ISCO or Analogix purification system
using ISCO pre-packed SiO2 cartridges. Preparative reverse-phase
chromatography was carried out on Phenomenex Gemini C18 col-
umns, solvent A: water–0.1% TFA, solvent B: AcN–0.1% TFA, gradi-
In order to confirm in vivo activity of our bioisosteres, a few
analogs were studied in oral glucose tolerance tests (oGTT) in mice
(Fig. 6 and SI). Single oral administration of compounds from 0.3 to
10 mg/kg 60 min prior to glucose challenge reduced the ensuing
plasma glucose excursion in a dose-dependent manner compared
to the control group, which received no drug. Efficacy of our mole-
cules in this rodent oGTT assay was comparable with the data
reported in the literature31–34 for the marketed drugs shown in
Figure 1B. We took this result as a sign that the overall pharma-
cokinetic properties of molecules prepared on the basis of our
bioisosteric scaffolds are compatible with the oral dosing, and
therefore remain comparable with xanthine core.
ent 5–95% of solvent
B in 20 min. NMR spectroscopy was
conducted on Bruker Avance 400 NMR spectrometer. Chemical
shifts are reported in ppm using the residual solvent peak of the
indicated solvent as the standard, and coupling constants J in Hz.
All compounds were purified to meet or exceed 95% purity thresh-
old by HPLC prior to biological testing. Purity assessment of final
compounds was carried on an Agilent-1200 HPLC system using
Zobrax SB-C18, 3 ꢁ 50 mm, 1.8
lM column, solvent A: water–
0.1% TFA, solvent B: AcN–0.1% TFA, flow 1.0 mL/min, gradient
5–95% of solvent B in 6.5 min followed by hold, monitoring UV
absorbance at both 220 and 254 nm. Compounds were named
using CambridgeSoft ChemBioDraw v.13.0.2. software.
Please cite this article in press as: Pissarnitski, D. A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.007

6
D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.1. 2-Chloro-9-((2-(trimethylsilyl)ethoxy)methyl)-1H-purin-6
(9H)-one (7)
2H), 0.89–0.96 (m, 2H), 0.00 (s, 9H); LCMS (442.3, M+H), rt
2.07 min.
2,6-Dichloro-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-purine
628 (10.37 g, 32.48 mmol) was added to an aqueous sodium
hydroxide solution (2.78 g, 69.5 mmol of NaOH and 350 mL of
water). The solution was warmed to 95 °C with vigorous stirring
for 2 h. The hot solution was gravity-filtered through paper filter
to remove the red oil. The filtrate was cooled, and precipitated
white crystals of 7 (7.42 g, 76%) were isolated by filtration. 1H
NMR (CD3OD) d: 7.96 (s, 1H), 5.54 (s, 2H), 3.66 (t, J = 8.0 Hz, 2H),
0.94 (t, J = 8.0 Hz, 2H), 0.01 (s, 9H); LCMS (301.1, M+H), rt 1.99 min.
4.1.4. 2-Bromo-5-(4-methoxybenzyl)-5,7,8,9-tetrahydropyrimi-
do[2,1-b]purin-4(3H)-one (12)
Compound 10 (470 mg, 1.06 mmol) was dissolved in 11.0 mL of
CH2Cl2 and N-bromosuccinimide (212 mg, 1.19 mmol) was added.
The reaction mixture was stirred at room temperature for 4 h and
quenched by partitioning between 50 mL of saturated sodium thio-
sulfate, 120 mL of CH2Cl2, and 100 mL of sat. sodium bicarbonate
solution. The aqueous layer was extracted with CH2Cl2
(2 ꢁ 100 mL). The combined organic phases were dried over
sodium sulfate, filtered and concentrated to furnish 550 mg of bro-
mide 11 as a solid, LCMS (391.3, 393.3, M+H), rt 1.69 min, which
was used without purification.
4.1.2. 2-Chloro-1-(4-methoxybenzyl)-9-((2-(trimethylsilyl)
ethoxy)methyl)-1H-purin-6(9H)-one (8)
Compound 7 (6.15 g, 20.4 mmol) was dissolved in DMF (50 mL).
Anhydrous K2CO3 (5.95 g, 43.1 mmol) was added, followed by
addition of 4-methoxybenzyl chloride (5.6 mL, 40.3 mmol) and
potassium iodide (440 mg, 2.66 mmol). The mixture was stirred
at room temperature overnight followed by addition of 100 mL of
water and extraction with 1:1 ethyl acetate–hexanes solution
(6 ꢁ 150 mL). The combined organic phase was dried over Na2SO4
and concentrated. The compound were purified by chromatogra-
phy on SiO2 (gradient from 0% to 50% of ethyl acetate in hexanes
over 50 min, followed by hold) to furnish the title compound as
an oil (4.81 g, 56%). 1H NMR (400 MHz, CDCl3) d: 7.90 (s, 1H),
7.40 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 5.38–5.71 (m, 4H),
3.82 (s, 3H), 3.43–3.67 (m, 2H), 0.82–1.08 (m, 2H), 0.00 (s, 9H);
LCMS (421.2, M+H), rt 2.37 min.
A mixture of compound 11 (545.8 mg, 1.049 mmol), CH2Cl2
(5 mL) and TFA (10 mL) stirred at room temperature for 1 h. The
reaction was cooled to 0 °C and quenched with aqueous K2CO3
solution, prepared from 8.7 g of K2CO3 and 80 mL of water. The
solution was extracted with CH2Cl2 (5 ꢁ 80 mL). The organic
phases were combined, dried over Na2SO4, and purified by chro-
matography on SiO2 (gradient 0–10% of methanol in CH2Cl2) to fur-
nish 323 mg (79%) of 12, 1H NMR (400 MHz, CD3OD) d: 7.12 (d,
J = 8.2 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 5.19 (br s, 2H), 4.18 (t,
J = 5.9 Hz, 2H), 3.73 (s, 3H), 3.53 (t, J = 5.7 Hz, 2H), 2.16 (quin,
J = 5.7 Hz, 2H); LCMS (391.3, 393.3), rt 1.69 min.
4.1.5. (R)-2-(1-(3-(But-2-yn-1-yl)-5-(4-methoxybenzyl)-4-oxo-
3,4,5,7,8,9-hexahydropyrimido[2,1-b]purin-2-yl)piperidin-3-yl)
isoindoline-1,3-dione (15)
4.1.3. 5-(4-Methoxybenzyl)-1-((2-(trimethylsilyl)ethoxy)
methyl)-5,7,8,9-tetrahydropyrimido[2,1-b]purin-4(1H)-one (10)
Compound 8 (3.43 g, 8.15 mmol) was dissolved in 10.0 mL of
NMP. 3-Amino-1-propanol (2.0 mL, 26.0 mmol) was added to the
solution followed by diisopropylethylamine (1.5 mL, 8.64 mmol).
The reaction was heated to 150 °C for 30 min in a microwave reac-
tor followed by cooling. The reaction mixture was partitioned
between water (100 mL) and 1:1 ethyl acetate–hexanes mixture
(120 mL). The aqueous phase was extracted with 1:1 ethyl acet-
ate–hexanes mixture (2 ꢁ 120 mL). The organic layer was dried
over Na2SO4, filtered and concentrated. Thus obtained intermedi-
ate 9 was purified by chromatography on SiO2 (gradient from 0%
to 10% of methanol in CH2Cl2 over 50 min, to furnish 3.18 g, LCMS
(460.0, M+H), rt 2.02 min.
Compound 12 (217.5 mg, 0.557 mmol) was dissolved in 2.75 mL
of DMF. Diisopropylethylamine (106 lL, 0.613 mmol) was added
followed by 1-bromo-2-butyne (81.5 mg, 0.613 mmol). The reac-
tion was stirred at room temperature for 21 h. Additional diiso-
propylethylamine (27 lL, 0.16 mmol) and 1-bromo-2-butyne
(15 mg, 0.17 mmol) were added and stirring continued for 3 h.
The reaction was quenched with 10% K2CO3 solution (20 mL), and
40 mL of 1:1 ethyl acetate–hexanes mixture was used to extract
the aqueous phase. The organic phase was washed with 20 mL of
water. The combined aqueous phase was re-extracted with 1:1
ethyl acetate–hexanes mixture (3 ꢁ 40 mL). The combined organic
phases were dried over Na2SO4, filtered and concentrated to fur-
nish 247.2 mg of 13 (LCMS (442.0, M+H), rt 1.96 min) which was
used without purification.
Intermediate 9 (1.80 g, 3.92 mmol) was dissolved in 40 mL of
CH2Cl2 and cooled to 0 °C. Methanesulfonyl chloride (445 lL,
The bromide 13 (247.2 mg, 0.559 mmol) was dissolved in NMP
(3 mL), and treated with 3-(R)-piperidinyl phthalimide hydrochlo-
ride 14 (181.5 mg, 0.682 mmol) and diisopropylethylamine
5.88 mmol) followed by triethylamine (1.65 mL, 11.8 mmol) was
added. The solution was stirred at room temperature for 2.5 h.
The reaction was washed with saturated NaHCO3 solution
(150 mL) and the aqueous phase was extracted with CH2Cl2
(3 ꢁ 150 mL). The combined organic phases were dried over Na2-
SO4, filtered, and concentrated to furnish 3.26 g of intermediate
mesylate, which was used in the following step without any fur-
ther purification.
The mesylate (3.26 g) was dissolved in dioxane (15 mL) then
diisopropylethylamine (2.20 mL, 12.8 mmol) was added. The reac-
tion was warmed to 80 °C and stirred overnight. After cooling, the
reaction was quenched with 100 mL of 0.5 M potassium carbonate
solution and extracted with 100 mL of ethyl acetate twice. The
combined organic phases were dried over sodium sulfate, filtered
and concentrated. The product was purified by chromatography
on SiO2 (gradient from 0% to 100% of ethyl acetate in hexanes over
50 min, followed by hold) to furnish 10 as an oil (1.59 g, 52% from
9) 1H NMR (400 MHz, CDCl3) d: 7.51 (d, J = 8.2 Hz, 2H), 7.25 (br s,
1H), 6.80 (d, J = 8.6 Hz, 2H), 5.36 (s, 2H), 5.28 (br s, 2H), 4.19 (br
s, 2H), 3.77 (s, 3H), 3.59 (br s, 2H), 3.50–3.56 (m, 2H), 1.67 (br s,
(301 lL, 1.73 mmol). The reaction mixture was heated to 100 °C
overnight, cooled to room temperature, quenched with saturated
sodium bicarbonate solution (50 mL) and extracted with 1:1 mix-
ture of EtOAc and hexanes. The combined organic phases were
dried over sodium sulfate, filtered and concentrated. The product
was purified by chromatography on SiO2 (gradient 0–10% of
methanol in CH2Cl2 over 50 min, to furnish 15 (250.4 mg, 76% from
12). 1H NMR (400 MHz, CDCl3) d: 7.81–7.86 (m, 2H), 7.72 (dd,
J = 5.3, 2.9 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H),
5.17 (s, 2H), 4.77–4.91 (m, 2H), 4.55 (tt, J = 11.7, 4.0 Hz, 1H), 3.92
(t, J = 5.5 Hz, 2H), 3.70–3.80 (m, 5H), 3.53–3.64 (m, 3H), 3.02 (td,
J = 12.0, 2.9 Hz, 1H), 2.48 (qd, J = 12.3, 4.9 Hz, 1H), 1.83–1.98 (m,
5H), 1.77 (s, 3H); LCMS (592.3, M+H), rt 2.15 min.
4.1.6. (R)-2-(1-(3-(But-2-yn-1-yl)-4-oxo-3,4,5,7,8,9-hexahydrop-
yrimido[2,1-b]purin-2-yl)piperidin-3-yl)isoindoline-1,3-dione
(16)
A mixture of compound 15 (185 mg, 0.313 mmol) and 2.0 mL of
TFA was heated in a sealed tube at 60 °C for 3.5 h. Reaction mixture
Please cite this article in press as: Pissarnitski, D. A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.007

D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
was concentrated on a rotovap and treated with a solution of 3.0 g
K2CO3 in 40 mL of water. The mixture was extracted with CH2Cl2
(3 ꢁ 40 mL), organic phase was dried over Na2SO4 and concen-
trated. The product was purified by chromatography on SiO2 using
a gradient of 0–10% of 7 N ammonia/MeOH in CH2Cl2 to furnish
104 mg (70%) of 16, 1H NMR (400 MHz, CD3Cl) d: 7.80 (m, 2H),
7.68 (m, 2H), 4.98 (m, 2H), 4.58 (m, 1H), 4.02, (m, 2H), 3.73 (m,
2H), 3.62 (m, 3H), 3.00 (m, 1H), 2.44 (m, 1H), 2.09–2.01 (m, 2H),
1.96–1.82 (m, 3H), 1.74 (t, J = 2.3 Hz, 3H); LCMS (472.3, M+H), rt
2.06 min.
J = 6.0 Hz, 2H), 2.12 (m, 3H), 2.01 (t, J = 6.0 Hz, 2H), 1.85–1.22 (m,
2H), 1.80 (t, J = 1.9 Hz, 3H), 1.75 (m, 1H); LCMS (460.3 M+H) rt
1.63 min.
4.1.11. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-6-(2-
oxo-2-phenylethyl)-6,7,8,9-tetrahydropyrimido[2,1-b]purin-4
(3H)-one (19e)
Obtained from 16 according to the procedure for synthesis of
19a in 37% yield using 2-bromo-1-phenylethanone as the alkylat-
ing agent. 1H NMR (400 MHz, CD3OD) d: 8.05 (d, J = 7.0 Hz, 2H),
7.68 (m, 1H), 7.55 (m, 2H), 5.29 (s, 2H), 4.84 (s, 2H), 4.33 (t,
J = 5.8 Hz, 2H), 3.91 (m, 1H), 3.64 (t, J = 5.8 Hz, 2H), 3.58 (m, 2H),
3.4–3.2 (m, 3H), 2.33 (m, 2H), 2.17 (m, 1H), 1.94 (m, 1H), 1.79 (t,
J = 1.9 Hz, 3H), 1.74 (m, 1H), 1.36 (m, 1H); LCMS (460.3 M+H), rt
1.64 min.
4.1.7. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-6-((4-m-
ethylquinazolin-2-yl)methyl)-6,7,8,9-tetrahydropyrimido[2,1-
b]purin-4(3H)-one (19a)
A solution of compound 16 (64.8 mg, 0.137 mmol) in 2.0 mL of
DMF was treated with sodium hydride (60% dispersion in mineral
oil, 11 mg, 0.275 mmol) and the mixture was warmed at 55 °C over
3 h. After cooling to room temperature, 2-(chloromethyl)-4-
methylquinazoline30 (52.8 mg, 0.275 mmol) was added and the
mixture was stirred overnight. To the reaction was added of
methanol (0.1 mL) and hydrazine monohydrate (137 mg,
2.7 mmol), and stirring was continued for 8 h. The reaction mixture
was filtered concentrated, and purified by reverse phase HPLC.
After concentration of the fractions containing the product, the
material was desalted by workup using CH2Cl2 and sat. sodium
bicarbonate to furnish 28.6 mg (42%) of compound 19a. 1H NMR
(400 MHz, CD3OD) d: 8.24 (d, J = 8.3 Hz, 1H), 7.92–7.89 (m, 2H),
7.68 (m, 1H), 5.49 (s, 2H), 5.20 (m, 2H), 4.29 (t, J = 6.0 Hz, 2H),
3.76 (t, J = 3.6 Hz, 2H), 3.70 (m, 1H), 3.68 (m, 1H), 3.22 (m, 1H),
3.19 (m, 1H), 3.16 (m, 1H), 3.13 (m, 1H), 3.11 (m, 1H), 3.08 (m,
1H), 3.94 (s, 3H), 2.34 (m, 2H), 7.17 (t, J = 2.2, 3H); LCMS (498.3,
M+H), rt 1.43 min.
4.1.12. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-(4-
methoxybenzyl)-5,7,8,9-tetrahydropyrimido[2,1-b]purin-4(3H)-
one (20b)
Phthalimide 15 (89.3 mg, 0.151 mmol) was dissolved in CH2Cl2
(1.0 mL) and MeOH (1.0 mL). Hydrazine (30
added and the reaction mixture was stirred at room temperature
for 3.5 h. An additional portion of hydrazine (30 L, 0.603 mmol)
lL, 0.603 mmol) was
l
was added and the reaction was stirred at room temperature over-
night. The reaction was concentrated, filtered and purified by
reverse phase HPLC to furnish 77 mg (78%) of 20b. Prior to NMR
analysis, the product was desalted by workup using CH2Cl2 and
sat. sodium bicarbonate. 1H NMR (400 MHz, CDCl3) d: 7.37 (d,
J = 8.6 Hz, 2H), 6.72 (d, J = 8.6 Hz, 2H), 5.11 (s, 2H), 4.70–4.82 (m,
2H), 3.86 (t, J = 5.7 Hz, 2H), 3.69 (s, 3H), 3.50 (t, J = 5.1 Hz, 3H),
3.34–3.43 (m, 1H), 2.90–3.01 (m, 2H), 2.75 (dd, J = 11.7, 8.6 Hz,
1H), 1.78–1.94 (m, 5H), 1.74 (br s, 3H), 1.14–1.32 (m, 3H); LCMS
(462.3 M+H), rt 1.72 min.
4.1.8. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-6-(4-me-
thoxybenzyl)-6,7,8,9-tetrahydropyrimido[2,1-b]purin-4(3H)-
one (19b)
4.1.13. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-
(naphthalen-1-ylmethyl)-5,7,8,9-tetrahydropyrimido[2,1-b]
purin-4(3H)-one (20c)
Isolated as a minor regioisomer (15% yield) during the prepara-
tion of 19c. 1H NMR (400 MHz, CD3Cl) d: 8.12 (m, 1H), 7.80 (m,
1H), 7.69 (m, 1H), 7.51 (m, 2H), 7.42 (m, 1H), 7.31 (m, 1H), 7.10
(m, 1H), 5.73 (s, 2H), 4.91 (m, 1H), 4.85 (m, 1H), 4.1 (m, 2H), 3.60
(m, 1H), 3.50–3.30 (m, 2H), 3.3–3.1 (m, 2H), 2.0–1.8 (m, 4H), 1.71
(s, 3H), 1.70–1.45 (m, 2H); LCMS (482.3 M+H), rt 1.78 min.
Obtained from 16 according to the procedure for synthesis of
19a in 62% yield using 4-methoxybenzyl chloride as the alkylating
agent. 1H NMR (400 MHz, CD3OD) d: 7.29 (d, J = 8.5 Hz, 2H), 6.85 (d,
J = 8.5 Hz, 2H), 4.98 (m, 2H), 4.82 (s, 2H), 4.11 (t, J = 5.8 Hz, 2H),
3.75 (s, 3H), 3.63 (m, 1H), 3.54 (m, 1H), 3.40 (t, J = 5.8 Hz, 2H),
3.10–2.99 (m, 2H), 2.87 (m, 1H), 2.08 (m, 2H), 1.99 (m, 1H), 1.86
(m, 1H), 1.78 (t, J = 1.9 Hz, 3H), 1.75 (m, 1H), 1.39 (m, 1H); LCMS
(462.3 M+H), rt 1.68 min.
4.1.14. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-6-(3-
phenylpropyl)-6,7,8,9-tetrahydropyrimido[2,1-b]purin-4(3H)-
one (20d)
Isolated as a minor regioisomer (7% yield) during preparation of
19d. 1H NMR (600 MHz, CD3OD) d: 7.21 (m, 4H), 7.10 (m, 1H), 4.95
(s, 2H), 4.21 (t, J = 5.8 Hz, 2H), 1.95 (m, 1H), 1.80 (t, J = 1.9 Hz, 3H),
1.75 (m, 1H), 1.3 (m, 3H); LCMS (460.3 M+H), rt 1.74 min.
4.1.9. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-6-
(naphthalen-1-ylmethyl)-6,7,8,9-tetrahydropyrimido[2,1-b]
purin-4(3H)-one (19c)
Obtained from 16 according to the procedure for synthesis of
19a in 48% yield using 1-bromomethylnaphthalene as the alkylat-
ing agent. 1H NMR (400 MHz, CD3Cl) d: 8.1 (m, 1H), 7.85 (m, 1H),
7.78 (m, 1H), 7.47 (m, 2H), 7.42–7.35, (m, 2H), 5.40 (s, 2H), 5.10
(m, 1H), 5.0 (m, 1H), 4.05 (m, 2H), 3.52 (m, 1H), 3.38 (m, 1H),
3.18 (m, 2H), 3.28–3.10 (m, 2H), 2.91 (m, 1H), 1.97 (m, 2H),
1.92–1.82 (m, 2H), 1.80 (s, 3H), 1.68 (m, 1H), 1.45 (m, 1H); LCMS
(482.3 M+H), rt 1.78 min.
4.1.15. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-6,7,8,9-
tetrahydropyrimido[2,1-b]purin-4(3H)-one (20f)
Dissolved compound 16 (50 mg, 0.106 mmol) in a mixture of
1.0 mL of methanol and 1.0 mL of CH2Cl2. Added hydrazine mono-
hydrate (40.3 lL, 0.83 mmol) and stirred the reaction mixture
4.1.10. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-6-(3-
phenylpropyl)-6,7,8,9-tetrahydropyrimido[2,1-b]purin-4(3H)-
one (19d)
Obtained from 16 according to the procedure for synthesis of
19a in 34% yield using (3-bromopropyl)benzene as the alkylating
agent. 1H NMR (600 MHz, CD3OD) d: 7.21 (m, 4H), 7.13 (m, 1H),
4.95 (s, 2H), 4.21 (t, J = 5.8 Hz, 2H), 3.80 (m, 1H), 3.72 (t,
J = 6.0 Hz, 2H), 3.62 (m, 1H), 3.55 (m, 1H), 3.52 (t, J = 5.8 Hz, 2H),
3.35–3.28 (m, 2H, overlapped with solvent signal), 2.69 (t,
overnight. Concentrated the reaction mixture and isolated the pro-
duct by reverse phase HPLC. Re-purified the product by preparative
TLC on SiO2 using a mixture of CH2Cl2 and 7 N ammonia/methanol
as the solvent in 9:1 ratio, to furnish 24.3 mg (67%) of the title
material. 1H NMR (400 MHz, CD3OD) d: 7.96 (br, 1H), 4.96 (m,
1H), 4.73 (s, 1H), 4.11 (t, J = 6.0 Hz, 1H), 8.62 (m, 1H), 8.51 (m,
1H), 3.44 (t, J = 6.0, 2H), 3.35 (s, 2H), 3.18–3.07 (m, 2H), 2.88 (m,
1H), 2.11 (m, 2H), 2.02 (m, 1H), 1.89 (m, 1H), 1.78 (t, J = 2.3 Hz,
3H), 1.48 (m, 1H); LMCS (342.2 M+H), rt 0.79 min.
Please cite this article in press as: Pissarnitski, D. A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.007

8
D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.16. 2-(2-Chloro-7-(4-methoxybenzyl)-6-oxo-6,7-dihydro-
1H-purin-1-yl)acetonitrile 22
methanesulfonyl chloride (0.19 mL, 2.4 mmol). The mixture was
stirred 5 h, washed with satd sodium carbonate, brine, and organic
phase was dried over sodium sulfate. The product was chro-
matographed on SiO2 using a gradient 0–10% of methanol in CH2-
Cl2 to afford 330 mg (59%) of 26a. 1H NMR CDCl3 d 8.02 (m, 1H),
7.87 (m, 1H), 7.76 (m, 1H), 7.53 (m, 1H), 7.43 (s, 1H), 7.30 (d,
J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 5.60 (s, 2H), 5.45 (s, 2H),
4.03 (m, 2H), 3.78 (s, 3H), 3.45 (m, 2H), 2.91 (s, 3H), 1.90 (m,
2H); m/z = 468.3 (M+H)+.
To 2-chloro-7-(4-methoxybenzyl)-1H-purin-6(7H)-one 2129
(5.98 g, 20.62 mmol) in DMF (90.0 mL) at 0 °C was added NaH
(1.072 g, 26.81 mmol). The mixture was stirred at room tempera-
ture for 10 min, and at 55 °C for 3 h. Chloroacetonitrile (1.57 mL,
24.74 mmol) was added at 0 °C, and the reaction mixture was stir-
red at ambient temperature overnight. Another 0.65 mL
(10.31 mmol) of chloroacetonitrile was added, and mixture was
stirred for another overnight period. The reaction mixture was
quenched with water at 0 °C, extracted with CH2Cl2, organic phase
was washed with water and brine, dried (MgSO4) and concen-
trated. The crude product was purified by flash chromatography
(ethyl acetate/hexanes) to afford the title product (4.88 g, 72%).
1H NMR CDCl3 d 7.878 (s, 1H), 7.269 (d, J = 8.8 Hz, 2H), 6.880, (d,
J = 8.8 Hz, 2H), 5.465 (s, 2H), 5.189 (s, 2H), 3.789 (s, 3H); m/
z = 330.2 (M+H)+.
4.1.21. 3-(4-Methoxybenzyl)-5-((4-methylquinazolin-2-yl)
methyl)-7,8-dihydro-3H-imidazo[2,1-b]purin-4(5H)-one (27a)
Following the procedure described for 26a, using 2-(2-hydrox-
yethylamino)-7-(4-methoxybenzyl)-1-((4-methylquinazolin-2-yl)
methyl)-1H-purin-6(7H)-one 25a as the starting material, com-
pound 27a was synthesized in a 58% yield. 1H NMR d 8.03 (d,
J = 7.81, 1H), 7.87 (d, J = 7.81, 1H), 7.78 (t, J = 7.03, 1H), 7.55 (t,
J = 7.03, 1H), 7.46 (s, 1H), 7.28 (d, J = 8.59, 2H), 6.85 (d, J = 8.59,
2H), 5.51 (s, 2H), 5.38 (s, 2H), 4.20 (m, 2H), 3.93 (m, 2H), 3.78 (s,
3H), 2.89 (s, 3H); m/z = 454.3 (M+H)+.
4.1.17. 2-Chloro-7-(4-methoxybenzyl)-1-((4-methylquinazolin-
2-yl)methyl)-1H-purin-6(7H)-one (23a)
To 2-aminoacetophenone (2.50 g, 18.49 mmol) in dioxane at
0 °C was added HCl (4 M in dioxane, 30 mL, 120 mmol). The reac-
tion mixture was stirred for 7 h. To the resulting mixture was
added 2-(2-chloro-7-(4-methoxybenzyl)-6-oxo-6,7-dihydro-1H-
purin-1-yl)acetonitrile 22 (5.598 g, 17.0 mmol) in dioxane
(15 mL) at 0 °C. The reaction was stirred overnight, at which period
it was allowed to warm up to room temperature. The crude mix-
ture was added in portions to a solution of NaOH (10.36 g in
30 mL water, 259 mmol), and was stirred for 45 min. The mixture
was extracted with CH2Cl2, the organic phase was washed with
water and brine, dried (MgSO4) and concentrated. The crude pro-
duct was purified by flash chromatography (ethyl acetate/hexanes)
to afford 4.41 (58%) of the title product. 1H NMR CDCl3 d 8.04 (m,
1H), 7.86 (m, 1H), 7,78 (m, 1H), 7.54 (m, 2H), 7.30 (d, J = 8.7 Hz,
2H), 6.86 (d, J = 8.7 Hz, 2H), 5.55 (s, 2H), 5.41 (s, 2H), 3.79 (s, 3H),
2.90 (s, 3H); m/z = 447.3 (M+H)+.
4.1.22. 5-((4-Methylquinazolin-2-yl)methyl)-5,7,8,9-tetrahydro-
pyrimido[2,1-b]purin-4(3H)-one (28a)
A mixture of 26a (330 mg, 0.71 mmol) in TFA (10.0 mL) was
heated at 70 °C in a sealed tube overnight. TFA was removed in
vacuo. The product was purified by SiO2 chromatography using
0–15% gradient of solvent B in solvent A, where solvent A was CH2-
Cl2, and solvent B was 10% NH3 in methanol to furnish 245 mg
(99%) of the title product. 1H NMR d 8.25 (m, 1H), 7.92 (m, 1H),
7.91 (m, 2H), 7.68 (m, 1H), 5.42 (s, 2H), 3.95 (m, 2H), 3.43 (m,
2H), 2.82 (s, 3H), 1.85 (m, 2H); m/z = 348.2 (M+H+).
4.1.23. 5-((4-Methylquinazolin-2-yl)methyl)-7,8-dihydro-3H-i-
midazo[2,1-b]purin-4(5H)-one (29a)
A mixture of 27a (215 mg, 0.475 mmol) and TFA (5.0 mL) was
heated in a sealed tube at 70 °C overnight. Volatiles were removed
in vacuo and the product was purified by chromatography on silica
gel chromatography using 0–15% gradient of solvent B in solvent A,
where solvent A was CH2Cl2, and solvent B was 10% NH3 in metha-
nol to furnish 155 mg (98%) of 29a. 1H NMR d 8.26 (m, 1H), 7.93 (s,
1H), 7.96–7.86 (m, 2H), 7.69 (m, 1H), 5.44 (s, 2H), 4.32 (m, 2H),
3.96 (m, 2H), 2.95 (s, 3H); m/z = 334.2 (M+H+).
4.1.18. 2-((3-Hydroxypropyl)amino)-7-(4-methoxybenzyl)-1-
((4-methylquinazolin-2-yl)methyl)-1H-purin-6(7H)-one (24a)
To 2-chloro-7-(4-methoxybenzyl)-1-((4-methylquinazolin-2-
yl)methyl)-1H-purin-6(7H)-one 23a (558 mg, 1.25 mmol) in diox-
ane (13.0 mL) in a microwave tube was added 3-aminopropanol
(564 mg, 7.51 mmol) and diisopropylethylamine (0.65 mL,
3.75 mmol). The reaction was heated in a microwave reactor at
125 °C for 30 min. The mixture was partitioned between CH2Cl2
and water, the organic phase was washed with water and brine,
dried (MgSO4) and concentrated to afford the title product
(523 mg, 77%). 1H NMR CDCl3 d 8.09 (m, 1H), 7.98 (m, 1H), 7.90
(m, 1H), 7.67 (m, 1H), 7.63 (s, 1H), 7.30 (d, J = 8.7 Hz, 2H), 6.85
(d, J = 8.7 Hz, 2H), 5.58 (s, 2H), 5.45 (s, 2H), 3.88 (m, 2H), 3.65 (m,
2H), 3.78 (s, 3H), 3.79 (m, 2H), 2.95 (s, 3H); m/z = 486.2 (M+H)+.
4.1.24. 2-Bromo-3-(but-2-yn-1-yl)-5-((4-methylquinazolin-2-yl)
methyl)-5,7,8,9-tetrahydropyrimido[2,1-b]purin-4(3H)-one
(30a)
A solution of 28a (278 mg, 0.835 mmol) in 10.0 mL of DMF was
treated at 0 °C with 193 mg (1.08 mmol) of N-bromosuccinimide,
and the reaction mixture was stirred for 2.5 h. The yellow solution
was concentrated in vacuo and passed through a silica gel plug
using a solution of 25% MeOH and 1% triethylamine in CH2Cl2.
The crude bromide (m/z = 426.1, 428.1, M+H+) was dissolved in
DMF (5 mL) and treated with diisopropylethylamine (0.3 mL,
1.71 mmol) and 1-bromo-2-butyne (0.145 mL, 1.6 mmol). The
mixture was stirred overnight. Another 0.103 mL (1.14 mmol) of
1-bromo-2-butyne was added and stirring continued for another
overnight period. The reaction mixture was partitioned between
CH2Cl2 and satd sodium bicarbonate, aqueous phase was extracted
with CH2Cl2 twice, combined organic phase was dried over MgSO4
and concentrated. The crude product was purified by flash chro-
matography (MeOH/CH2Cl2) to afford 221 mg (55%) of the title
product. 1H NMR d 8.02 (d, J = 7.81 Hz, 1H), 7.90 (d, J = 7.81 Hz,
1H), 7.78 (t, J = 8.59 Hz, 1H), 7.54 (t, J = 8.59 Hz, 1H), 5.50 (s, 2H),
5.58 (s, 2H), 5.11 (s, 2H), 4.00 (m, 2H), 3.41 (m, 2H), 2.89 (s, 3H),
1.86 (m, 2H); m/z = 478.2, 480.2 (M+H+).
4.1.19. 2-((2-Hydroxyethyl)amino)-7-(4-methoxybenzyl)-1-((4-
methylquinazolin-2-yl)methyl)-1H-purin-6(7H)-one (25a)
Using procedure described for compound 24a and ethanola-
mine as the reagent, compound 25a was obtained in a 58% yield.
1H NMR CDCl3 d 8.09 (m, 1H), 8.02 (m, 1H), 7.89 (m, 1H), 7.65
(m, 1H), 7.62 (s, 1H), 7.30 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz,
2H), 5.58 (s, 2H), 5.45 (s, 2H), 3.88 (m, 2H), 3.78 (s, 3H), 3.79 (m,
2H), 2.95 (s, 3H); m/z = 472.2 (M+H)+.
4.1.20. 3-(4-Methoxybenzyl)-5-((4-methylquinazolin-2-yl)
methyl)-5,7,8,9-tetrahydropyrimido[2,1-b]purin-4(3H)-one
(26a)
To a solution of 24a (583 mg, 1.2 mmol) in CH2Cl2 (23.0 mL)
was added triethylamine (0.507 mL, 3.61 mmol) followed by
Please cite this article in press as: Pissarnitski, D. A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.007

D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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4.1.25. 2-Bromo-3-(but-2-yn-1-yl)-5-((4-methylquinazolin-2-yl)
methyl)-7,8-dihydro-3H-imidazo[2,1-b]purin-4(5H)-one (31a)
Starting from 29a, the title compound was obtained in a 59%
yield using procedures described for the synthesis of 30a. 1H
NMR d 8.02 (d, J = 7.81 Hz, 1H), 7.90 (d, J = 7.81 Hz, 1H), 7.78 (t,
J = 8.59 Hz, 1H), 7.54 (t, J = 8.59 Hz, 1H), 5.50 (s, 2H), 5.30 (s, 2H),
5.08 (q, J = 2.34 Hz, 2H), 4.12 (t, J = 9.38 Hz, 2H), 3.92 (t,
J = 9.38 Hz, 2H), 2.89 (s, 3H), 1.79 (t, J = 2.34 Hz, 3H); m/z = 464.2,
466.2 (M+H+).
J = 9.37 Hz, 2H), 3.64 (d, J = 11.72 Hz, 1H), 3.52 (d, J = 12.5 Hz,
1H), 3.09 (m, 2H), 2.90 (m, 1H), 1.99 (m, 1H), 1.87 (m, 1H), 1.70
(m, 1H), 1.38 (m, 1H); LCMS (484.2, M+H), rt 1.66 min.
4.1.30. (R)-2-(3-Aminopiperidin-1-yl)-3-methyl-5-((4-methylq-
uinazolin-2-yl)methyl)-7,8-dihydro-3H-imidazo[2,1-b]purin-4
(5H)-one (34b)
Synthesized from 29a analogously to 34a, except iodomethane
was substituted for 1-bromo-2-butyne as the N-alkylating agent.
1H NMR d 8.01 (d, J = 7.81 Hz, 1H), 7.90 (d, J = 8.59 Hz, 1H), 7.76
(t, J = 3.09 Hz, 1H), 7.52 (t, J = 2.90 Hz, 1H), 5.48 (s, 2H), 4.10 (t,
J = 8.59 Hz, 2H), 3.89 (t, J = 9.37 Hz, 2H), 3.70 (s, 3H), 3.47 (m,
1H), 3.33 (m, 1H), 3.01 (m, 2H), 2.90 (s, 3H), 2.78 (m, 1H), 2.0 (m,
1H), 1.80 (m, 4H), 1.32 (m, 1H); LCMS (446.1, M+H), rt 1.57 min.
4.1.26. tert-Butyl (R)-(1-(3-(but-2-yn-1-yl)-5-((4-methylquina-
zolin-2-yl)methyl)-4-oxo-3,4,5,7,8,9-hexahydropyrimido[2,1-b]
purin-2-yl)piperidin-3-yl)carbamate (32a)
To compound 30a (135 mg, 0.28 mmol) in NMP (2.0 mL) was
added diisopropylethylamine (0.10 mL, 0.58 mmol) and (R)-3-
Boc-aminopiperidine (70 mg, 0.35 mmol). The reaction mixture
was stirred overnight at 100 °C, cooled, diluted with CH2Cl2,
washed with satd sodium carbonate, water, and brine. Organic
phase was dried (MgSO4) and concentrated. The crude product
was purified by flash chromatography (MeOH/CH2Cl2) to afford
121 mg (71%) of the title compound. NMR d 8.01 (d, J = 7.81 Hz,
1H), 7.92 (d, J = 7.81 Hz, 1H), 7.76 (t, J = 8.59 Hz, 1H), 7.53 (t,
J = 8.59 Hz, 1H), 5.49 (s, 2H), 4.90 (d, J = 15 Hz, 1H), 4.81 (d,
J = 15 Hz, 1H), 3.89 (m, 2H), 3.50 (m, 1H), 3.32 (m, 4H), 3.21 (m,
1H), 2.85 (s, 3H), 1.86 (m, 5H), 1.71 (m, 3H), 1.59 (m, 2H), 1.49
(s, 9H); m/z = 598.3 (M+H+), 542.3 (Mꢂt-Bu)+.
4.1.31. (R)-2-(2-(3-Aminopiperidin-1-yl)-5-((4-methylquinaz-
olin-2-yl)methyl)-4-oxo-4,5,7,8-tetrahydro-3H-imidazo[2,1-b]
purin-3-yl)acetonitrile (34c)
Synthesized from 29a analogously to 34a, except bromoace-
tonitrile was substituted for 1-bromo-2-butyne as the N-alkylating
agent. 1H NMR d 7.97 (d, J = 7.81 Hz, 1H), 7.86 (d, J = 8.59 Hz, 1H),
7.72 (t, J = 3.09 Hz, 1H), 7.47 (t, J = 2.90 Hz, 1H), 5.41 (s, 2H), 5.04
(s, 2H), 4.10 (t, J = 8.59 Hz, 2H), 3.85 (t, J = 9.37 Hz, 2H), 3.45 (m,
1H), 3.33 (m, 1H), 3.06 (m, 2H), 2.84 (s, 3H), 2.80 (m, 1H), 1.91
(m, 2H), 1.75 (m, 1H), 1.60 (br, 2H), 1.33 (m, 1H); LCMS (471.1,
M+H), rt 1.59 min.
4.1.27. (R)-tert-Butyl (1-(3-(but-2-yn-1-yl)-5-((4-methylquina-
zolin-2-yl)methyl)-4-oxo-3,4,5,7,8,9-hexahydropyrimido[2,1-b]
purin-2-yl)piperidin-3-yl)carbamate (33a)
4.1.32. (R)-2-(3-Aminopiperidin-1-yl)-3-(cyclopropylmethyl)-5-
((4-methylquinazolin-2-yl)methyl)-7,8-dihydro-3H-imidazo
[2,1-b]purin-4(5H)-one (34d)
Prepared from 31a using procedure described for the synthesis
of 32a. 1H NMR d 8.02 (d, J = 7.81 Hz, 1H), 7.90 (d, J = 7.81 Hz, 1H),
7.78 (t, J = 8.59 Hz, 1H), 7.54 (t, J = 8.59 Hz, 1H), 5.50 (s, 2H), 5.46
(br, 1H), 4.95 (d, J = 15 Hz, 1H), 4.79 (d, J = 15 Hz, 1H), 4.09 (m,
2H), 3.89 (m, 2H), 3.50 (m, 1H), 3.32 (m, 2H), 3.23 (m, 1H), 2.89
(s, 3H), 1.86 (m, 3H), 1.71 (m, 3H), 1.59 (m, 2H), 1.45 (s, 9H); m/
z = 584.3 (M+H+), 528.3 (Mꢂt-Bu)+.
Synthesized from 29a analogously to 34a, except (bro-
momethyl)cyclopropane was substituted for 1-bromo-2-butyne
as the N-alkylating agent. 1H NMR d 7.68 (d, J = 7.81 Hz, 1H), 7.56
(d, J = 8.59 Hz, 1H), 7.43 (t, J = 3.09 Hz, 1H), 7.19 (t, J = 2.90 Hz,
1H), 5.41 (s, 2H), 3.79 (t, J = 8.59 Hz, 2H), 3.65 (d, J = 7.2 Hz, 2H),
3.59 (t, J = 9.37 Hz, 2H), 3.08 (m, 1H), 2.95 (m, 1H), 2.66 (m, 2H),
2.56 (s, 3H), 2.44 (m, 1H), 2.21 (m, 1H), 1.66 (m, 2H), 1.53 (m,
1H), 1.38 (m, 1H), 1.24 (br, 2H), 0.98 (m, 2H), 0.13 (m, 2H), 0.03
(m, 2H); LCMS (486.1, M+H), rt 1.67 min.
4.1.28. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-((4-
methylquinazolin-2-yl)methyl)-5,7,8,9-tetrahydropyrimido
[2,1-b]purin-4(3H)-one (20a)
4.1.33. 2-((R)-3-Aminopiperidin-1-yl)-3-((2,2-difluorocyclopro-
pyl)methyl)-5-((4-methylquinazolin-2-yl)methyl)-7,8-dihydro-
3H-imidazo[2,1-b]purin-4(5H)-one (34e)
To a solution of 32a (121 mg, 0.202 mmol) in CH2Cl2 (2 mL) was
added TFA (1.5 mL, 20.7 mmol). The mixture was stirred at room
temperature for 1.5 h. To the reaction flask was added water
(10 mL), then solid K2CO3 (5.7 g, 41.5 mmol) in portions at 0 °C
with vigorous stirring. The mixture was extracted with CH2Cl2,
organic phase was washed with water and brine, dried (MgSO4)
and concentrated. The crude product was purified by flash chro-
matography on SiO2 (MeOH/CH2Cl2, containing 1% Et3N) to afford
Synthesized from 29a analogously to 34a, except 2-(bro-
momethyl)-1,1-difluorocyclopropane was substituted for 1-
bromo-2-butyne as the N-alkylating agent. 1H NMR d 8.02 (d,
J = 7.81 Hz, 1H), 7.88 (d, J = 8.59 Hz, 1H), 7.78 (t, J = 3.09 Hz, 1H),
7.53 (t, J = 2.90 Hz, 1H), 5.48 (AB, 2H), 4.19 (d, J = 7.2 Hz, 2H),
4.12 (t, J = 8.59 Hz, 2H), 3.92 (t, J = 9.37 Hz, 2H), 3.37 (m, 1H),
3.24 (m, 1H), 3.01 (m, 2H), 2.88 (s, 3H), 2.80 (m, 1H), 2.25 (m,
1H), 2.0 (m, 1H), 1.86 (m, 1H), 1.63 (m, 4H), 1.48–1.10 (m, 2H);
LCMS (522.3, M+H), rt 1.69 min.
87.2 mg (87%) of the title compound. 1H NMR
d 7.98 (d,
J = 7.81 Hz, 1H), 7.87 (d, J = 7.81 Hz, 1H), 7.72 (t, J = 8.59 Hz, 1H),
7.48 (t, J = 8.59 Hz, 1H), 5.54 (s, 2H), 4.84 (m, 2H), 3.95 (t,
J = 5.9 Hz, 2H), 3.59 (m, 1H), 3.48 (m, 1H), 3.38 (t, J = 5.9 Hz, 2H),
3.04 (m, 2H), 2.85 (s, 3H), 2.85 (m, 1H), 1.96 (m, 1H), 1.83 (m,
4H), 1.77 (t, J = 5.9 Hz, 2H), 1.71 (m, 2H); m/z = 498.6 (M+H+), rt
1.68 min.
4.1.34. (R)-2-(3-Aminopiperidin-1-yl)-3-(cyclobutylmethyl)-5-
((4-methylquinazolin-2-yl)methyl)-7,8-dihydro-3H-imidazo
[2,1-b]purin-4(5H)-one (34f)
Synthesized from 29a analogously to 34a, except (bro-
momethyl)cyclobutane was substituted for 1-bromo-2-butyne as
the N-alkylating agent. 1H NMR d 7.97 (d, J = 7.81 Hz, 1H), 7.82
(d, J = 8.59 Hz, 1H), 7.72 (t, J = 3.09 Hz, 1H), 7.47 (t, J = 2.90 Hz,
1H), 5.41 (s, 2H), 5.04 (s, 2H), 4.05 (m, 4H), 3.45 (t, J = 9.5 Hz,
2H), 3.33 (m, 1H), 3.20 (m, 2H), 2.95 (m, 2H), 2.82 (s, 2H), 2.71
(m, 2H), 1.91 (m, 3H), 1.76 (m, 3H), 1.66 (m, 3H), 1.24 (m, 1H);
LCMS (500.3, M+H), rt 1.71 min.
4.1.29. (R)-2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-5-((4-
methylquinazolin-2-yl)methyl)-7,8-dihydro-3H-imidazo[2,1-b]
purin-4(5H)-one (34a)
Prepared from 33a using the procedure described for the prepa-
ration of 20a. 1H NMR d 8.01 (d, J = 7.81 Hz, 1H), 7.90 (d, J = 8.59 Hz,
1H), 7.76 (t, J = 3.09 Hz, 1H), 7.52 (t, J = 2.90 Hz, 1H), 5.50 (s, 2H),
4.84 (q, J = 2.34 Hz, 2H), 4.09 (t, J = 8.59 Hz, 2H), 3.90 (t,
Please cite this article in press as: Pissarnitski, D. A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.007

10
D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.35. (R)-2-(3-Aminopiperidin-1-yl)-3-(3-methylbut-2-en-1-
yl)-5-((4-methylquinazolin-2-yl)methyl)-7,8-dihydro-3H-imid-
azo[2,1-b]purin-4(5H)-one (34g)
4.1.40. (R)-2-((2-(3-Aminopiperidin-1-yl)-5-((4-methylquinaz-
olin-2-yl)methyl)-4-oxo-4,5,7,8-tetrahydro-3H-imidazo[2,1-b]
purin-3-yl)methyl)benzonitrile (34l)
Synthesized from 29a analogously to 34a, except 1-bromo-3-
methylbut-2-ene was substituted for 1-bromo-2-butyne as the
N-alkylating agent. 1H NMR d 8.02 (d, J = 7.81 Hz, 1H), 7.90 (d,
J = 8.59 Hz, 1H), 7.76 (t, J = 3.09 Hz, 1H), 7.52 (t, J = 2.90 Hz, 1H),
5.48 (s, 2H), 5.41 (m, 1H), 4.67 (d, J = 6.7 Hz, 1H), 4.10 (t,
J = 9.3 Hz, 2H), 3.89 (t, J = 8.9 Hz, 2H), 3.47 (m, 1H), 3.33 (m, 1H),
3.01 (m, 1H), 2.94 (m, 1H) 2.88 (s, 3H), 2.75 (m, 1H), 2.0 (m, 1H),
1.84 (m, 4H), 1.71 (s, 3H), 1.70 (s, 3H), 1.59 (br, 3H), 1.28 (m,
1H); LCMS (500.3, M+H), rt 1.73 min.
Synthesized from 29a analogously to 34a, except 2-cyanoben-
zylbromide was substituted for 1-bromo-2-butyne as the N-alky-
lating agent. 1H NMR
d 7.96 (d, J = 7.81 Hz, 1H), 7.84 (d,
J = 8.59 Hz, 1H), 7.74 (t, J = 3.09 Hz, 1H), 7.59 (t, J = 2.90 Hz, 1H),
6.99 (m, 1H), 7.46 (m, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.15 (d,
J = 7.9 Hz, 1H), 5.50 (AB, 2H), 5.40 (s, 2H), 4.09 (t, J = 8.59 Hz, 2H),
3.87 (t, J = 9.37 Hz, 2H), 3.26 (m, 1H), 3.11 (m, 1H), 2.88 (m, 2H),
2.82 (s, 3H), 2.64 (m, 1H), 1.87 (m, 1H), 1.69 (m, 2H), 1.60 (m,
2H), 1.18 (m, 1H); LCMS (547.3, M+H), rt 1.69 min.
4.1.36. (R)-2-(3-Aminopiperidin-1-yl)-3-(2-cyclopropyl-2-oxoe-
thyl)-5-((4-methylquinazolin-2-yl)methyl)-7,8-dihydro-3H-imi-
dazo[2,1-b]purin-4(5H)-one (34h)
4.1.41. (R)-2-(3-Aminopiperidin-1-yl)-3-((4-fluorothiophen-2-
yl)methyl)-5-((4-methylquinazolin-2-yl)methyl)-7,8-dihydro-
3H-imidazo[2,1-b]purin-4(5H)-one (34m)
Synthesized from 29a analogously to 34a, except 2-chloro-1-
cyclopropylethan-1-one was substituted for 1-bromo-2-butyne as
the N-alkylating agent. 1H NMR d 8.01 (d, J = 7.81 Hz, 1H), 7.90
(d, J = 8.59 Hz, 1H), 7.76 (t, J = 3.09 Hz, 1H), 7.52 (t, J = 2.90 Hz,
1H), 5.44 (s, 2H), 5.10 (s, 2H), 4.11 (t, J = 8.59 Hz, 2H), 3.89 (t,
J = 9.37 Hz, 2H), 3.34 (m, 1H), 3.19 (m, 1H), 2.98 (m, 2H), 2.88 (s,
3H), 2.78 (m, 1H), 1.92 (m, 2H), 1.81 (m, 1H), 1.69 (m, 3H), 1.32
(m, 1H), 1.12 (m, 2H), 0.96 (m, 2H); LCMS (514.3, M+H), rt
1.65 min.
Synthesized from 29a analogously to 34a, except 2-(bro-
momethyl)-4-fluorothiophene was substituted for 1-bromo-2-
butyne as the N-alkylating agent. 1H NMR d 8.03 (d, J = 7.81 Hz,
1H), 7.91 (d, J = 8.59 Hz, 1H), 7.78 (t, J = 3.09 Hz, 1H), 7.54 (t,
J = 2.90 Hz, 1H), 7.08 (dd, J = 4.0, 5.6 Hz, 1H), 6.71 (d, J = 5.6 Hz,
1H), 5.51 (s, 2H), 5.42 (s, 2H), 4.10 (t, J = 8.59 Hz, 2H), 3.90 (t,
J = 9.37 Hz, 2H), 3.41 (m, 1H), 3.29 (m, 1H), 3.02 (m, 2H), 2.89 (s,
3H), 2.76 (m, 1H), 1.98 (m, 1H), 1.84 (m, 1H), 1.75 (m, 3H), 1.28
(m, 1H); LCMS (545.3, M+H), rt 1.70 min.
4.1.37. (R)-2-(3-Aminopiperidin-1-yl)-3-benzyl-5-((4-methylqu-
inazolin-2-yl)methyl)-7,8-dihydro-3H-imidazo[2,1-b]purin-4
(5H)-one (34i)
4.1.42. (R)-2-((2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-4-
oxo-3,4,7,8-tetrahydro-5H-imidazo[2,1-b]purin-5-yl)methyl)
benzonitrile (34n)
Synthesized from 29a analogously to 34a, except benzyl bro-
mide was substituted for 1-bromo-2-butyne as the N-alkylating
agent. 1H NMR d 8.01 (d, J = 7.81 Hz, 1H), 7.90 (d, J = 8.59 Hz, 1H),
7.76 (t, J = 3.09 Hz, 1H), 7.52 (t, J = 2.90 Hz, 1H), 7.31–7.20 (m,
5H), 5.48 (s, 2H), 5.35 (AB, 2H),4.12 (t, J = 8.59 Hz, 2H), 3.90 (t,
J = 9.37 Hz, 2H), 3.33 (m, 1H), 3.23 (m, 1H), 2.93 (m, 2H), 2.88 (s,
3H), 2.70 (m, 1H), 1.94 (m, 1H), 1.76 (m, 2H), 1.64 (m, 2H), 1.26
(m, 1H); LCMS (522.3, M+H), rt 1.71 min.
Synthesized analogously to 34a from 21, using 2-cyanobenzyl-
bromide as the alkylating agent. 1H NMR d 7.64 (d, J = 7.7 Hz,
1H), 7.48 (t, J = 7.7 Hz, 1H), 7.28 (m, 2H), 5.37 (s, 2H), 4.77 (m,
2H), 4.08 (t, J = 8.59 Hz, 2H), 3.93 (t, J = 9.37 Hz, 2H), 3.62 (m,
1H), 3.52 (m, 1H), 3.06 (m, 2H), 2.76 (m, 1H), 1.99 (m, 1H), 1.88
(m, 1H), 1.80 (t, J = 2.2 Hz, 3H), 1.72 (m, 1H), 1.50 (br, 2H), 1.33
(m, 1H); LCMS (443.3, M+H), rt 1.62 min.
4.1.43. (R)-3-((2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-4-
oxo-3,4,7,8-tetrahydro-5H-imidazo[2,1-b]purin-5-yl)methyl)
benzonitrile (34o)
Synthesized analogously to 34a from 21, using 3-cyanobenzyl-
bromide as the alkylating agent. 1H NMR d 7.70 (d, J = 7.7 Hz,
1H), 7.69 (s, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.39 (d, J = 7.7 Hz, 1H),
5.49 (br, 2H), 5.17 (s, 2H), 4.81 (m, 2H), 4.11 (t, J = 9.0 Hz, 2H),
3.97 (t, J = 9.0 Hz, 2H), 3.62 (m, 1H), 3.45 (m, 1H), 3.37 (m, 2H),
2.76 (m, 1H), 1.99 (m, 1H), 1.88 (m, 2H), 1.80 (t, J = 2.2 Hz, 3H),
1.72 (m, 1H); LCMS (443.3, M+H), rt 1.63 min.
4.1.38. (R)-2-(3-Aminopiperidin-1-yl)-3-(3-fluorobenzyl)-5-((4-
methylquinazolin-2-yl)methyl)-7,8-dihydro-3H-imidazo[2,1-b]
purin-4(5H)-one (34j)
Synthesized from 29a analogously to 34a, except 3-fluoroben-
zyl bromide was substituted for 1-bromo-2-butyne as the N-alky-
lating agent. 1H NMR d 8.01 (d, J = 7.81 Hz, 1H), 7.90 (d, J = 8.59 Hz,
1H), 7.76 (t, J = 3.09 Hz, 1H), 7.52 (t, J = 2.90 Hz, 1H), 7.23 (m, 1H),
7.02 (d, J = 8.0 Hz, 1H), 6.92 (m, 2H), 5.47 (s, 2H), 5.32 (AB, 2H), 4.12
(t, J = 8.59 Hz, 2H), 3.90 (t, J = 9.37 Hz, 2H), 3.33 (m, 1H), 3.23 (m,
1H), 2.94 (m, 2H), 2.86 (s, 3H), 2.71 (m, 1H), 1.94 (m, 1H), 1.76
(m, 2H), 1.64 (m, 2H), 1.26 (m, 1H); LCMS (540.3, M+H), rt
1.71 min.
4.1.44. (R)-4-((2-(3-Aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-4-
oxo-3,4,7,8-tetrahydro-5H-imidazo[2,1-b]purin-5-yl)methyl)
benzonitrile (34p)
Synthesized analogously to 34a from 21, using 4-cyanobenzyl-
bromide as the alkylating agent 1H NMR d 7.54 (AB, 4H), 5.14 (s,
2H), 4.76 (m, 2H), 4.03 (t, J = 9.0 Hz, 2H), 3.92 (t, J = 9.0 Hz, 2H),
3.58 (m, 1H), 3.48 (m, 1H), 3.03 (m, 2H), 2.83 (m, 1H), 1.96 (m,
1H), 1.84 (m, 1H), 1.80 (t, J = 2.2 Hz, 3H), 1.69 (m, 1H), 1.41 (br,
2H), 1.31 (m, 1H); LCMS (443.3, M+H), rt 1.62 min.
4.1.39. (R)-2-(3-Aminopiperidin-1-yl)-3-(2,5-difluorobenzyl)-5-
((4-methylquinazolin-2-yl)methyl)-7,8-dihydro-3H-imidazo
[2,1-b]purin-4(5H)-one (34k)
Synthesized from 29a analogously to 34a, except 2,5-difluo-
robenzyl bromide was substituted for 1-bromo-2-butyne as the
N-alkylating agent. 1H NMR d 8.01 (d, J = 7.81 Hz, 1H), 7.90 (d,
J = 8.59 Hz, 1H), 7.76 (t, J = 3.09 Hz, 1H), 7.52 (t, J = 2.90 Hz, 1H),
6.99 (m, 1H), 6.89 (m, 1H), 6.83 (m, 1H), 5.47 (s, 2H), 5.37 (AB,
2H), 4.14 (t, J = 8.59 Hz, 2H), 3.92 (t, J = 9.37 Hz, 2H), 3.35 (m,
1H), 3.19 (m, 1H), 2.93 (m, 2H), 2.88 (s, 3H), 2.71 (m, 1H), 1.94
(m, 1H), 1.76 (m, 2H), 1.64 (m, 2H), 1.26 (m, 1H). LCMS (558.3, M
+H), rt 1.72 min.
4.2. X-ray crystallography and molecular modeling
DPP4 protein expression, purification and crystallization, and all
crystallographic methods have been previously reported.32 The
structures of DPP4 in complex with inhibitors 34a, 19a, 34p, and
Please cite this article in press as: Pissarnitski, D. A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.007

D. A. Pissarnitski et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
34n have been determined to atomic resolution. The coordinates
have been deposited and are available at the Protein Data Bank,
codes 5T4B, 5T4E, 5T4F, and 5T4H. Figures were generated using
PyMol.35 Molecular modeling was carried out on Merck supercom-
puter cluster using MaestroTM environment (Schrödinger, LLC, 120
West 45th Street, New York, NY 10036) with AMBER force field.
and dosed orally with vehicle (0.4% methyl cellulose) or treatment
compounds (n = 8 per group), and gently put back to home cages.
60 min later, mice were orally challenged with either water (con-
trol) or 50% dextrose (5.0 g/kg), immediately after measuring
0 min blood glucose reading. Upon dextrose challenge, ensuing
blood glucose measurements at various time points were recorded
and analyzed.
4.3. In vitro assays
Acknowledgments
DPP4 activity was measured according to a modified literature
protocol36 using the peptide substrate, Gly-Pro-AMC, which is
cleaved by DPP4 to release the fluorescent AMC group. Release of
AMC was monitored in real time in a continuous fluorescence
intensity measurement (kinetic mode) using the PHERAstar Plus
plate reader (BMG Lab. Tech.) at an excitation wavelength of
360 nm and emission wavelength of 460 nm. All assay components
and materials were pre-incubated to the assay temperature (37 °C)
prior to starting the reaction. Enzyme, test compound, and sub-
strate working solutions were prepared in assay buffer (100 mM
HEPES, 100 mM NaCl, 0.1 mg/mL BSA, pH 7.5). To determine
The authors are grateful to Alexei Buevich, T.-M. Chan, Rebecca
Osterman, and Mary Senior for interpretation of NMR spectra, and
Drs. William Greenlee and Andrew Stamford for fruitful
discussions.
A. Supplementary data
Supplementary data (summary of mouse oGTT results for com-
pounds 34a and 34p) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.09.007.
enzyme inhibition potency (IC50), 10 lL DPP4 and 10 lL of test
compound (11 concentrations from a 3-fold serial dilution) were
mixed in 384-well plate and pre-incubated at 37 °C for 30 min.
References and notes
Substrate (10
concentrations: 20 pM DPP4, 50
trations of test compound starting at 10
l
L) was then added to start the reaction (final assay
M Gly-Pro-AMC, varying concen-
M, 1% DMSO in assay
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