Synthesis and biological evaluation of 7-(aminoalkyl)pyrazolo[1,5-a]pyrimidine derivatives as cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.11.029

A series of novel 7-aminoalkyl substituted pyrazolo[1,5-a]pyrimidine derivatives were synthesized and tested for inhibition of cathepsin K. The synthetic methodology comprises cyclization of 5-aminopyrazoles with N-Boc-α-amino acid-derived ynones followed by transformation of the ester and the Boc-amino functions. It allows for easy diversification of the pyrazolo[1,5-a]pyrimidine scaffold at various positions. Molecular docking studies with pyrazolo[1,5-a]pyrimidine derivatives were also performed to elucidate the binding mode in the active site of cathepsin K. The synthesized compounds exhibited moderate inhibition activity (K_i ≥ 77 μM).

Full text of DOI:10.1016/j.bioorg.2018.11.029

Accepted Manuscript
Synthesis and biological evaluation of 7-(aminoalkyl)pyrazolo[1,5-a]pyrimi-
dine derivatives as cathepsin K inhibitors
Nejc Petek, Bogdan Štefane, Marko Novinec, Jurij Svete
PII:
S0045-2068(18)31124-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.029
YBIOO 2637
Reference:
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Bioorganic Chemistry
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Revised Date:
Accepted Date:
3 October 2018
13 November 2018
17 November 2018
Please cite this article as: N. Petek, B. Štefane, M. Novinec, J. Svete, Synthesis and biological evaluation of 7-
(
aminoalkyl)pyrazolo[1,5-a]pyrimidine derivatives as cathepsin K inhibitors, Bioorganic Chemistry (2018), doi:
https://doi.org/10.1016/j.bioorg.2018.11.029
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Synthesis and biological evaluation of 7-(aminoalkyl)pyrazolo[1,5-a]pyrimidine
derivatives as cathepsin K inhibitors.
Nejc Petek, Bogdan Štefane, Marko Novinec *, and Jurij Svete*
University of Ljubljana, Faculty of Chemistry and Chemical Technology, Večna pot 113, 1000
Ljubljana, Slovenia. E-mail: jurij.svete@fkkt.uni-lj.si
Abstract. A series of novel 7-aminoalkyl substituted pyrazolo[1,5-a]pyrimidine derivatives were
synthesized and tested for inhibition of cathepsin K. The synthetic methodology comprises cyclization
of 5-aminopyrazoles with N-Boc-α-amino acid-derived ynones followed by transformation of the ester
and the Boc-amino functions. It allows for easy diversification of the pyrazolo[1,5-a]pyrimidine
scaffold at various positions. Molecular docking studies with pyrazolo[1,5-a]pyrimidine derivatives
were also performed to elucidate the binding mode in the active site of cathepsin K. The synthesized
compounds exhibited moderate inhibition activity (K ≥ 77 μM).
i
Keywords: pyrazoles; ynones; cyclisation; pyrazolo[1,5-a]pyrimidines; cathepsin K; enzyme
inhibition; molecular docking.
1
. Introduction
Heterocyclic building blocks represent useful scaffolds for applications in medicinal chemistry,
catalysis, and materials science [1]. For example, heterocyclic systems that can simulate β-turn
structures can serve as replacements of dipeptide motifs in a given native (or natural) peptidic substrate
1

[2]. An important group of such conformationally constrained U-shaped heterocyclic dipeptide
analogues are azabicycloalkane amino acids, which comprise fused heterocycles with a bridgehead
nitrogen atom [3].
Among 5–6-fused systems, pyrazolo[1,5-a]pyrimidine is an important heterocycle, due to biological
activity of many of its derivatives [4]. A SciFinder [5] Substructure search shows around 175,000
known pyrazolo[1,5-a]pyrimidine derivatives with over 7,000 references and with preparation,
biological study, and uses as the predominant substance roles. For 2017 alone, over 2,000 references
can be found for pyrazolo[1,5-a]pyrimidines. Examples of bioactive pyrazolo[1,5-a]pyrimidines
include hepatitis C virus inhibitors [6], antagonists of serotonin 5-HT6 receptors [7], kinase inhibitors
[8], PET tumour imaging agents [9], and inhibitors of amyloid β-peptide 1–42 aggregation [10].
Sedative agents zaleplon and indiplon and the anxiolytic agent ocinaplon are approved drugs
containing a pyrazolo[1,5-a]pyrimidine core (Fig. 1).
O
CN
O
N
S
N
N
N
N
N
N
N
N
N
O
O
N
N
N
Ocinaplon
Indiplon
Zaleplon
Fig. 1. Approved drugs containing a pyrazolo[1,5-a]pyrimidine.
Cathepsin K is a cysteine protease, which is selectively and abundantly expressed within osteoclasts,
and which is believed to be crucial for the resorption of bone matrix [11–15]. The ability to degrade
type I collagen allows cathepsin K to contribute importantly to the balance between bone resorption
and bone formation [16, 17]. It plays an important role in bone resorption and therefore in degenerative
bone diseases such as osteoporosis [18].
2

In contrast to osteoporosis treatments, which mainly utilize estrogen receptor modulators or
bisphosphonates accumulation [19], cathepsin K inhibition offers a more controlled treatment.
Selective reduction of bone resorption rather than both resorption and formation is possible by
inhibition of cathepsin K. Inhibitors of cathepsin K could prevent bone resorption and may provide a
promising approach for the treatment of osteoporosis, therefore inhibition of cathepsin K has been
proposed as a promising strategy for the treatment of osteoporosis, cancer, and other diseases [11–13].
Several inhibitors have progressed into clinical trials for the treatment of osteporosis as well as other
bone and cartilage related diseases but there are, as yet, no inhibitors on the market [19, 20]. The most
successful inhibitor was odanacatib (Merck & Co.) USA) [21] which showed robust efficacy in a phase
III clinical trial aimed at evaluating osteoporosis-related risk of bone fractures [22]. Unfortunately, it
was later terminated due to side effects. Another cathepsin K inhibitor, MIV-711 (Medivir AB,
Sweden) [23], is currently in phase II clinical trials for the treatment of osteoarthritis.
Pyrazolo[1,5-a]pyrimidines are commonly available by cyclisation of a 3-aminopyrazole derivative
with a 1,3-dicarbonyl compound or its synthetic equivalent [24]. Due to this ease of access, a plethora
of known pyrazolo[1,5-a]pyrimidine derivatives is not surprising. Nevertheless, a more detailed
literature search also reveals that only a handful of 7-aminoalkylpyrazolo[1,5-a]pyrimidine-3-
carboxamides are known [25].
Recently, we focused our attention on the synthesis and transformations of derivatives of
pyrazolo[1,5-a]pyrimidine-3-carboxamide. Within this context, we reported (parallel) syntheses of
libraries of novel 7-heteroarylpyrazolo[1,5-a]pyridine-3-carboxamides [26], 7-oxopyrazolo[1,5-
a]pyrimidine-3-carboxamides
[27],
7-(1-aminoethyl)pyrazolo[1,2-a]pyrimidines
[28],
tetrahydropyrazolo[1,5-c]pyrimidine-3-carboxamides [29], and 5-(N-Boc-N-benzyl-2-aminoethyl)-7-
oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-3-carboxamides [30]. The latter derivatives also exhibited
moderate inhibition of cathepsins B and K [30]. In extension, we elaborated the synthesis of 7-
3

aminoalkyl substituted pyrazolo[1,5-a]pyrimidine derivatives from α-amino acids [28] in terms of
scope and structural diversity of the products. A library of pyrazolo[1,5-a]pyrimidine derivatives with
variable substituents at positions 2, 3, and 7 and with variable degree of saturation of the pyrimidine
ring was synthesised and the products were tested for inhibition of cathepsin K. Herein, we report the
results of this study.
2
. Results and Discussion
2
.1.
Synthesis of pyrazolo[1,5-a]pyrimidine derivatives
Starting 5-amino-1H-pyrazoles 5a–g were prepared by cyclisation of β-ketonitriles 4a–g with
hydrazine hydrate following general literature procedures [31–34]. The β-ketonitriles 4a–f in turn, were
obtained by base-catalysed condensation of alkyl benzoates 1a,b with acetonitrile [35], by acylation of
benzyl cyanide 2 [36], and by cyanide displacement of phenacyl bromides 3a,b following literature
procedures [37] (Scheme 1, Table 1).
4

H
N
O
H N
2
CO Me
N
2
CN
i
ii
R
R
R
1a,b
4a,b
5a,
b
R
O
CN
H
N
H N
2
N
iii
iv
ii
CN
Ph
R
2a,b
4c,d
5
c,d
Br
O
CN
O
H
N
H N
2
N
ii
R
R
R
5e,f
4e,f
3a,b
H
N
H N
2
N
MeO C
CN
v, ii
2
4g
MeO C
2
5g
Scheme 1. Synthesis of 5-amino-1H-pyrazoles 5a–f from β-keto nitriles 4a–f. Reaction conditions: i)
MeCN, t-BuOK, toluene, 0 °C; ii) N H ·H O, EtOH, reflux; iii) RCO Me, EtOH, EtONa, reflux; iv)
2
4
2
2
KCN, EtOH, H O, reflux; v) CH(OEt) , Ac O, reflux.
2
3
2
5

Table 1. Experimental data for β-keto nitriles 4a–f and 5-aminopyrazoles 5a–f.
Transformation
R
Yield (%)a
Ref.
4
5
1
1
2
2
3
3
a→4a→5a
b→4b→5b
a→4c→5c
b→4d→5d
a→4e→5e
b→4f→5f
g→5g
H
46
33
65
96
70
62
-
75 (89)b
59
38
39
40
41
42
43
44
OMe
Me
Bn
NO2
CF3
-
68
71 (62)b
72
77 (82)b
79c
4
a
Isolated yield. b Under microwave irradiation. c The yield of 5g obtained in our laboratory; the
literature [44] yield is not provided.
The starting ynones 7a–c were obtained from N-Boc-glycine (6a), (S)-N-Boc-alanine, and (S)-N-
Boc-3-phenylalanine (6c) following known literature protocol [45]. Also subsequent cyclizations of
7
a–c with 5-amino-1H-pyrazoles 5a–g and 3-aminoindazole (5h) were carried out as described
previously for close analogues [28] to afford 7-aminoalkylpyrazolo[1,5-a]pyrimidine-3-carboxylates
0a–l in 20–93% yields. The reaction mechanism can be explained by initial Michael addition of the
1
aminopyrazole 5 to the conjugated triple bond of 7 to give the intermediate enaminone 8 followed by
cyclisation to 9 and elimination of water to furnish title compound 10. Notably, cyclizations of 7a–c
with aminopyrazole 5g in aqueous methanol in the presence of acetic acid (Method B) gave the
respective products 10b,i,l, in better yields than the reactions in methanol (Method A). This is
explainable by the lower nucleophilicity and reactivity of aminopyrazole 5g in the reactions with 1,3-
dielectrophiles 7, which is due to electron-withdrawing ester group at position 4. Acetic acid activates
6

the ynone 7 in the Michael addition step and, hence, compensates the lower reactivity of aminopyrazole
g. In contrast to our previous observations [28], the cyclizations were regioselective to furnish
isomerically pure products 10 isolated upon workup by filtration or by flash chromatography [46]
5
(Scheme 2 and Table 2).
NHBoc
H
O
N
H N
2
R1
N
i
HN
NHBoc
+
R3
O
R3
a–g
R2
R1
7a–c
NH
5
N
R2
8
a–l
R3
N
H
N
R3
3
4
N
-
H O
5
6
2
R2
R2
2
N
N
N
1
7
NHBoc
HO
R1
R1
7' NHBoc
9a–l
10a–l
Scheme 2. Cyclizations of ynones 6a–c with 5-aminopyrazoles 5a–g. Reaction conditions: i) MeOH,
r.t. (Method A) or MeOH, H O, AcOH, r.t. (Method B).
2
7

Table 2. Experimental data for 7-(1-aminoalkyl)pyrazole derivatives 10a–l.
Reaction
R1
R2
Ph
H
R3
H
Yield (%)a
Ref.
b
5
5
5
5
5
5
5
5
5
5
5
5
a+7a→10a
g+7a→10b
a+7b→10c
b+7b→10d
c+7b→10e
d+7b→10f
e+7b→10g
f+7b→10h
g+7b→10i
h+7b→10j
a+7c→10k
g+7c→10l
H
43
74c
93
86
60
59
57
75
70c
46
48
20c
b
b
b
b
b
b
b
H
CO Me
2
Me
Me
Me
Me
Me
Me
Me
Me
Bn
Bn
Ph
H
2-MeOC H
H
6
4
Me
Ph
Ph
H
Bn
3-O NC H
4
2
6
3-F CC H
4
H
3
6
H
CO Me
28
2
b
–CH=CH–CH=CH–
Ph
b
b
H
H
CO Me
2
a
b
c
Isolated yield. This paper. Cyclisation was performed in the presence of water and acetic acid.
Base-catalyzed hydrolysis of the pyrazolo[1,5–a]pyrimidine-esters 10b,i,l afforded the
corresponding carboxylic acids 11a–c in 54–89% yields. Next, amidation of 11a,b with primary (12a–
e) and secondary amines 12f–h using 1,1’-carbonyldiimidazole (CDI) as activating reagent furnished
carboxamides 13a–i (Scheme 3, Table 3).
8

R2
3N
O
MeO C
HO2C
2
R
N
N
N
i
ii, iii
N
N
N
N
N
N
R1
NHBoc
R1
NHBoc
R1
NHBoc
10b,i,l
11a–c
13a–i
2
3
Amines R R NH (12a–h):
nPr NH2
Me NH2
2a
Bn NH2
HO
NH2
NH
1
12b
12c
12d
NH2
NH
NH
O
12
12e
12f
12g
12h
Scheme 3. Synthesis of carboxamides 13a–i. Reaction conditions: i) LiOH·H O, MeOH, r.t.; ii) CDI,
2
THF, r.t.; iii) amine 12, THF, r.t.
9

Table 3. Experimental data for compounds 11a–c and 13a–i.
Reaction
R1
R2
R3
-
Yield (%)a
1
1
1
1
1
1
1
1
1
1
1
0b→11a
H
-
65
89
54
49
70
79
100
89
60
90
99
97
0i→11b
Me
Bn
H
-
-
0l→11c
-
-
1a+12a→13a
1b+12a→13b
1b+12b→13c
1b+12c→13d
1b+12d→13e
1b+12e→13f
1b+12f→13g
1b+12g→13h
1b+12h→13i
Isolated yield.
Me
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
n-Pr
Bn
(CH ) OH
2
3
n-C H
31
1
5
–(CH ) –
2
4
–(CH ) –
2
5
1
–(CH ) –O–(CH ) –
2 2 2 2
a
Next, free amines 14a–i were prepared from the N-Boc analogues 10, 11, and 13 by acidolytic
deprotection of amino group with HCl–EtOAc. The amines 14 were then N-acylated to give
compounds 15a–d in moderate yields. Quarternization of 14f and 14j with excess methyl iodide in the
presence of potassium carbonate afforded the quaternary salts 16a and 16b in 60% yields (Scheme 4,
Table 4).
1
0

R2
N
N
R1
N
NHBoc
10, 11, 13
i
R2
N
N
R1
N
ii
iii
N
NH Cl
3
14a–i
R2
R2
N
R1
R1
N
N
N
N
NHCOR3
NMe3 I
6a,b
1
15a–d
Scheme 4. Synthesis of amines 14a–i, 16a,b and carboxamides 15a–d. Reaction conditions: i) EtOAc,
HCl, r.t.; ii) R COCl, Et N,CH Cl , r.t.; iii) MeI, K CO , r.t.
3
3
2
2
2
3
1
1

Table 4. Experimental data for compounds 14a–i and 15a–d.
Reaction
R1
Ph
R2
H
R3
Yield (%)a
32
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0b→14a
0d→14b
0e→14c
0f→14d
0h→14e
0i→14f
2-MeOC H
H
54
6
4
Me
Ph
Ph
H
32
Bn
20
3-F CC H
4
100
88
3
6
H
H
H
H
H
H
H
H
H
H
CO Me
2
1b→14g
3b→14h
3f→14i
CO H
51
2
CONHMe
70
CONH(CH ) CH
3
62
2
14
4f→15a
4f→15b
4f→15c
4h→15d
4f→16a
4j→16b
Isolated yield.
CO Me
Me
Ph
48
2
CO Me
85
2
CO Me
Bn
Bn
68
2
CONHMe
99
CO Me
60
2
1
CONH(CH ) CH
3
60
2
14
a
Somewhat expectedly [28], catalytic hydrogenation of 10a,b, 11b, and 13b in the presence of Pd–C
under 3 bar of hydrogen afforded the corresponding 4,5;6,7-tetrahydropyrazolo[1,5-a]pyrimidines 17a
and 17b–d/17'b–d in almost quantitative yields. Subsequent chromatographic purification of mixtures
of diastereomers 17b–d/17'b–d then furnished pure major (1'S,7R)-isomers 17b–d in 50–93% yields.
Notably, catalytic hydrogenations took place only with the above substrates 10b,i, 11b, and 13b
1
2

bearing an electron-withdrawing carbonyl group at position 3, whereas attempted hydrogenations of
other substrates (10c–h) were not successful, neither under these reaction conditions, nor in the
presence of acetic acid. Diastereoselectivity of hydrogenation is explainable by the less hindered Si-
attack of hydrogen provided that the substrates 10b, 11b, and 13b adopt a conformation, which is
stabilized by an intramolecular hydrogen bond between the amide NH group and the ring nitrogen atom
N(1) (Scheme 5, Table 5).
O
O
O
R2
R2
R2
H
H
N
N
N
i
+
N
N
N
N
N
N
R1
NHBoc
R1
NHBoc
R1
NHBoc
1
7a–d
17'a–d
1'S,7S
10a,b, 11b, 13b
1'S,7R
(not isolated)
less hindered Si-approach
O
R2
N
O
R2
N
N
N
N
N
H
H
H
N
Boc
N
CH3
CH3
Boc
10'b, 11'b, 13'b
Scheme 5. Synthesis of compounds 17a–d. Reaction conditions: i) H (3 bar), 10% Pd-C, MeOH, r.t.
2
1
3

Table 5. Experimental data for compounds 17a–d.
Compound
R1
R2
d.r.a
/
Yield (%)b
Ref.
c
1
1
1
1
7a
7b
7c
H
OMe
OMe
OH
96
71
93
Me
Me
Me
84:16
92:8
83:17
[28]
c
c
7d
NHMe
50
a
1
b
c
Determined from the H NMR spectrum of the crude reaction mixture. Isolated yields. This
paper.
2
.2.
Structure determination
The structures of novel compounds 10, 11, and 13–17 were determined by spectroscopic methods
1
13
(
IR, H- and C-NMR, and MS-HRMS) and by elemental analyses for C, H, and N. NMR data for
compounds 10, 11, and 13–17 were in agreement with the literature data for related pyrazolo[1,5-
a]pyrimidines [4, 28]. The structure of compound 17b was also determined by X-ray diffraction
1
13
analysis (Figure 2). The detailed structure determination including copies of H and C NMR spectra
are given in the Supporting Information.
1
4

Fig. 2. ORTEP view of molecular structure of compound 17b and two molecules of cocrystallized
water. Displacement ellipsoids are drawn with 40% probability level and the hydrogen atoms are
shown as small spheres of arbitrary radii.
2
.3. Inhibition of cathepsin K
The synthesized pyrazolo[1,5-a]pyrimidines were tested for inhibition of cathepsin K. Due to the
absorptive properties of some of the compounds enzymatic activity was followed photometrically using
the substrate Z-Phe-arg-pNA (benzyloxycarbonyl-Phe-arg-p-nitroanilide). Compounds 10a,c-h,j,k and
1
3c,d were insoluble in the buffer solution used for kinetic measurements due to their higher
lipophilicity. Therefore, we removed the Boc protecting group and synthesized more soluble
compounds 14. A preliminary screen revealed that compounds 10i, 11b, 13b,e-I and 15a,c were weak
inhibitors of cathepsin K, while compounds 14a-h showed stronger inhibitory activity. Compound 14f
acted as a stronger inhibitor than its precursor 10i, indicating additional benefit from removal of the
Boc protecting group. Titration curves were recorded for compounds 14a,b,d,e,f,h and showed that all
compounds acted as linear (full) inhibitors, which is consistent with their binding into the active site of
1
5

the protease. Their inhibition constants (K ) were determined from the titration curves by non-linear
i
regression analysis using the model for linear competitive inhibition (Table 6). The strongest affinity
was determined for compound 14d with a K value of 77±5 µM (Table 6, Entry 3).
i
Table 6. Enzyme inhibition data for compounds 14a,b,d,e,f,h.
a
Entry
Compound
14a
R1
Ph
R2
H
K [µM]
i
1
2
3
4
5
236±15
288±27
77±5
14b
2-MeOC H
H
6
4
14d
Bn
Ph
H
14e
3-F CC H
4
241±27
714±53
595±80
3
6
14f
H
H
CO Me
2
6
14h
CONHMe
a
Determined by non-linear regression analysis using GraphPad Prism 5.0 with the model for linear
competitive inhibition.
All pyrazolo[1,5-a]pyrimidines 10, 11, and 13–17 were subjected to molecular docking into the
active site of cathepsin K [47]. The compounds bound preferentially into the cleft forming the non-
primed sites (sites S1 through S3) which is the narrowest part of the active site. At the same time, site
S2 which forms a well-defined pocket is also the primary specificity determinant in cathepsin K and
other related proteases. Specifically, cathepsin K shows preference for hydrophobic residues as well as
Pro at the corresponding P2 position of the substrate [48]. The binding modes of compounds 14, which
exhibited quantifiable inhibitory activity, are shown in Figure 3. In all cases, the S2 pocket was
occupied by a hydrophobic group which is in accordance with the specificity of cathepsin K.
Interestingly, the binding pose of compound 14d, which had the highest affinity for cathepsin K in vitro
1
6

and the second-best docking score in silico, is the only compound that occupies all three non-primed
sites.
1
7

S2
S3
S2
S1
S1
S3
S2
1
4a (–3.85)
14b (–3.18)
S2
S1
S1
S3
S3
1
4d (–5.62)
14e (–5.60)
S2
S2
S1
S1
S3
S3
1
4f (–3.83)
14h (–4.49)
Fig. 3. Molecular docking of compounds 14a,b,d,e,f,h into the active site of cathepsin K with
corresponding docking scores. The S1, S2 and S3 sites of the active site are marked in the
representations. Hydrogen bonds of the amino group are marked yellow.
1
8

4
.
Conclusions
We developed a general synthetic route towards 7-(1-aminoalkyl)pyrazolo[1,5-a]pyrimidine
derivatives starting with regioselective cyclocondensation of 3-aminopyrazoles with aminoalkyl
ethynyl ketones, which in turn are easily accessible from α-amino acids. The method has a broad scope
in terms of diversity of substituents and functional groups attached to the pyrazolo[1,5-a]pyrimidine
core. Beside this, also the degree of saturation of the heterocyclic scaffold, i.e. it’s 3D character can be
increased by stereoselective catalytic hydrogenation. In summary, this allows for easy preparation of
structurally diverse pyrazolo[1,5-a]pyrimidine derivatives. The synthesised compounds were then
tested for inhibition of cathepsin K. The strongest affinity was determined for the free amino compound
1
4d with a K value of 77±5 µM. Subsequent molecular docking of the above compounds showed that
i
the S2 pocket was occupied by a hydrophobic group which is in accordance with the specificity of
cathepsin K. The binding pose of compound 14d with the highest affinity for cathepsin K in vitro is the
only one that occupies all three non-primed sites, S1–S3. In summary, compound 14d could represent a
lead for further development of cathepsin K inhibitors, in particular because of viability of synthetic
approach that enables the preparation of structurally diverse compounds based on pyrazolo[1,5-
a]pyrimidine scaffold.
3
.
Experimental
3
.1.
Materials and methods
1
9

Melting points were determined on a Kofler micro hot stage and on a Stanford Research Systems
MPA100 OptiMelt automated melting point system. The NMR spectra were recorded in CDCl and
3
DMSO-d using Me Si as the internal standard on a Bruker Avance DPX 300 and Bruker Avance III
6
4
1
13
UltraShield 500 plus instruments at 300 and 500 MHz for H and at 75.5 and 126 MHz for C nucleus,
respectively. Mass spectra were recorded on Agilent 6224 Accurate Mass TOF LC/MS spectrometer
and IR spectra on a Bruker FTIR Alpha Platinum spectrophotometer. Microanalyses were performed
by combustion analysis on a Perkin-Elmer CHN Analyzer 2400 II. Flash column chromatography (FC),
column chromatography (CC), and dry-vacuum flash chromatography (DVFC) were performed on
silica gel (particle size: 35–70 m).
Esters 1a,b, nitriles 2a,b, bromoketones 3a,b, 3-amino-1H-indazole (5h), N-Boc-amino acids 6a–c,
and amines 12a–i are commercially available. 5-Aminopyrazoles 5a–g [29–32] and ynones 7a–c [45]
were prepared following the literature procedures.
Molecular docking was performed with Glide, a part of a Schrödinger suite software [49].
3
.1.1. Enzyme assays
Kinetic measurements were performed in a 10 X 10-mm quartz cuvette at 25 °C with constant
stirring on a Varian Cary 50 Bio UV/VIS spectrophotometer in a reaction volume of 3 mL of 100 mM
sodium acetate buffer pH 5.50 containing 1 mM EDTA, 5 mM DTT, 20 microM of the substrate Z-FR-
pNA and respective concentrations of tested compounds. The determined value of the Michaelis
constant K for this substrate under the described experimental conditions was 16 microM. Bulk
m
solutions of potential inhibitors were made in DMSO. Absorbance was observed for 60 s at 405 nm and
initial reaction rates determined from the progress curves using linear regression. Materials used:
2
0

recombinant human cathepsin K, made in biochemistry department of UL FKKT; DTT, Sigma-
Aldrich, ZDA; Z-FR-pNA, Bachem, Switzerland.
Kinetic analyses. Titration curves of cathepsin K with the tested compounds (I) in the presence of
substrate (S) were analysed with the model for linear competitive inhibition in which the reaction rate
in the presence of inhibitor v is defined by equation 1:
i
[
푆]
푣 1 +
0
(
)
퐾푚
Eq. 1
푣푖 =
[
푆] [퐼]
1
+
+
퐾푚 퐾푖
where ν is the reaction rate in the absence of inhibitor and Ki is the inhibition constant, i.e. the
o
equilibrium dissociation constant of the EI complex. All analyses were performed using GraphPad
Prism 5.0 Software (GraphPad Software, La Jolla, CA, USA).
3
3
.2.
Synthesis of compounds 10.
.2.1. General procedure for the synthesis of compounds 10 A (G.P.A).
A mixture of 5-aminopyrazole 5 (1.1 mmol), ynone 7 (1 mmol), and MeOH (5 mL) was stirred at r.t.
for 7 days. The product was, either collected by filtration or washed with MeOH (2 mL), or the solvent
was evaporated in vacuo and the residue was purified by CC (EtOAc/hexanes). Fractions containing the
product were combined and evaporated in vacuo to give 10.
3
.2.2. General procedure for the synthesis of compounds 10 B (G.P.B).
A solution of 5-aminopyrazole 5g (1 mmol) and ynone 7 (1 mmol) in MeOH (1 mL) was added
dropwise to a mixture of AcOH (0.5 mL) and water (10 mL) and the mixture was stirred at r.t. for 7
2
1

days. The product was extracted by CH Cl (3 × 25 mL) and the combined organic phases were dried
2
2
over anhydrous Na SO , filtered, and the filtrate was evaporated in vacuo. The residue was purified by
2
4
CC (EtOAc/hexanes). Fractions containing the product were combined and evaporated in vacuo to give
1
0.
3
.2.3.
7-{[(tert-Butoxycarbonyl)amino]methyl}-2-phenylpyrazolo-[1,5-a]pyrimidine
(10a).
Prepared from 5a (175 mg, 1.1 mmol) and 7a (183 mg, 1.0 mmol), G.P.A, CC (EtOAc-hexanes, 1:2).
–
1
Yellow solid (70 mg, 43%); mp 76–80 °C; max/cm (ATR) 3208, 2975, 1738, 1712, 1617, 1548,
463, 1444, 1407, 1392, 1365, 1281, 1251, 1163, 1083, 1049, 944, 860, 814, 770, 742, 697, 640; δH
500 MHz; CDCl ; Me Si) 1.46 (s, 9H), 4.85 (d, J = 6.5 Hz, 2H), 5.65 (s, 1H), 6.82 (d, J = 4.2 Hz, 1H),
1
(
3
4
7
.03 (s, 1H), 7.39–7.45 (m, 1H), 7.46–7.51 (m, 2H), 7.99–8.05 (m, 2H), 8.45 (d, J = 4.2 Hz, 1H); δC
(
126 MHz; CDCl ; Me Si) 28.3, 40.0, 80.4, 93.8, 105.6, 126.6, 128.8, 129.1, 132.7, 145.0, 149.2,
3 4
+
+
1
50.0, 155.7, 155.9; HRMS (ESI): MH , found 325.1659. [C H N O ] requires 325.1659.
18 21 4 2
3
.2.4.
Methyl
7-{[(tert-butoxycarbonyl)amino]methyl}pyrazolo[1,5-a]pyrimidine-3-
carboxylate (10b). Prepared from 5g (706 mg, 5.0 mmol) and 7a (915 mg, 5.0 mmol), G.P.B, CC
–
1
(
EtOAc-hexanes, 1:1). White solid (1.13 g, 74%); mp 160–164 °C; max/cm (ATR) 3260, 2965, 1703,
1
9
618, 1547, 1531, 1475, 1432, 1400, 1385, 1364, 1354, 1281, 1246, 1229, 1167, 1085, 1050, 1030,
30, 894, 867, 834, 803, 783, 745, 722, 667, 642, 626; δ (500 MHz; CDCl ; Me Si) 1.45 (s, 9H), 3.98
H
3
4
(
s, 3H), 4.83 (d, J = 6.5 Hz, 2H), 5.51 (s, 1H), 7.04 (d, J = 4.3 Hz, 1H), 8.61 (s, 1H), 8.77 (d, J = 4.3
Hz, 1H); δ (126 MHz; CDCl ; Me Si) 28.3, 40.0, 51.8, 80.7, 103.2, 107.5, 146.8, 147.7, 147.8, 152.6,
C
3
4
+
+
1
5
55.6, 163.0; HRMS (ESI): MH , found 307.1396. [C H N O ] requires 307.1401; (Found: C,
14 19 4 4
4.98; H, 5.94; N, 18.23. C H N O requires C, 54.89; H, 5.92; N, 18.29%).
1
4
18
4
4
2
2

3
.3.5.
(1’S)-7-{1-[(tert-Butoxycarbonyl)amino]ethyl}-2-phenylpyrazolo[1,5-a]pyrimidine
(10c). Prepared from 5a (175 mg, 1.1 mmol) and 7b (197 mg, 1.0 mmol), G.P.A, CC (EtOAc-hexanes,
–1
1
3
1
1
:1). Pale brown solid (319 mg, 93%); mp 127–133 °C; [α]D22 –64.6 (c 0.17, CH Cl );  /cm (ATR)
2
2
max
345, 3114, 1680, 1614, 1525, 1463, 1443, 1391, 1366, 1331, 1306, 1269, 1248, 1219, 1162, 1112,
080, 1059, 1021, 937, 919, 866, 846, 822, 812, 766, 747, 687, 658, 640; δ (500 MHz; CDCl ; Me Si)
H
3
4
.43 (s, 9H), 1.75 (d, J = 7.1 Hz, 3H), 5.36–5.49 (m, 1H), 5.97 (s, 1H), 6.75 (d, J = 4.2 Hz, 1H), 7.00
s, 1H), 7.39–7.44 (m, 1H), 7.48 (dd, J = 6.7, 8.3 Hz, 2H), 8.00–8.06 (m, 2H), 8.42 (d, J = 4.2 Hz, 1H);
δ (126 MHz; CDCl ; Me Si) 18.7, 28.4, 48.0, 80.2, 93.5, 104.5, 126.5, 126.7, 128.8, 129.1, 132.8,
(
C
3
4
+
+
1
49.5, 150.5, 154.9, 155.7; HRMS (ESI): MH , found 339.1814. [C H N O ] requires 339.1816;
19 23 4 2
(
Found: C, 66.93; H, 6.88; N, 16.00. C H N O ·⅕H O requires C, 66.73; H, 6.60; N, 16.38%).
1
9
23
4
2
2
3
.3.6.
(1’S)-7-{1-[(tert-Butoxycarbonyl)amino]ethyl}-2-(2-methoxyphenyl)pyrazolo[1,5-
a]pyrimidine (10d). Prepared from 5b (208 mg, 1.1 mmol) and 7b (197 mg, 1.0 mmol), G.P.A, CC
EtOAc-hexanes, 1:1). Pale yellow solid (316 mg, 86%); mp 124–126 °C; [α]D22 –71.1 (c 0.19,
(
–
1
CH Cl );  /cm (ATR) 3370, 2974, 1678, 1613, 1583, 1538, 1516, 1479, 1443, 1392, 1369, 1331,
2
2
max
1
304, 1247, 1161, 1114, 1060, 1020, 938, 862, 848, 822, 797, 783, 746, 689, 658, 637; δ (500 MHz;
H
CDCl ; Me Si) 1.42 (s, 9H), 1.75 (d, J = 7.1 Hz, 3H), 3.97 (s, 3H), 5.32–5.48 (m, 1H), 6.11 (s, 1H),
3
4
6
.72 (d, J = 4.2 Hz, 1H), 7.05 (dd, J = 8.4, 1.0 Hz, 1H), 7.09 (td, J = 7.5, 1.1 Hz, 1H), 7.29 (s, 1H), 7.40
(
ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 8.15 (dd, J = 7.8, 1.8 Hz, 1H), 8.41 (d, J = 4.2 Hz, 1H); δ (126 MHz;
C
CDCl ; Me Si) 18.7, 28.4, 48.2, 55.6, 80.1, 97.9, 104.4, 111.5, 120.8, 121.7, 129.4, 130.1, 148.1, 148.9,
3
4
+
+
1
50.0, 152.7, 154.9, 157.6; HRMS (ESI): MH , found 369.1921. [C H N O ] requires 369.1920.
20 25 4 3
3
.3.7.
(1’S)-7-{1-[(tert-Butoxycarbonyl)amino]ethyl}-2-methyl-3-phenylpyrazolo[1,5-
a]pyrimidin-7-yl)ethyl]carbamate (10e). Prepared from 5c (191 mg, 1.1 mmol) and 7b (197 mg, 1.0
2
3

mmol), G.P.A, CC (EtOAc-hexanes, 1:4). Yellow solid (210 mg, 60%); mp 143–146 °C; [α]D22 –60.5
–
1
(
c 0.20, CH Cl );  /cm (ATR) 3357, 3983, 1678, 1608, 1552, 1521, 1476, 1441, 1391, 1367, 1345,
2
2
max
1
325, 1302, 1253, 1208, 1164, 1115, 1077, 1062, 1029, 1007, 908, 863, 825, 773, 748, 697, 652, 633;
δ (500 MHz; CDCl ; Me Si) 1.44 (s, 9H), 1.70 (d, J = 7.1 Hz, 3H), 2.65 (s, 3H), 5.32–5.43 (m, 1H),
H
3
4
6
8
1
.01 (s, 1H), 6.72 (d, J = 4.2 Hz, 1H), 7.27–7.35 (m, 1H), 7.48 (t, J = 7.7 Hz, 2H), 7.62–7.75 (m, 2H),
.43 (d, J = 4.2 Hz, 1H); δ (126 MHz; CDCl ; Me Si) 14.3, 18.8, 28.4, 47.8, 80.1, 104.1, 109.4, 126.4,
C
3
4
+
28.6, 128.9, 132.3, 146.8, 148.5, 149.3, 152.2, 154.9; HRMS (ESI): MH , found 353.1973.
+
[
C H N O ] requires 353.1972; (Found: C, 68.32; H, 6.80; N, 15.88. C H N O requires C, 68.16;
2
0
25
4
2
20 24
4
2
H, 6.86; N, 15.90%).
3
.3.8.
(1’S)-7-{1-[(tert-Butoxycarbonyl)amino]ethyl}-2-benzyl-3-phenylpyrazolo[1,5-
a]pyrimidin-7-yl)ethyl]carbamate (10f). Prepared from 5d (274 mg, 1.1 mmol) and 7b (197 mg, 1.0
mmol), G.P.A, CC (EtOAc-hexanes, 1:4). Pale yellow solid (253 mg, 59%); mp 131–139 °C; [α]D22
–
–
1
4
1
6
2
8.0 (c 0.18, CH Cl );  /cm (ATR) 3351, 2981, 1677, 1611, 1556, 1522, 1493, 1442, 1391, 1366,
2
2
max
328, 1297, 1265, 1249, 1213, 1161, 1112, 1074, 1060, 1027, 1007, 859, 829, 779, 755, 737, 720, 694,
62, 625, 606; δ (500 MHz; CDCl ; Me Si) 1.43 (s, 9H), 1.70 (d, J = 7.0 Hz, 3H), 4.33 (d, J = 1.4 Hz,
H
3
4
H), 5.35 (dq, J = 8.3, 8.8 Hz, 1H), 6.74 (d, J = 4.2 Hz, 1H), 6.05 (s, 1H), 7.18–7.55 (m, 10H), 8.44 (d,
J = 4.2 Hz, 1H); δ (126 MHz; CDCl ; Me Si) 18.8, 28.4, 33.8, 48.1, 80.1, 104.6, 110.0, 126.2, 126.7,
C
3
4
+
1
4
28.4, 128.6, 128.7, 129.4, 131.9, 139.3, 146.9, 148.4, 149.4, 154.1, 154.9; HRMS (ESI): MH , found
+
29.2283. [C H N O ] requires 429.2285; (Found: C, 72.32; H, 6.44; N, 12.97. C H N O ·¼H O
2
6
29
4
2
26 28
4
2
2
requires C, 72.11; H, 6.63; N, 12.94%).
3
.3.9.
(1’S)-7-{1-[(tert-Butoxycarbonyl)amino]ethyl}-2-(3-nitrophenyl)pyrazolo[1,5-
a]pyrimidine (10g). Prepared from 5e (225 mg, 1.1 mmol) and 7b (197 mg, 1.0 mmol), G.P.A, isolated
2
4

by filtration. Pale yellow solid (219 mg, 57%); mp 218–223 °C; [α]D22 –30.3 (c 0.15, CH Cl );  /cm–
2
2
max
1
(ATR) 3350, 2979, 1679, 1611, 1526, 1464, 1394, 1368, 1344, 1328, 1303, 1268, 1251, 1218, 1164,
1
113, 1061, 1023, 957, 815, 864, 845, 800, 777, 761, 743, 698, 659, 636; δ (500 MHz; DMSO-d ;
H
6
Me Si) 1.40 (s, 9H), 1.54 (d, J = 7.1 Hz, 3H), 5.49 (p, J = 7.1 Hz, 1H), 7.00 (d, J = 4.4 Hz, 1H), 7.52 (s,
4
1
7
1
1
5
H), 7.83 (t, J = 8.0 Hz, 1H), 7.92 (d, J = 7.7 Hz, 1H), 8.29 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H), 8.54 (dt, J =
.8, 1.3 Hz, 1H), 8.61 (d, J = 4.3 Hz, 1H), 8.86 (t, J = 2.0 Hz, 1H); δ (126 MHz; DMSO-d6; Me Si)
C
4
8.3, 28.6, 45.6, 79.1, 94.6, 104.6, 120.8, 124.1, 131.1, 133.1, 134.7, 148.9, 150.3, 150.9, 152.3, 152.9,
+
+
55.5; HRMS (ESI): MH , found 384.1661. [C H N O ] requires 384.1666; (Found: C, 58.83; H,
1
9
22
5
4
.34; N, 17.79. C H N O ·¼H O requires C, 58.83; H, 5.59; N, 18.05%).
1
9
21
5
4
2
3
.3.10.
(1’S)-7-{1-[(tert-Butoxycarbonyl)amino]ethyl}-2-(3-trifluorophenyl)pyrazolo[1,5-
a]pyrimidine (10h). Prepared from 5f (250 mg, 1.1 mmol) and 7b (197 mg, 1.0 mmol), G.P.A, isolation
by filtration. White solid (303 mg, 75%); mp 196–197 °C; [α]D22 –33.2 (c 0.16, CH Cl );  /cm–1
2
2
max
(ATR) 3351, 2990, 1679, 1617, 1524, 1466, 1450, 1395, 1369, 1331, 1303, 1270, 1253, 1226, 1158,
1
112, 1100, 1080, 1060, 1021, 951, 917, 853, 824, 798, 782, 749, 694, 657, 635; δ (500 MHz; CDCl ;
H
3
Me Si) 1.44 (s, 9H), 1.74 (d, J = 7.1 Hz, 3H), 5.48 (p, J = 7.2 Hz, 1H), 5.66–5.86 (m, 1H), 6.81 (d, J =
4
4
8
9
.2 Hz, 1H), 7.04 (s, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 8.19 (d, J = 7.7 Hz, 1H),
.26 (d, J = 2.2 Hz, 1H), 8.46 (d, J = 4.2 Hz, 1H); δ (126 MHz; CDCl ; Me Si) 18.7, 28.3, 47.6, 80.4,
C
3
4
3.8, 104.6, 123.4 (q, J = 3.8 Hz), 124.1 (q, J = 273.4 Hz), 125.5 (q, J = 3.8 Hz), 129.3, 129.9, 131.2
+
(
q, J = 32.7 Hz), 133.7, 149.5, 149.7, 150.6, 164.2, 154.8; HRMS (ESI): MH , found 407.1689.
+
[
C H F N O ] requires 407.1683; (Found: C, 59.20; H, 4.99; N, 13.82. C H F N O requires C,
2
0
22
3
4
2
20 21
3
4
2
5
9.11; H, 5.21; N, 13.79%).
2
5

3
.3.11.
carboxylate (10i). Prepared from 5g (1.425 g, 10.1 mmol) and 7b (2.00 g, 10.1 mmol), G.P.B, CC
EtOAc-hexanes, 2:1). Pale yellow solid (2.27 g, 70%); physical and spectral data were in agreement
with the literature data [28].
Methyl
(1’S)-7-{1-[(tert-butoxycarbonyl)amino]ethyl}pyrazolo[1,5-a]pyrimidine-3-
(
3
.3.12.
(4’S)-4-[1-[(tert-Butoxycarbonyl)amino]ethyl]pyrimido[1,2-b]indazole (10j). Prepared
from 5h (146 mg, 1.1 mmol) and 7b (197 mg, 1.0 mmol), G.P.A, CC (EtOAc-hexanes, 1:1). Brown
solid (133 mg, 46%); mp 131–135 °C; [α]D22 –78.6 (c 0.16, CH Cl );  /cm (ATR) 3357, 2978,
–1
2
2
max
1
8
5
680, 1635, 1595, 1519, 1441, 1391, 1366, 1344, 1324, 1301, 1247, 1162, 1125, 1078, 1055, 1013,
61, 831, 752, 735, 645; δ (500 MHz; CDCl ; Me Si) 1.26–1.55 (m, 9H), 1.78 (d, J = 7.1 Hz, 3H),
H
3
4
.57 (p, J = 7.7 Hz, 1H), 6.16–6.38 (m, 1H), 7.13–7.26 (m, 1H), 7.31 (d, J = 8.2, 6.7 Hz, 1H), 7.64
(
ddd, J = 8.3, 6.7, 1.1 Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 8.33 (d, J = 8.3 Hz, 1H), 8.62 (d, J = 4.4 Hz,
H); δ (126 MHz; CDCl ; Me Si) 18.2, 28.3, 48.4, 80.2, 108.4, 113.3, 116.3, 120.8, 120.9, 130.0,
1
1
3
1
C
3
4
+
+
44.5, 145.5, 147.6, 150.9, 154.9; HRMS (ESI): MH , found 313.1659. [C H N O ] requires
1
7
21
4
2
13.1654; (Found: C, 64.33; H, 6.52; N, 17.37. C H N O ·¼H O requires C, 64.44; H, 6.52; N,
1
7
20
4
2
2
7.68%).
3
.3.13.
(1’S)-7-({1-[(tert-Butoxycarbonyl)amino]-2-phenyl}ethyl)-2-phenyl-1-(2-
phenylpyrazolo[1,5-a]pyrimidine (10k). Prepared from 5a (175 mg, 1.1 mmol) and 7c (274 mg, 1.0
mmol), G.P.A, CC (EtOAc-hexanes, 1:2). Pale yellow solid (200 mg, 48%); mp 139–142 °C; [α]D22
–
1
+
24.5 (c 0.13, CH Cl );  /cm (ATR) 3351, 2978, 1680, 1613, 1543, 1513, 1462, 1443, 1391, 1365,
2
2
max
1
318, 1268, 1250, 1222, 1163, 1084, 1052, 1020, 938, 918, 889, 851, 817, 788, 764, 732, 691, 636; δH
(
500 MHz; CDCl ; Me Si) 1.40 (s, 9H), 3.37 (dd, J = 13.6 Hz, 7.2 Hz, 1H), 3.63 (dd, J = 14.2 Hz, 8.3
3
4
Hz, 1H), 5.48–5.59 (m, 1H), 6.01–6.17 (m, 1H), 6.49 (d, = J 4.3 Hz, 1H), 7.01 (d, J = 6.9 Hz, 2H), 7.11
2
6

(s, 1H), 7.18–7.25 (m, 3H), 7.44–7.48 (m, 1H), 7.53 (dd, J = 8.3, 6.7 Hz, 2H), 8.06–8.10 (m, 2H) 8.31
(
d, J = 4.3 Hz, 1H); δ (126 MHz; CDCl ; Me Si) 28.3, 38.1, 53.9, 80.3, 93.6, 106.2, 126.7, 127.0,
C
3
4
+
1
4
28.6, 128.8, 129.0, 129.2, 132.7, 136.6, 146.4, 149.1, 150.4, 155.0, 155.8; HRMS (ESI): MH , found
+
15.2124. [C H N O ] requires 415.2129.
2
5
27
4
2
3
.3.14.
Methyl
(1’S)-7-{1-[(tert-butoxycarbonyl)amino]-2-phenylethyl}pyrazolo[1,5-
a]pyrimidine-3-carboxylate (10l). Prepared from 5g (706 mg, 5.0 mmol) and 7c (1.365 g, 5.0 mmol),
G.P.B, CC (EtOAc-hexanes, 1:1). Pale yellow solid (400 mg, 20%); mp 143–146 °C; [α]D22 +36.1 (c
–
1
0
1
7
1
6
.23, CH Cl );  /cm (ATR) 3336, 2974, 1704, 1683, 1620, 1553, 1526, 1487, 1455, 1436, 1377,
2
2
max
336, 1317, 1276, 1255, 1219, 1199, 1161, 1100, 1049, 1031, 1001, 951, 910, 891, 850, 823, 800, 781,
55, 728, 702, 683, 642, 627; δ (500 MHz; CDCl ; Me Si) 1.39 (s, 9H), 3.30 (dd, J = 13.7 Hz, 7.3 Hz,
H
3
4
H), 3.49 (dd, J = 13.7 Hz, 7.6 Hz, 1H), 3.99 (s, 3H), 5.52 (q, J = 8.0 Hz, 1H), 6.02 (d, J = 9.0 Hz, 1H),
.68 (d, J = 4.4 Hz, 1H), 6.90–7.00 (m, 2H), 7.13-7.25 (m, 3H), 8.60 (d, J = 4.4 Hz, 1H), 8.68 (s, 1H);
δ (126 MHz; CDCl ; Me Si) 28.3, 38.2, 51.8, 53.7, 80.5, 102.9, 108.1, 127.2, 128.7, 128.9, 136.0,
C
3
4
+
+
1
3
1
47.5, 148.2, 148.5, 152.5, 154.9, 163.0; HRMS (ESI): MH , found 397.187 [C H N O ] requires
21 25 4 4
97.187; (Found: C, 63.42; H, 6.08; N, 13.81. C H N O ·⅛ H O requires C, 63.26; H, 6.13; N,
2
1
24
4
4
2
4.05%).
3
.4.
General procedure for the synthesis of compounds 11.
A mixture of the ester 10 (0.5 mmol), MeOH (7 mL), water (5 mL), and LiOH·H O (46 mg, 1.1
2
mmol) was stirred at r.t. for 4 days. Saturated aq. NaHCO (20 mL) was added, the non-polar
3
impurities were extracted with CH Cl (3 × 10 mL) and the aqueous phase was acidified with 1 M aq.
2
2
2
7

NaHSO (15 mL). The product was extracted with CH Cl (3 × 15 mL), the combined organic phases
4
2
2
were dried over anhydrous Na SO , filtered, and the filtrate was evaporated in vacuo to give 11.
2
4
3
.4.1.
7-{[(tert-Butoxycarbonyl)amino]methyl}pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
(
11a). Prepared from 10b (148 mg, 0.5 mmol) and LiOH·H O following general procedure. Pale pink
2
–
1
solid (92 mg, 65%); mp 113–141 °C (decomp.); max/cm (ATR) 2977, 1683, 1620, 1554, 1531, 1410,
1
392, 1367, 1284, 1251, 1158, 1095, 938, 861, 821, 785, 761, 668, 637, 614; δ (500 MHz; DMSO-d ;
H
6
Me Si) 1.44 (s, 9H), 4.68 (d, J = 5.2 Hz, 2H), 7.08 (d, J = 4.4 Hz, 1H), 7.76 (t, J = 6.0 Hz, 1H), 8.65 (s,
4
1
H), 8.80 (d, J = 4.4 Hz, 1H), 12.38 (s, 1H); δ (126 MHz; DMSO-d ; Me Si) 28.6, 55.9, 79.3, 103.2,
C 6 4
+
+
1
06.8, 147.7, 147.8, 149.0, 153.1, 156.3, 163.7; HRMS (ESI): MH , found 293.1240. [C H N O ]
13 17 4 4
requires 293.1244.
3
.4.2.
(1’S)-7-{1-[(tert-Butoxycarbonyl)amino]ethyl}pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid (11b). Prepared from 10i (1.10 g, 3.4 mmol) and LiOH·H O following general procedure. Pale
2
yellow solid (930 mg, 89%); mp 77–80 °C; [α]D22 –25.0 (c 0.08, CH Cl );  /cm (ATR) 3307,
–1
2
2
max
2
8
5
4
1
3
978, 1685, 1618, 1547, 1520, 1452, 1392, 1366, 1323, 1250, 1162, 1108, 1058, 1010, 953, 909, 862,
36, 786, 756, 657, 632, 614; δ (500 MHz; DMSO-d ; Me Si) 1.39 (s, 9H); 1.48 (d, J = 7.1 Hz, 3H);
H
6
4
.41 (p, J = 7.1 Hz, 1H); 7.16 (d, J = 4.4 Hz, 1H); 7.97 (d, J = 7.5 Hz, 1H); 8.67 (s, 1H); 8.83 (d, J =
.3 Hz, 1H); 12.45 (s, 1H); δ (126 MHz; CDCl ; Me Si) 18.4, 28.6, 45.8, 79.2, 103.2, 105.6, 147.8,
C
3
4
+
+
47.9, 153.4, 153.8, 155.4, 163.6; HRMS (ESI): MH , found 307.1401. [C H N O ] requires
1
4
19
4
4
07.1401.
3
.4.3.
(1’S)-7-{1-[(tert-Butoxycarbonyl)amino]-2-phenylethyl}pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid (11c). Prepared from 10l (198 mg, 0.5 mmol) and LiOH·H O following general
2
2
8

procedure. Pale brown solid (103 mg, 54%); mp 86–95 °C (decomp.); [α]D22 –20.5 (c 0.14, CH Cl );
2
2
–
1
max/cm (ATR) 3340, 2978, 1683, 1619, 1548, 1520, 1494, 1454, 1392, 1366, 1274, 1250, 1160,
1
9
5
106, 1048, 1017, 919, 889, 852, 821, 780, 750, 730, 698, 645; δ (500 MHz; CDCl ; Me Si) 1.39 (s,
H 3 4
H), 3.31 (dd, J = 13.8 Hz, 7.4 Hz, 1H), 3.50 (dd, J = 13.6 Hz, 7.9 Hz, 1H), 5.54 (d, J = 8.4 Hz, 1H),
.88 (d, J = 8.8 Hz, 1H), 6.74 (s, 1H), 6.97 (d, J = 6.4 Hz, 2H), 7.18–7.31 (m, 3H), 8.57 (s, 1H), 8.74
(
s, 1H); δ (126 MHz; CDCl ; Me Si) 28.3, 38.1, 53.7, 80.7, 102.4, 108.2, 127.3, 128.8, 128.9, 135.8,
C 3 4
+
+
1
3
47.6, 148.4, 149.4, 152.4, 154.9, 165.3; HRMS (ESI): MH , found 383.1713. [C H N O ] requires
20 23 4 4
83.1714.
3
.5.
General procedure for the synthesis of compounds 13.
Under Ar CDI (0.5 mmol) was added to a solution of 11 (0.4 mmol) in anhydrous THF (5 mL) and
the mixture was stirred at r.t. for 2 h. Amine 12 (2.0 mmol) or its respective salt (0.44 mmol) and
triethylamine (0.44 mmol) was added and stirring at r.t. was continued for 16 h. The mixture was
acidified with 1 M aq. NaHSO (10 mL) and the product was extracted with CH Cl (2 × 10 mL). The
4
2
2
combined organic phases are washed with saturated NaHCO (10 mL), dried over anhydrous Na SO ,
3
2
4
filtered, and the filtrate was evaporated in vacuo. The residue was purified by CC (EtOAc/hexanes).
Fractions containing the product were combined and evaporated in vacuo to give 13.
3
.5.1.
7-[(tert-Butoxycarbonyl)aminomethyl]pyrazolo[1,5-a]pyrimidine-3-(N-
methylcarboxamide) (13a). Prepared from 11b (65 mg, 0.22 mmol), 12a·HCl (8 mg, 0.24 mmol), and
triethylamine (34 μL, 0.24 mmol) following general procedure. Yellow solid (33 mg, 49%); mp 168–
–
1
1
1
70 °C; max/cm (ATR) 3308, 2931, 1718, 1635, 1618, 1557, 1511, 1415, 1365, 1329, 1288, 1250,
158, 1126, 1094, 1053, 968, 938, 906, 869, 845, 802, 779, 659, 643, 620; δ (500 MHz; CDCl ;
H
3
2
9