Development of Scalable Synthesis of 5-Butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine (WQE-134), a Dual Inhibitor of Nitric Oxide and Prostaglandin E2Production

Article abstract of DOI:10.1021/acs.oprd.0c00324

We report a scalable efficient synthesis of 5-butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine (WQE-134), a dual inhibitor of nitric oxide and prostaglandin E2 production with potent anti-inflammatory properties. The original five-step synthesis (40% overall yield) was based on two Suzuki-Miyaura reactions and required chromatographic purification of both the intermediate and final products. The novel synthetic pathway is based on cyclization of 2-butyl-1-(4-methoxyphenyl)-3-phenylpropane-1,3-dione with guanidinium methanesulfonate using Eaton's reagent (P2O5/MsOH) at 50 °C. The two-step synthesis (34% overall yield) can be performed on a multigram to kilogram scale, and purification of the final product consists of simple extraction (dichloromethane) and crystallization (ethyl acetate and methanol).

Full text of DOI:10.1021/acs.oprd.0c00324

Subscriber access provided by University of Rochester | River Campus & Miner Libraries
Full Paper
Development of scalable synthesis of 5-butyl-4-(4-
methoxyphenyl)-6-phenylpyrimidin-2-amine (WQE-134), a
2
dual inhibitor of nitric oxide and prostaglandin E production
Pavel Dosoudil, Vladislav Kotek, Viktor Kolman, Ond#ej Baszczy#ski,
Martin Maxmilian Kaiser, Zlatko Janeba, and Miroslav Havránek
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00324 • Publication Date (Web): 28 Aug 2020
Downloaded from pubs.acs.org on August 28, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 17
Organic Process Research & Development
1
2
3
4
5
6
Development of scalable synthesis of 5-butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-
amine (WQE-134), a dual inhibitor of nitric oxide and prostaglandin E2 production
7
8
9
Pavel Dosoudil,† Vladislav Kotek,† Viktor Kolman,‡ Ondřej Baszczyňski,‡ Martin Maxmilian
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Kaiser,‡ Zlatko Janeba,‡,* and Miroslav Havránek†,*
†APIGENEX s.r.o., Poděbradská 173/5, 190 00 Prague 9, Czech Republic
‡Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences,
Flemingovo nám. 2, 166 10, Prague 6, Czech Republic
1
ACS Paragon Plus Environment

Organic Process Research & Development
Page 2 of 17
1
2
3
4
Table of Contents graphic
5
6
7
8
9
NH2
O
original method
new method
O
O
N
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EtO
OEt
O
34% overall
2 steps
no chromatography
40% overall
5 steps
2x chromatography
WQE-134
2
ACS Paragon Plus Environment

Page 3 of 17
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
Abstract: We report a scalable efficient synthesis of 5-butyl-4-(4-methoxyphenyl)-6-
phenylpyrimidin-2-amine (WQE-134), a dual inhibitor of nitric oxide and prostaglandin E2
production with potent anti-inflammatory properties. The original five-step synthesis (40%
overall yield) was based on two Suzuki-Miyaura reactions and required a chromatographic
purification of both intermediate and final product. The novel synthetic pathway is based on
cyclization of 2-butyl-1-(4-methoxyphenyl)-3-phenylpropane-1,3-dione with guanidinium
methanesulfonate using Eaton’s reagent (P2O5/MsOH) at 50 °C. The two-step synthesis (34%
overall yield) can be performed on a multigram to kilogram scale and purification of the final
product consists of simple extraction (dichloromethane) and crystallization (ethyl acetate and
methanol).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Keywords: Pyrimidines, Eaton’s reagent, upscale, C-acylation
1. INTRODUCTION
Recently, polysubstituted pyrimidines were discovered to possess interesting anti-
inflammatory properties.1-6 As for the mode of action, it was demonstrated that many 5-alkyl-
4,6-diarylpyrimidin-2-amines were able to inhibit the production of nitric oxide and/or
prostaglandin E2. 5-Butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine5 (WQE-134, 5,
Scheme 1) was selected for further preclinical development as an anti-inflammatory agent,
especially for its potential to treat ulcerative colitis. The original synthetic route towards
compound 5 was based on 2-amino-5-butyl-4,6-dichloropyrimidine (3),5 a common precursor
for the whole series of polysubstituted pyrimidines, which was conveniently prepared by
condensation of diethyl 2-butylmalonate (1) with guanidine under basic conditions, followed
by chlorination with Vilsmeier-Haack-Arnold reagent7 and subsequent removal of the
dimethylaminomethylene moiety (Scheme 1). Tandem Suzuki-Miyaura cross-coupling
reactions8 of obtained 4,6-dichloropyrimide intermediate 3 with (4-methoxyphenyl)boronic
and phenylboronic acids gave the desired product 5 in a satisfactory overall (5 steps) yield of
40% (Scheme 1).5
3
ACS Paragon Plus Environment

Organic Process Research & Development
Page 4 of 17
1
2
3
4
Scheme 1: Original synthesis of WQE-1345
5
6
7
8
NH
.HCl
NH2
Cl-
NH2
N
NH2
N
O
O
N+ Cl
1.
H2N
, CHCl3
N
N
EtO
OEt
EtONa, EtOH
96%
crystallization
2. HCl, EtOH
87% (2 steps)
extraction/crystallization
HO
OH
Cl
Cl
9
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
3
NH2
NH2
N
(HO)2B
B(OH)2
N
N
N
O
Cl
Pd(PPh3)4, Na2CO3
toluene, EtOH
52%
Pd(PPh3)4, Na2CO3
1,4-dioxane, H2O
92%
O
O
4
5
WQE-134
chromatography
chromatography
1
40% from
The above-mentioned procedure requires a column chromatography purification step after
each Suzuki-Miyaura cross-coupling reaction. Such a synthetic methodology is suitable for
the synthesis of a larger series of bisaryl-substituted pyrimidines for their biological
evaluation (SAR study), but it is not applicable for the scale-up preparation of a selected
preclinical candidate. Thus, in order to supply enough material for extensive animal studies, it
became unavoidable to develop a more efficient procedure with attempts to eliminate
problematic chromatographic purifications.
2. RESULTS AND DISCUSSION
A more effective approach, involving only one Suzuki-Miyaura coupling, was based on the
cyclization of ß-ketoester 7 (Scheme 2) which was prepared in a 92% yield by alkylation of
commercially available ethyl benzoylacetate (6) with 1-bromobutane. Treatment of ß-
ketoester 7 with guanidine under basic conditions afforded polysubstituted pyrimidine 8 in a
65% yield. Subsequent conversion of hydroxypyrimidine 8 into chloropyrimidine 9 using
Vilsmeier-Haack-Arnold reagent, followed by Suzuki-Miyaura cross-coupling with (4-
methoxyphenyl)boronic acid, gave target compound 5 in a 48% overall yield (5 steps).
Although the overall yield of Method A was slightly improved (by some 8%) compared to the
original synthesis (Scheme 1) and one Suzuki-Miyaura coupling/chromatography step was
eliminated, the total number of reaction steps remained the same, large excess of Vilsmeier-
Haack-Arnold reagent (10 equiv.) had to be used for the efficient chlorination step, and
4
ACS Paragon Plus Environment

Page 5 of 17
Organic Process Research & Development
1
2
3
4
5
6
column chromatography of the final product was still required. Thus, we decided to look for a
more convenient synthetic methodology with a reduced number of reaction steps and with
completely eliminated chromatographic purification.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2: Synthesis of WQE-134 (5) from ketoester 6 (Method A)
NH2
NH2
O
O
O
O
.HCl
NH
N
N
Br
H2N
O
O
OH
6
EtONa, EtOH, reflux
92%
MeOH, MeONa, reflux
65%
precipitation/filtration
7
8
evaporation
NH2
NH2
Cl-
(HO)2B
1.
Cl
N+
N
N
N
N
O
CHCl3, reflux
Cl
Pd(PPh3)4, Na2CO3
1,4-dioxane, H2O
92%
chromatography
crystallization
2. HCl, EtOH
50 °C
extraction/crystallization
O
9
88%
5
6
48% from
The retrosynthetic study of WQE-134 (5) identified hexanophenone (10, Figure 1) as a cheap
starting material and 1,3-diketone 11 as a potential synthetic intermediate. Thus, we
concentrated our efforts on optimization of two reaction steps involved: a) preparation of 2-
butyl-1,3-diketone 11; and b) cyclization of 2-butyl-1,3-diketone 11 with guanidine to give
product 5.
O
NH2
O
O
N
N
base
guanidine
10
+
O
O
11
O
5
Cl
WQE-134
O
Figure 1. Retrosynthesis of WQE-134 (5) leading to 1,3-diketone intermediate 11 and starting
hexanophenone (10).
The first goal was an efficient preparation of 2-butyl-1,3-diketone 11 starting from
commercially available hexanophenone (10) and p-anisoyl chloride. In general, acylation of
5
ACS Paragon Plus Environment

Organic Process Research & Development
Page 6 of 17
1
2
3
4
5
6
7
8
9
enolates suffers from a competition between the C- and O-acylation resulting in the formation
of a mixture of C- and O-acylated products which are difficult to separate.9 Wiles et al.10
reported a simple procedure for the regioselective C-acylation of ketones from their respective
lithium enolates (prepared with LiHMDS in THF) and silyl enol ethers using various
acylating reagents. Later, direct acylation of lithium enolates was described,11 and it was
found that toluene was even better reaction solvent for selective C-acylation than THF, with
no homocondensation observed.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Treatment of hexanophenone (10, Scheme 3) with p-anisoyl chloride using LiHMDS in
toluene afforded desired 2-butyl-1,3-diketone 11 quantitatively. It is necessary to note,
however, that 1,3-diketone 11 undergoes keto-cleavage when treated with a base, e.g. sodium
hydroxide, in a similar way as acylenolates. Thus, hexanophenone (10), butyllithium,
hexamethyldisilazane (HMDS), and p-anisoyl chloride were used in an equimolar ratio,
despite the fact that an excess of ketone and base (2 equiv.) was recommended in the
literature.11 Also, since the acidic hydrogen of formed 1,3-diketone can potentially react with
hexanophenone-enolate under the reaction conditions, we changed the setup of the reaction:
the solution of lithium enolate was slowly added to the solution of p-anisoyl chloride at low
temperature (-20 °C). This setup allowed for the quantitative conversion of all reagents used
in the equimolar ratio.
Scheme 3. Synthesis of 2-butyl-1,3-diketone 11 and confirmation of its structure by
transformation to pyrazine derivative 12
O
O
O
1. LiHMDS, toluene, -20 °C
O
2.
Cl
O
O
11
98%
H
10
O
C-acylated product
N2H4
AcOH
O
O
toluene
O-acylated product
-20 °C to -5 °C
12 h
not observed
N
HN
O
nBu
12
MW: 306,4
1
Since the H NMR analysis of product 11 (Scheme 3) was not conclusive enough due to the
multiplicity of hydrogens, we needed another proof that compound 11 was the C-acylated
product. Thus, subsequent reaction of compound 11 with hydrazine in acetic acid led to the
6
ACS Paragon Plus Environment

Page 7 of 17
Organic Process Research & Development
1
2
3
4
5
6
unambiguous formation of 4-butyl-3-(4-methoxyphenyl)-5-phenyl-1H-pyrazole (12, Scheme
3), giving us the proof of the correct structure of 1,3-diketone 11.
7
8
9
The next step was the cyclization reaction of 2-butyl-1,3-diketone 11 with guanidine. Despite
the high reactivity of compound 11 with hydrazine (Scheme 3), the reaction of 11 with
guanidine proved to be very slow. We observed only traces of product (2-5%) when
compound 11 was treated with guanidine carbonate or with free guanidine in triflurorethanol
at elevated temperature. Reactions using a combination of other solvents (water, DMSO,
THF, HFIP or ethanol) and bases (Cs2CO3, DBU, EtONa, iPrONa) led mostly to the cleavage
of 1,3-diketone without a formation of the desired product. Acid mediated reaction conditions
(AcOH, DMF/AcOH, HCl in EtOH, HCl in iPrOH or 1,4-dioxane, H2SO4 in TFE or EtOH)
did not afford any product either, although the decomposition process seemed to be slower.
The reaction of compound 11 with Boc-protected guanidine under various reaction conditions
did not work either. Next, we decided to explore the acid-catalyzed cyclization using urea
instead of guanidine.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The condensation of crude 1,3-diketone 11 with urea and HCl (4 N) in 1,4-dioxane under
heating afforded desired 2-hydroxypyrimidine derivative 13 (Scheme 4) in a 34% yield (from
compound 10). Subsequently, treatment of compound 13 with Arnold’s reagent under reflux
gave 2-chloropyrimidine analogue 14 in a 66% yield. Nevertheless, the final amination of
compound 14 to yield 5 proved to be problematic. Simple aromatic nucleophilic substitution
with ammonia did not afford any product, while reaction with sodium amide gave only traces
of the desired product (UPLC-MS). The solution offered Buchwald-Hartwig reaction:
treatment of 2-chloropyrimidine 14 with formamide resulted in the mixture of 2-
aminopyrimidine and 2-formamidopyrimidine derivatives, which upon basic conditions
(deformylation) afforded final compound 5 in a 96% yield. Thus, although chromatography
was completely eliminated, Method B gave compound 5 in much lower overall yield
compared to Method A (22% from hexanophenone (10) compared to 48% from ketoester 6,
respectively). The result led us to rethink the direct conversion of 1,3-diketone 11 into
compound 5 in one step.
Scheme 4: Synthesis of WQE-134 (5) from hexanophenone (Method B)
7
ACS Paragon Plus Environment

Organic Process Research & Development
Page 8 of 17
1
2
3
4
5
6
7
8
9
O
LiHMDS, toluene
-20 °C to RT, 16 h
OH
O
O
urea
N
N
O
Cl
HCl, 1,4-dioxane
75 °C, 4 days
34% from
extraction
precipitation
10
O
11
10
13
O
O
Cl
NH2
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1. HCOONH2, Pd2dba3. CHCl3,
Xantphos, K2CO3, 1,4-dioxane
DMF, (COCl)2
N
N
N
DCM
reflux, 48 h
66%
2. NaOH, MeOH, H2O, 1,4-dioxane
100 °C
O
O
crystallization
5
96%
filtration
through
silica gel
14
10
22% from
After thorough literature search, we decided to exploit Eaton’s reagent12 for the desired
condensation. We were able to detect product 5, when 1,3-diketone 11 and guanidinium
carbonate were dissolved in a solution of phosphorous pentoxide in methanesulfonic acid and
the mixture was heated to 80 °C, but the reaction was slow. In order to increase the
conversion and purity of the reaction, it was necessary to maintain a high concentration of the
reagents and keep the reaction mixture at 80 °C for several days. In order to estimate the right
ratio of starting compounds, it was important to consider the fact that Eaton’s reagent is a
phosphorous pentoxide/methanesulfonic acid (1:10) mixture and that two equivalents of water
are released during the reaction. Subsequently, we changed the ratio to 1:5 (P2O5/MsOH) and
reaction temperature to 50 °C. Guanidine carbonate during the reaction also releases one
equivalent of water. In order to eliminate water formation and keep maximum possible
concentration of the reagents, guanidinium mesylate (15, Scheme 5) was prepared
quantitatively by suspending guanidine carbonate in methanol and slow addition of
methanesulfonic acid. Guanidinium mesylate (15) as such can be used in excess without the
necessity to increase the amount of Eaton’s reagent.
Thus, treatment of 1,3-diketone 11 with guanidinium mesylate (15) at 50 °C for 8 days
(Method C, Scheme 5) afforded the desired compound 5 in a 35% yield. By Method C, we
were able to prepare the final compound 5 from cheap hexanophenone (10), p-anisoyl
chloride, and pre-prepared guanidinium mesylate (15) in an overall 34% yield in two reaction
steps using only extraction and crystallization as purification methods (chromatography was
completely eliminated). The crude product 5 can be conveniently purified by subsequent
crystallization from cyclohexane and methanol. (Note 1: Crude compound 11 (93% HPLC
purity) was used in the subsequent cyclization step (Scheme 5, Method C) and purity of the
8
ACS Paragon Plus Environment

Page 9 of 17
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
final product 5 was 98.6% (HPLC). Purity of product 5 can be increased up to 99.2% when
purified 1,3-diketone 11 (99.4% HPLC purity after crystallization from cyclohexane) is used
for the cyclization, but the crystallization process decreases the yield of compound 11 to 50%.
Note 2: In order to decrease the reaction time of the cyclization step (8 days), several
experiments were carried out with increased reaction temperature, but such reaction
conditions led to lower yields and lower purity of product, due to the fasrter formation of by-
products).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 5: Two-step synthesis of WQE-134 (5) from hexanophenone 10 (Method C)
HN
NH2
MsOH, MeOH
2-
+
2 CO3
96%
filtration
HN
NH3
NH2
N
NH2
MsO-
O
O
O
1. LiHMDS,
toluene, -20 °C
+
15
N
NH3
P2O5, MsOH
50 °C, 8 days
35%
O
2.
Cl
O
10
11
5
O
extraction
crystallization
O
10
98% from
WQE 134
10
toluene, -20 °C
34% from
During the process development and analytical control, about 0.5% of impurities were
observed in final product 5. The side products were difficult to detect using reverse phase
HPLC methodology as the peaks of the impurities had almost identical Rf values as the main
1
product. Similarly, the impurities could not be easily detected by H NMR analysis because
their peaks had signals overlaid by the signals of the main product. Later, normal phase HPLC
method was developed with sufficient separation of peaks of the product and both impurities,
which were then identified (by the means of UPLC/MS) as symmetrically bis-arylated
pyrimidine compounds 16 and 17 (Scheme 6). Furthermore, compounds 16 and 17 were
synthesized by the original procedure5 and thus, the identity of the side products was
unambiguously confirmed by comparison of HPLC Rf values and NMR spectra. The
impurities were produced during the first reaction step when the product, 1,3-diketone 11, was
cleaved by lithium enolate 18 and, consequently, symmetrical diketones 19 and 20 (Scheme
6) were formed in trace amounts. Diketones 19 and 20 then underwent cyclization with
guanidinium mesylate (15) to give compounds 16 and 17, respectively.
9
ACS Paragon Plus Environment

Organic Process Research & Development
Page 10 of 17
1
2
3
4
Scheme 6: Formation of impurities 16 and 17
5
6
7
NH2
N
NH2
N
N
N
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
16
17
O-Li+
O
O
O
O
O
O
18
toluene
+
UPLC/MS
(compounds
not isolated)
O
+ other by-products
O
O
20
19
11
As a proof, isolated 1,3-diketone 11 was treated with lithium enolate 18 in toluene and
symmetrical diketones 19 and 20 were identified (UPLC/MS) in the reaction mixture.
However, those products were not stable and could not be isolated. The total amount of
impurities after the first reaction step was always below 5 mol%. The most successful method
of reducing the amount of impurities in the final product was the crystallization of crude 1,3-
diketone 11 from cyclohexane. Using this technique, 1,3-diketone 11 was obtained in high
purity (up to 99%), which in the following reaction step afforded cyclized product 5
containing less than 0.1% of impurities.
3. CONCLUSIONS
In conclusion, we developed several alternative approaches for the preparation of 5-butyl-4-
(4-methoxyphenyl)-6-phenylpyrimidin-2-amine (5, WQE-134), starting from either ethyl
benzoylacetate (6) or hexanophenone (10). The goal was to reduce the number of reaction
steps (5 steps in the original synthesis) and to eliminate chromatographic purifications (2
chromatographies in the original procedure). In the best method we optimized the synthesis of
compound 5 using Eaton’s reagent in order to achieve highly acidic conditions in the critical
cyclization step. Compound 5 was prepared from cheap starting hexanophenone (10) in a 34%
overall yield in two steps and the use of column chromatography was avoided completely.
This methodology was scaled up for the preparation of 1175 g of compound 5 in high purity
(> 99.9%).
10
ACS Paragon Plus Environment

Page 11 of 17
Organic Process Research & Development
1
2
3
4
5
6
4. EXPERIMENTAL SECTION
7
8
9
Unless otherwise stated, solvents were evaporated at 40 °C/2 kPa and the compounds were
dried at 40 °C/2 kPa. NMR spectra were recorded on Bruker Avance (300 MHz or 400 MHz)
spectrometers referenced to the residual solvent signal or a specified additive (TMS).
Chemical shifts (δ) are reported in ppm and interaction constants (J) in Hz. The spectra were
referenced to tetramethylsilane (TMS) or the residual solvent signal. High-resolution mass
spectra (HRMS) of the products were obtained using GCT Premier (Waters, USA)
spectrometer for electrospray ionization (ESI). Melting points were measured on the capillary
Büchi apparatus. Purity of the prepared compounds was >95%; the purity was determined by
the combination of HPLC (H2O/CH3CN, linear gradient), UPLC-MS and NMR.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-Butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine (5, WQE-134). Method A: A
mixture of compound 9 (3.30 g, 13 mmol), 4-methoxyphenylboronic acid (1.90 g,
15.6 mmol), tetrakis(triphenylphosphine)palladium (0.75 g, 0.65 mmol) and potassium
carbonate (2.76 g, 26.0 mmol) in 1,4-dioxane (100 mL) and water (30 mL) was heated to 100
°C for 12 h. After cooling to room temperature the solvents were removed in vacuo. The
residue was dissolved in ethyl acetate (100 mL) and adsorbed on silica gel. After the removal
of the organic solvent in vacuo, the product was purified by silica gel column chromatography
using hexane/ethyl acetate mixture (4:1). The product was crystallized from hexanes to give 5
(3.9 g, 92%) as white solid. Analytical data corresponds to the literature.5 Method B: A
mixture of compound 14 (684 g, 1.94 mol), formamide (170 g, 3.88 mol), Xantphos (6.73 g,
11.6 mmol), Pd2(dba)3·CHCl3 (6.02 g, 5.80 mmol), potassium carbonate (347 g, 2.52 mol)
and cesium carbonate (18.9 g, 58.0 mmol) in 1,4-dioxane (1.9 L) was degassed and put under
the nitrogen atmosphere. The reaction mixture was heated to 100 °C for 20 h. (gaseous
ammonia was formed, the exhaust is necessary via narrow tubing). The conversion was
monitored by TLC (cyclohexane/AcOEt, 2:1). After the completion, the reaction mixture was
cooled to ambient temperature and the mixture was filtered and the solid material was washed
with 1,4-dioxane (300 mL). The aqueous solution of sodium hydroxide (310 g, 7.75 mol in
0.5 L of water) was added and the mixture was heated to 100 °C. The conversion of 2-
formamidopyrimidine derivative was monitored by TLC (cyclohexane/AcOEt, 2:1). After 16
h, the reaction mixture was cooled to ambient temperature, water (5 L) was added and the
mixture was extracted with dichloromethane (3 x 2.5 L). Combined organic layers were
11
ACS Paragon Plus Environment

Organic Process Research & Development
Page 12 of 17
1
2
3
4
5
6
7
8
9
filtered through a pad of silica gel (0.5 kg) which was washed with ethyl acetate (1 L).
Solvents were removed in vacuo and cyclohexane was added. Precipitated material was
crystallized from a dichloromethane/cyclohexane mixture to give 5 (432 g, 67%). Method C:
The reactor (50 L) was charged with methanesulfonic acid (10 L) and phosphorus pentoxide
(1.95 kg, 13.7 mol). The mixture was heated to 100 °C and vigorously stirred. When
phosphorus pentoxide was dissolved, the reactor was adjusted to 50 °C and guanidinium
methanesulfonate (15, 3.53 kg, 22.7 mol) was added. Crude diketone 11 was added (3.53 kg,
11.4 mol), the reaction mixture was stirred at 50 °C for 8 days and then cooled down to room
temperature. Ice/water mixture (25 L) was added and the mixture was extracted with
dichloromethane (1 x 7.5 L). The organic layer was washed with water (2 x 10 L) and with a
saturated aqueous solution of sodium bicarbonate (1 x 5 L), dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo (until dry, about 7.5 L of dichloromethane was
removed). The crude product was dissolved in dichloromethane (2.0 L) and precipitated by
the addition of cyclohexane (5.0 L), dichloromethane was removed in vacuo (about 2.0 L),
filtered and washed with cyclohexane (3.0 L). The precipitate was dissolved in excess of hot
ethyl acetate (10 L, 70 °C) and crystallized by evaporation of solvent (about 1.0 L of solvent
removed) along with cooling. Crystals were filtered and recrystallized from ethyl acetate (5.0
L) by the same procedure and from methanol (8.0 L). The product was dried in vacuo to give
5 (1.18 kg, 35%) as white crystals. M.p. 114 °C (methanol), m.p. 119-120 °C (cyclohexane).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Ethyl 2-butyl-3-oxo-3-phenylpropanoate (7).13 Ethyl 3-oxo-3-fenylpropanoate (6, 200
mL, 1.15 mol) was added dropwise to a solution of sodium ethoxide (140 g, 2.07 mol) in
anhydrous ethanol (600 mL). 1-Bromobutane (220 mL, 2.30 mol) was added dropwise and
the mixture was heated to reflux for 16 h. The mixture was left to cool to ambient temperature
and water (400 mL) was added. The mixture was concentrated in vacuo to half of the volume
and extracted with ethyl acetate (3 x 200 mL). The organic phase was combined and dried
with sodium sulfate. The desiccant was filtered off and the solvent was removed in vacuo to
give 7 (238 g, 92%) as a whitish solid. 1H NMR, 13C NMR and HRMS data are in agreement
with the literature.13 The crude product was used in the next step.
2-Amino-5-butyl-6-phenylpyrimidin-4-ol (8). Ethyl 2-benzoylhexanoate (7, 100 g, 0.40
mol) was added a solution of sodium methoxide (59.0 g, 1.10 mol) in anhydrous methanol
(800 mL). Guanidine hydrochloride (42.0 g, 0.44 mol) was added in portions over 15 min and
the mixture was heated to reflux for 12 h. Methanol was removed in vacuo and water (400
mL) was added to the residue. The mixture was neutralized with acetic acid (pH 6) and the
12
ACS Paragon Plus Environment

Page 13 of 17
Organic Process Research & Development
1
2
3
4
5
6
precipitated product was filtered, washed with hexane and dried in vacuo to give 8 (63.5 g,
1
65%) as a white solid. H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 7.43 – 7.33 (m, 5H),
6.47 (s, 2H), 2.23 – 2.17 (m, 2H), 1.33 (tt, J = 6.2, 7.7 Hz, 2H), 1.13 (h, J = 7.3 Hz, 2H), 0.73
7
8
9
13
(t, J = 7.3 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 158.73, 156.28, 153.70, 128.46,
128.34, 128.28, 112.44, 97.96, 31.48, 25.69, 22.57, 14.10. HRMS (ESI) m/z [M+H]+ calcd for
C14H18N3O: 244.1444, found: 244.1447.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-Butyl-4-chloro-6-phenylpyrimidin-2-amine (9). A mixture of 2-amino-5-butyl-6-
phenylpyrimidin-4-ol (8, 20.0 g, 82.0 mmol) and Arnold’s reagent (105 g, 0.82 mol) in
chloroform (1 L) was heated to reflux for 16 h. The reaction mixture was poured onto crushed
ice (500 g). A saturated solution of sodium bicarbonate (500 mL) was added carefully. Phases
were separated and the aqueous phase was washed with chloroform (2 x 200 mL). Combined
organic layers were concentrated in vacuo and the residue was dissolved in ethanol (500 mL).
Concentrated hydrochloric acid (10 mL) was added (pH 1–2) and the mixture was heated to
50 °C for 16 h. The solvent was removed in vacuo and water (500 mL) was added to the
residue and the mixture was neutralized with a saturated aqueous solution of sodium hydrogen
carbonate. The mixture was extracted with ethyl acetate (3 x 200 mL). Combined organic
extracts were dried over anhydrous sodium sulfate, filtered; solvent was removed in vacuo.
The crude product was crystallized from methanol to afford 9 (18.79 g, 88%) as a whitish
solid. 1H NMR (400 MHz, CDCl3) δ 7.51 – 7.37 (m, 5H), 5.23 (s, 2H), 2.61 – 2.54 (m, 2H),
1.52 – 1.42 (m, 2H), 1.24 (h, J = 7.3 Hz, 2H), 0.82 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 169.02, 162.42, 160.08, 138.46, 129.01, 128.38, 127.94, 121.14, 31.63, 28.22,
22.48, 13.59. HRMS (ESI) m/z [M+H]+ calcd for C14H17ClN3: 262.1106, found: 262.1109.
2-Butyl-1-(4-methoxyphenyl)-3-phenylpropane-1,3-dione (11).
A
mixture of
hexamethyldisilazane (1.65 kg, 10.2 mol) and dry toluene (2 L) in the reactor (20 L) was
tempered to 0 °C. Apparatus was flushed with nitrogen and a slight overpressure of nitrogen
was kept for the whole reaction time. A solution of n-butyllithium in hexanes (2.5 M, 4.00 L,
10.0 mol) was added dropwise (extensive cooling is required) and the reaction mixture was
cooled to -20 °C. A solution of hexanophenone (10, 1.76 kg, 10.0 mol) in toluene (3 L) was
added dropwise and the mixture was stirred at -20 °C for 30 min. The second reactor was
charged with p-anisoyl chloride (1.71 kg, 10.0 mol) and toluene (8 L), flushed with nitrogen
and tempered to -10 °C. The content of the first reactor was slowly transferred into the second
reactor using polyethylene tubing and stirred for 30 min. After addition, the reaction mixture
was kept at -5 °C for 12 h. The reaction mixture was washed with 1 M solution of hydrogen
13
ACS Paragon Plus Environment

Organic Process Research & Development
Page 14 of 17
1
2
3
4
5
6
7
8
9
chloride (2 x 10 L) and water (2 x 10 L). The organic layer was dried over anhydrous sodium
sulfate, filtered and concentrated in vacuo to afford 11 (3.1 kg, 98%) as a yellow-orange oil.
1H NMR (300 MHz, CDCl3) : 7.97 (d, J = 8.6 Hz, 4 H), 7.60 - 7.47 (m, 1 H), 7.50 - 7.35 (m,
2 H), 6.98 - 6.86 (m, 2 H), 5.13 (t, J = 6.6 Hz, 1 H), 3.85 (s, 3 H), 2.26 - 2.06 (m, 2 H), 1.50 -
1.26 (m, 4 H), 0.96 - 0.77 (m, 3 H). 13C NMR (75.5 MHz, CDCl3) : 196.26, 194.67, 163.72,
136.24, 133.26, 129.08, 130.90, 128.74, 128.47, 113.99, 57.30, 55.45, 30.46, 29.32, 22.71,
13.81. HPLC (Luna Silica 5 µm, 100 x 4.6 mm, hexane/2-propanol 9:1; UV 254 nm): 1,5
min, purity 93% (impurities 18 and 19: 1.33 min and 1.74 min, respectively). HRMS (ESI)
m/z [M+H]+ calcd for C20H23O3: 311.1642, found: 311.1648. Purification of diketone 11 by
crystallization. Dry diketone 11 was dissolved in cyclohexane at 45 °C in a ratio 1:10 (1 g of
diketone:10 mL of solvent). The solution was cooled down to 23-25 °C, seeded and slowly
cooled to 10 °C. Crystals were filtered, washed with precooled ExxsolTM D30 and re-
dissolved in cyclohexane again. The process was repeated two more times to afford 11 in a
99.4% purity (HPLC, 254 nm) and 50% yield. M.p. 49.2-50.1 °C (cyclohexane), 45.6-47,5 °C
(methanol).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-Butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-ol (13). Hexamethyldisilazane (856
g, 5.29 mol) was dissolved in anhydrous tetrahydrofuran (1.5 L) and toluene (2 L). The
mixture was cooled to -20°C and the solution of butyllithium (2.11 L, 5.29 mol) was added
dropwise via capillary under the nitrogen atmosphere. The solution of hexanophenone (10,
930 g, 5.29 mol) in toluene (2 L) was added over 30 min. The reaction mixture was stirred for
another 30 min at -20 °C. In another reactor, the p-anisoyl chloride (902 g, 5.29 mol) was
dissolved in anhydrous toluene (5 L) and cooled to -20 °C. The mixture of enolate was
transferred via tubing into the p-anisoyl chloride solution and the reaction mixture was stirred
for 16 h while the temperature was left to reach 25°C. The reaction was quenched by water
(10 L) containing hydrochloric acid (37%, 968 mL, 11.7 mol) and extracted with ethyl acetate
(2 x 5 L). Combined organic layers were dried over anhydrous sodium sulfate. The solvent
was removed in vacuo and the crude material was subjected to the next step. All material was
dissolved in 1,4-dioxane (13 L) and urea (684 g, 11.4 mol) was added. Gaseous hydrogen
chloride was bubbled into the mixture until the saturation of the solution. The mixture was
heated to 75 °C under nitrogen atmosphere for four days (TLC monitoring of diketone
content: cyklohexane/AcOEt, 9:1). The reaction mixture was quenched with water (10 L) and
extracted with dichloromethane (3 x 5 L). Combined organic layers were dried over
anhydrous sodium sulfate. After filtration of the desiccant, the solvent was removed in vacuo
14
ACS Paragon Plus Environment

Page 15 of 17
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
and the crude material was triturated with diethyl ether (10 L). Formed precipitated material
was filtered and washed with diethyl ether (500 mL) to give 13 (647 g, 34% yield) as an off-
white amorphous solid. (Material can form oil, not crystals. In that case, the diethyl ether was
removed and the crude material was used in next step). The analytical sample was purified by
column chromatography (silica gel, dichloromethane/methanol, 9:1) and crystallized from
diethyl ether. M.p. 180-181 °C (diethyl ether). 1H NMR (300 MHz, CDCl3) : 13.09 (br.s., 1
H); 7.63 - 7.41 (m, 7 H), 7.00 (d, J = 8.8 Hz, 2 H), 3.88 (s, 3 H), 2.65 - 2.36 (m, 2 H), 1.10 –
0.78 (m, 4 H), 0.71 - 0.32 (m, 3 H). 13C NMR (75.5 MHz, CDCl3) : 160.81, 158.09, 130.00,
129.45, 128.27, 128.09, 115.05, 113.93, 55.22, 31.91, 26.49, 21.82, 13.21. HRMS (ESI) m/z
[M+H]+ calcd for C21H23N2O2: 335.1768, found: 335.1765.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-Butyl-2-chloro-4-(4-methoxyphenyl)-6-phenylpyrimidin (14). Oxalyl chloride (615 g,
4.85 mol) was dissolved in anhydrous dichloromethane (2.5 L) and N,N-dimethylformamide
(354 g, 4.85 mol) was added dropwise. After 15 min, a solution of compound 13 (647 g,
1.938 mol) in anhydrous dichloromethane (3.3 L) was added and the reaction mixture was
heated to reflux until starting material was converted into product (TLC monitoring, silica gel,
DCM/MeOH, 95:5), usually 48 h. The reaction mixture was filtered through pad of silica gel
(3 kg) and eluted with dichloromethane (5 L) to give 14 (452 g, 66%) as a white solid. M.p.
81-82 °C (methanol). 1H NMR (300 MHz, CDCl3) : 7.64 - 7.42 (m, 7 H), 6.94 - 7.07 (m, 2
H), 3.88 (s, 3 H), 2.73 - 2.89 (m, 2 H), 1.19 – 1.03 (m, 2 H), 1.04 – 0.86 (m, 2 H), 0.57 (t, J =
7.2 Hz, 3 H). 13C NMR (75.5 MHz, CDCl3) : 170.06, 169.56, 160.56, 157.63, 137.84,
130.17, 130.10, 129.24, 129.45, 128.41, 113.86, 55.33, 31.47, 27.55, 22.13, 13.22. HRMS
(ESI) m/z [M+H]+ calcd for C21H22ClN2O: 353.1421, found: 353.1422.
Guanidinium methanesulfonate (15). Guanidine carbonate (3.0 kg, 33.3 mol) was
suspended in methanol (6 L) and methanesulfonic acid (3.3 kg, 34.3 mol) was slowly added
while carbon dioxide evolved. The suspension was stirred for 1 h and the solid was filtered,
washed with methanol (500 mL) and dried on air giving 15 (5.1 kg, 96%) as white solid.
AUTHOR INFORMATION
Corresponding Authors
*E-mail (Z. J.): janeba@uochb.cas.cz.
*E-mail (M. H.): havranek@apigenex.com.
15
ACS Paragon Plus Environment

Organic Process Research & Development
Page 16 of 17
1
2
3
4
Current address (O.B.): Department of Organic Chemistry, Faculty of Science, Charles
5
6
University, Hlavova 2030/8, 128 43 Prague, Czech Republic
7
8
9
ORCID
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Zlatko Janeba: 0000-0003-4654-679X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the subvention for development of research organization
(Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, RVO:
61388963) and by the Technology Agency of the Czech Republic (TE01020028).
REFERENCES
(1)
Jansa, P.; Holý, A.; Zídek, Z.; Kmoníčková, E.; Janeba, Z. Pyrimidine Compounds
Inhibiting the Formation of Nitric Oxide and Prostaglandin E2, Method of Production
Thereof and Use Thereof. WO2012/116666, 2012.
(2)
Jansa, P.; Holý, A.; Dračínský, M.; Kolman, V.; Janeba, Z.; Kostecká, P.; Kmoníčková,
E.; Zídek, Z. 5-Substituted 2-Amino-4,6-Dihydroxypyrimidines and 2-Amino-4,6-
Dichloropyrimidines: Synthesis and Inhibitory Effects on Immune-Activated Nitric
Oxide Production. Med. Chem. Res. 2014, 23, 4482–4490.
(3)
Jansa, P.; Holý, A.; Dračínský, M.; Kolman, V.; Janeba, Z.; Kmoníčková, E.; Zídek, Z.
Synthesis and Structure–activity Relationship Studies of Polysubstituted Pyrimidines as
Inhibitors of Immune-Activated Nitric Oxide Production. Med. Chem. Res. 2015, 24,
2154–2166.
(4)
(5)
Kolman, V.; Jansa, P.; Kalčic, F.; Janeba, Z.; Zídek, Z. Polysubstituted 4,6-
Bis(Hetero)Arylpyrimidines as Dual Inhibitors of Nitric Oxide and Prostaglandin E2
Production. Nitric Oxide 2017, 67, 53–57.
Kolman, V.; Kalčic, F.; Jansa, P.; Zídek, Z.; Janeba, Z. Influence of the C-5
Substitution in Polysubstituted Pyrimidines on Inhibition of Prostaglandin E 2
16
ACS Paragon Plus Environment

Page 17 of 17
Organic Process Research & Development
1
2
3
4
Production. Eur. J. Med. Chem. 2018, 156, 295–301.
5
6
7
8
(6)
Kalčic, F.; Kolman, V.; Ajani, H.; Zídek, Z.; Janeba, Z. Polysubstituted Pyrimidines as
mPGES‐1 Inhibitors: Discovery of Potent Inhibitors of PGE2 Production with Strong
Anti‐inflammatory Effects in Carrageenan‐Induced Rat Paw Edema. ChemMedChem
2020, doi.org/10.1002/cmdc.202000258.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(7)
(8)
Reichardt, C. Vilsmeier–Haack–Arnold formylations of aliphatic substrates with N-
chloromethylene-N, N-dimethylammonium salts. J. Prakt. Chem. 1999, 341, 609–615.
Miyaura, N.; Yamada, K.; Suzuki, A. A new stereospecific cross-coupling by the
palladium-catalyzed reaction of 1-alkenylboranes with 1-alkenyl or 1-alkynyl halides.
Tetrahedron Lett. 1979, 36, 3437–3440.
(9)
House, H.; Auerbach, R. A.; Gall, M.; Peet, N. P. The chemistry of carbanions. XXII.
C- vs. O-acylation of metal enolates. J. Org. Chem. 1973, 38, 514–522.
(10) Wiles, Ch.; Watts, P; Haswell, S. J; Pombo-Villar, E. The regioselective preparation of
1,3-diketones Tetrahedron Lett. 2002, 43, 2945–2948.
(11) Heller, S. T.; Natarajan, S. R. 1,3-Diketones from acid chlorides and ketones: A rapid
and general one-pot synthesis of pyrazoles. Org. Lett. 2006, 8, 2675–2678.
(12) Eaton, P. E.; Carlson, G. R.; Lee, J. T. Phosphorus pentoxide-methanesulfonic acid.
Convenient alternative to polyphosphoric acid. J. Org. Chem. 1973, 38, 4071–4073.
(13) Malkov, A. V.; Stončius, S.; Vranková, K.; Arndt, M.; Kočovský, P. Dynamic Kinetic
Resolution in the Asymmetric Synthesis of β-Amino Acids by Organocatalytic
Reduction of Enamines with Trichlorosilane. Chem. Eur. J. 2008, 14, 8082–8085.
17
ACS Paragon Plus Environment