Library synthesis of cardiomyogenesis inducing compounds using an efficient two-step-one-flow process

Article abstract of DOI:10.1007/s00706-015-1605-6

Within this work we telescoped a batch laboratory scale synthesis towards a two-step-one-flow process to synthesize cardiomyogenesis inducing compounds (4,6-diaminopyrimidines) in continuous manner. Special attention was put on a quick and robust screenin

Full text of DOI:10.1007/s00706-015-1605-6

Monatsh Chem (2016) 147:523–532
DOI 10.1007/s00706-015-1605-6
ORIGINAL PAPER
Library synthesis of cardiomyogenesis inducing compounds using
an efficient two-step-one-flow process
1
1
•
Michael Schon Dominik Dreier1 Michael Schnurch Marko D. Mihovilovic1
¨
•
•
¨
Received: 27 October 2015 / Accepted: 9 November 2015 / Published online: 24 November 2015
Ó Springer-Verlag Wien 2015
Abstract Within this work we telescoped a batch labo-
ratory scale synthesis towards a two-step-one-flow process
to synthesize cardiomyogenesis inducing compounds (4,6-
diaminopyrimidines) in continuous manner. Special atten-
tion was put on a quick and robust screening protocol and
subsequent UHPLC analysis. Finally, the robustness of the
method was proven by the multi-gram synthesis of VUT-
MK142 using a laboratory scale continuous flow reactor,
enabling to deliver enough material for extensive biologi-
cal testing.
Introduction
In nature, cell differentiation is controlled by molecular
processes which involve cell signaling [1]. Generally, cells
differentiate from totipotent stem cells into more special-
ized multipotent cells until the final faith of the cell is
reached with completely differentiated cells. This differ-
entiation is growth-factor induced and relies on molecular
level on signal transmission based on receptor binding.
Consequently, changes in protein conformation and phos-
phorylation take place, which is equivalent to cell
activation. This differential gene activity is responsible for
the switch in the type of a cell [2].
Graphical abstract
There exist a number of possibilities to influence cell
differentiation artificially. Complementary DNA fragments
can be transferred into cells by retroviral vectors resulting
in expression of the corresponding proteins. This technique
was exploited to transfer specific transcription factors
which consequently led to cell differentiation [3] and
reprogramming [4], respectively. However, the risk of
permanent changes in the genome and the usage of onco-
Keywords Continuous flow chemistry Á
Medicinal chemistry Á Library synthesis Á
Diaminopyrimidines
genic transcription factors make this approach
a
controversial one. Another possibility to change the fate of
a cell is nuclear transfer which is termed nuclear repro-
gramming [5, 6]. A very active field of research is the
reprogramming of non-pluripotent somatic cells into
induced pluripotent stem cells which was ultimately
awarded with the Nobel Prize in Physiology or Medicine in
2012. This can be achieved by the artificial expression of
several transcription factors wherefore a couple of methods
exist [7–12]. A very promising approach towards a thera-
peutic application is an induced cell differentiation [13]
and reprogramming [14] by synthetic small molecules
(SySMs). Such cell fate controlling molecules attracted
strong interest in recent years due to the potential and
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-015-1605-6) contains supplementary
material, which is available to authorized users.
& Michael Schnu¨rch
michael.schnuerch@tuwien.ac.at
1
TU Wien, Institute of Applied Synthetic Chemistry,
Getreidemarkt 9/163, 1060 Vienna, Austria
123

¨
M. Schon et al.
524
beneficial application for certain regenerative tissue ther-
apies [13]. Among other differentiation processes, the
transformation towards cardiomyocytes is particularly
interesting as the heart has naturally poor regenerating
features [15]. There are a couple of cardiomyogenesis
inducing compounds known to the literature. Early exam-
ples are given by Wu et al. They found four 2,4-
diaminosubstituted pyrimidines (cardiogenols A–D, Fig. 1)
to be potentially able to induce cardiomyogenesis in mur-
ine embryonic stem cells. Thus, stem cells can be
influenced to transform into cardiac muscle cells [16].
Other examples of cardiogenic SySMs include a 2,6-dis-
ubstituted 4-anilinoquinazoline derivative [17], Shz-1 [18],
XAV939 [19], and CHIR99021 [20] (Fig. 1).
cardiogenol C, the previously reported compound VUT-
MK142 (Fig. 2) was also found to be an efficient car-
diomyogenesis inducing agent [24]. The compound was
tested successfully on a mouse skeletal myoblast cell line
(C2C12) and a mouse embryonic carcinoma cell line (P19)
by three different assays giving remarkable transformation
results on C2C12 cells: they could be transformed into
functional cardiomyocytes, which was the first report of
induced cardiomyogenic effects on lineage committed cells
[25, 26]. The formation of cardiomyocytes from trans-
planted cells in the heart of a patient may improve the heart
function significantly, e.g., after cardiac infarction. There-
fore, such growth factors are of strong interest as heart
diseases are the leading causes of death in the United States
in 2011 [27].
The diamino substituted pyrimidines synthesized by Wu
et al. served as an inspiration for the preparation of similar
compounds in our laboratory. To synthesize cardiogenol
derivatives, a sequential nucleophilic substitution route was
reported [21]. Furthermore, also 4,6-diaminopyrimidines as
structural isomers to cardiogenols are known to derive
from an accelerated microwave-assisted protocol [22] and
flow-chemical synthesis of 2-aminopyrimidines was pre-
viously published [23]. Based on the structure of
To allow for extensive biological testing of 4,6-disub-
stituted pyrimidines and to evaluate the biological effects
of VUT-MK142 derivatives, the known batch protocol was
transferred into a continuous flow procedure which bears
the potential for automated synthesis. Following this
approach, a small library of differently substituted pyrim-
idines was synthesized, and a continuous multi-gram
synthesis of VUT-MK142 was developed.
Fig. 1 Cardiomyogenesis
inducing compounds
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Library synthesis of cardiomyogenesis inducing compounds using an efficient two-step-one-…
525
available in a Hastelloy version which provides optimal
chemical resistance, interchangeable reactor coils to influ-
ence residence times and, additionally, it allows very high
reaction temperatures up to 350 °C—temperatures
assumed to be beneficial in the second, less favored sub-
stitution step. Conversion was monitored to be incomplete
in all cases with a maximum UHPLC yield of 44 %.
Therefore, the concentration of the starting materials was
increased to 50 and 100 mM boosting the conversion to
completeness accompanied with an excellent maximum
UHPLC yield of 97 % at 160 °C reaction temperature and
8 min of residence time in the reaction coil (Fig. 3, details
see ‘‘Experimental’’ section).
Fig. 2 VUT-MK142 as an example for 4,6-disubstituted pyrimidines
inducing cardiomyogenesis
Results and discussion
The first milestone in the library synthesis of 4,6-disub-
stituted pyrimidines was the development of a model
reaction sequence leading to VUT-MK142 as target prod-
uct. Retrosynthetic analysis shows that VUT-MK142 could
be synthesized via two sequential nucleophilic substitu-
tions starting from 4,6-dichloropyrimidine (Scheme 1).
Due to the symmetry of 4,6-dichloropyrimidine, the
less-reactive aromatic amine could be reacted first, fol-
lowed by the second substitution reaction with an excess of
cyclohexylamine as more reactive reagent. Selectivity for a
mono-substitution should be achievable due to the de-ac-
tivating nature of the newly introduced arylamine
substituent. To allow for a quick, automation-assisted
optimization of the two sequential substitution reactions,
reference material was synthesized using a batch protocol
(see ‘‘Experimental’’ section). Furthermore, an ultra-quick
UHPLC method of 2 min was established to enable a rapid
analysis of the screened reaction parameters (details see
supplementary information).
Interestingly, the highest temperature did not turn out to
work best, but depending on the substrate concentration,
160 °C showed optimal reaction performance. Increasing
the substrate concentration from 10 to 100 mM concen-
tration increased the product yield from 44 % (160 °C,
10 mM) to 97 % (160 °C, 100 mM). An increase in reac-
tion temperature again led to a drop in yields.
Subsequently, also the second substitution reaction was
carried out in flow starting from purified intermediate of
the first step (10 mM) and cyclohexylamine (30 mM)
under base catalysis (Scheme 3). To compensate the
reduced reactivity of the mono-substituted chloropyrim-
idine, the reaction temperature range of choice was
increased to an interval of 200–300 °C with increments of
20 °C. Already in the first runs, the highest temperature of
300 °C was skipped due to repeated reactor cloggage.
Furthermore, a limited stability of the desired target
molecule at higher temperatures, as well as the occurrence
of dechlorinated intermediate as by-product (identified via
chromatography with a known reference compound as
In the first step, 4,6-dichloropyrimidine was reacted in
flow with p-anisidine at 10 mM concentration in NMP
under base catalysis at temperatures ranging from 150 to
200 °C in 10 °C increments (Scheme 2). The ThalesNano
X-Cube Flash was chosen as reactor system, as it is
Scheme 1
Scheme 2
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M. Schon et al.
526
Fig. 3 UHPLC yields of the first reaction step at different concentrations and temperatures
Scheme 3
standard) was monitored, explaining the poor maximum
yield of 25 % at 240 °C reaction temperature.
second nucleophilic substitution reaction. The reaction
time of 8 min reaction time (which equaled a flow rate of
0.5 cm3/min using a 4 cm3 coil) was kept the same for all
optimization reactions.
Following the strategy of the first substitution reaction,
the reaction concentration was increased to 50/150 mM
(chloropyrimidine/amine) and 100/300 mM giving signifi-
cantly improved UHPLC yield of 79 % at 50 mM
concentration (referenced to intermediate conversion) and
220 °C reaction temperature (Fig. 4). To boost the yield
even more, we also increased the molar ratio of cyclo-
hexylamine to intermediate from 3:1 up to a ratio of 10:1.
This approach enabled us to get 94 % UHPLC yield at the
lowest screened reaction temperature of 200 °C and 8 min
residence time in the reaction coil (Figs. 5, 6), which was
significantly higher than the yield of the corresponding
batch reaction (38 %). Unfortunately, the combination of
high substrate concentrations and high reaction tempera-
tures led to an increased number of system cloggages,
rendering a number of experiments impossible to perform
(for details see ‘‘Experimental’’ section).
To give access to a small library of mono-substituted
and 4,6-disubstituted pyrimidines, p-anisidine, m-anisidine,
m-chloroaniline, and o-toluidine were reacted with 4,6-
dichloropyrimidine according to the optimized reaction
conditions in good to very good isolated yields (Table 1).
Despite all optimization efforts, the reaction time had to be
doubled for the latter three amines doubling the size of the
reaction coil to 8 cm3 to give full conversion of starting
materials (see ‘‘Experimental’’ section).
In the synthesis of the desired target molecules, it was
exemplarily shown that the use of purified intermediate
gave VUT-MK142 in very good isolated yield of 84 %,
which equaled to a total yield over two steps of 68 % (see
‘‘Experimental’’ section). However, merging the two
reaction steps into a single reaction sequence without
intermediate purification could further streamline the syn-
thesis of the target compounds by reducing the purification
steps to a single one. After the first substitution reaction, a
To summarize the optimization series, optimal condi-
tions were identified to be 160 °C reaction temperature for
the first reaction step and 200 °C temperature for the
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Library synthesis of cardiomyogenesis inducing compounds using an efficient two-step-one-…
527
Fig. 4 UHPLC yields of the second reaction step at different concentrations and temperatures; 3 equiv. cyclohexylamine. Superscript a reaction
conditions led to system cloggage
Fig. 5 UHPLC yields of the second reaction step at different concentrations and temperatures; 5 equiv. cyclohexylamine. Superscript a reaction
conditions led to system cloggage
defined aliquot was mixed with the appropriate amount of
cyclohexylamine and subjected to the reactor system for a
second time. Due to this fact, synthesis of all 4,6-disub-
stituted pyrimidines was carried out following the single
reaction sequence giving products in acceptable to good
yields (Table 2, for details see ‘‘Experimental’’ section).
Finally, it was also demonstrated that the prevalent
method was also suitable to synthesize VUT-MK142 on
large scale. To double the amount of product keeping the
specific reaction parameters constant for this specific
reaction sequence, the coil size was doubled from 4 to
8 cm3, as well as the flow rate from 0.5 to 1.0 cm3/min.
Allowing the system to run for about 4 h, more than 5 g of
pure product could be produced still using a laboratory
scale flow reactor, which equals a productivity of
29.1 g/day.
Conclusion
In this work, we successfully transferred the previously
published synthesis of the cardiomyogenesis inducing
compound VUT-MK142 into a continuous flow process.
The reaction was optimized to enable a high yield synthesis
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M. Schon et al.
528
Fig. 6 UHPLC yields of the second reaction step at different concentrations and temperatures; 10 equiv. cyclohexylamine. Superscript a
reaction conditions led to system cloggage
Table 1 Library synthesis of mono-substituted chloropyrimidines in
flow
Experimental
Solvents: NMP, DMA, DMF, and DMSO were purchased
Product
Structure
Isolated yield /%
81
from Merck, Sigma-Aldrich, or Loba. Starting materials:
4,6-dichloropyrimidine, p-anisidine, m-anisidine, m-
chloroaniline, o-toluidine, N,N-diisopropylethylamine, and
cyclohexylamine were bought from Sigma-Aldrich, Fluka,
Merck, or Baker. During work-up, diisopropylether, chlo-
roform, sodium chloride, ammonium chloride, sodium
carbonate, and benzyl benzoate were supplied by Acros,
Neuber, and Merck. For chromatography, redistilled pet-
roleum ether and ethyl acetate were used, running on
1
60
51
53
2
3
4
1
Merck silica gel. H and 13C NMR spectra were recorded
using TMS as an internal standard on a Bruker Avance
200 MHz spectrometer. Chemical shifts were reported in
ppm. Reactions in flow were performed on ThalesNanoÒ
X-Cube Flash high temperature/high pressure reactor. As a
reaction coil, 4 and 8 cm3 HastelloyÒ coils were installed.
To precisely collect the product plug in the screening
process, the dead volume of the system and the heat
exchangers was determined for each coil using an inten-
sively colored solution. Reaction analysis was carried out
using a Shimadzu NexeraÒ UHPLC system using a Phe-
nomenex KinetexÒ PFP column for separation running on
an acetonitrile–water gradient (details see supplementary
information). To guarantee for robust measurements, an
internal standard protocol was established. Product purifi-
cation was performed on a Bu¨chi SepacoreÒ MPLC
system. Melting points were measured on a calibrated
Stanford Research Systems Optimelt MPA100 automated
melting point system.
with an effective residence time in the range of minutes.
The selection of a sequential amination approach allowed
for an increased yield compared to the batch protocol and
lowered the work-up effort. Consequently, the method was
telescoped to give a productivity of 29.1 g/day. To broaden
the scope of the reaction, a small library of intermediates
and diaminopyrimidine products was successfully synthe-
sized without major changes to the standard protocol.
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Library synthesis of cardiomyogenesis inducing compounds using an efficient two-step-one-…
Table 2 Library and large
scale synthesis of 4,6-
disubstituted pyrimidines in
flow
529
Product
Structure
Isolated yield /%
(single step)
70 (68 with purified
intermediate)
5
(VUT-MK-142)
48
42
51
6
7
8
General protocol A
starting material concentration of the first reaction was
increased to about 120 mM 4,6-dichloropyrimidine and
132 mM N,N-diisopropylethylamine as a compensation.
System parameters were equal to general protocol A.
The following general protocol A was used for the opti-
mization of the flow synthesis and preparation of
4-substituted pyrimidines in flow: reaction lines 1 and 2
were purged with pure NMP to get rid of air bubbles. Then,
reaction line 1 was changed to starting material solution.
The flow rate was set to 0.5 cm3/min and the backpressure
was set to 75 bar (to have sufficient tolerance in the case of
pressure fluctuations). Reaction temperature was set to the
desired value and all the conditions were allowed to sta-
bilize for 10 min running on solvent only. Then, the pumps
were switched to introduce the starting material solution
into the system. After completion, the pumps were swit-
ched back to NMP, dead volume was allowed to pass and
subsequent product collection was triggered. Using this
protocol, reaction temperature as well as concentration and
molar ratio of starting materials were optimized. In the case
of incomplete conversions, size of the reaction coil was
changed from 4 to 8 cm3 (instead of varying the flow rates)
so as not to affect the space–time-yield of the process. To
increase productivity and minimize product decomposition,
in some cases the flow rate was increased from 0.5 to
1.0 cm3/min.
Reference material synthesis
6-Chloro-N-(4-methoxyphenyl)pyrimidine-4-amine (1)
In batch: 317 mg 4,6-dichloropyrimidine (2.13 mmol, 1.0
equiv.), 262 mg p-anisidine (2.13 mmol, 1.0 equiv.), and
303 mg N,N-diisopropylethylamine
(2.34 mmol,
1.1
equiv.) were introduced into a high pressure glass vial,
equipped with a magnetic stirring bar and dissolved in
15 cm3 NMP. The reaction solution was heated conven-
tionally to 120 °C for 10 h. After cooling, 25 cm3 EtOAc
was added and the solution was extracted three times with
saturated solution of ammonium chloride (3 9 25 cm3).
The combined aqueous phases were extracted once again
with 50 cm3 EtOAc and the combined organic phases were
washed thoroughly with brine (5 9 25 cm3). After drying
the organic phase with anhydrous sodium sulfate, filtration,
and evaporation of the solvent, crude product was obtained
as a brown solid. Further purification using chromatogra-
phy gave pure product 1 as a white, fluffy solid in 75 %
yield (380 mg, 1.61 mmol). Spectral data were in accor-
dance with literature [25].
General protocol B
N4-Cyclohexyl-N6-(4-methoxyphenyl)pyrimidine-4,6-di-
amine (5)
In the case of 4,6-diaminopyrimidine derivative one-flow
synthesis, general protocol B was developed: cyclohexy-
lamine and base were added to the reaction solution after
the first step without further purification. To take account
for dilution effects caused by addition of reagents and
diffusion of the reaction plug during the first reaction,
In batch: 49 mg 4,6-dichloropyrimidine (0.33 mmol, 1.0
equiv.), 41 mg p-anisidine (0.33 mmol, 1.0 equiv.), and
47 mg N,N-diisopropylethylamine (0.37 mmol, 1.0 equiv.)
were reacted as described above. The reaction solution was
123

¨
M. Schon et al.
530
used for the second reaction step without further purifica-
tion. Cyclohexylamine (99 mg, 1.00 mmol, 3.0 equiv.) and
filled up to a volume of 12.7 cm3 with NMP. The solution
was reacted according to general procedure A at 160 °C
using a 4 cm3 coil at a flow rate of 0.5 cm3/min. Crude
product was purified as described for the batch synthesis
giving pyrimidine 1 as a white, fluffy solid in 81 % yield
(243 mg, 1.03 mmol).
129 mg N,N-diisopropylethylamine
(1.00 mmol,
3.0
equiv.) were added and the reaction solution was heated
to 200 °C for 17 h. Work-up and purification was carried
out as for the intermediate compound giving pure product 5
as a white, fluffy solid in 38 % yield (38 mg, 0.13 mmol).
Spectral data were in accordance with literature [25].
6-Chloro-N-(3-methoxyphenyl)pyrimidine-4-amine (2)
4,6-Dichloropyrimidine (190 mg, 1.28 mmol, 1.0 equiv.),
157 mg m-anisidine (1.27 mmol, 1.0 equiv.), and 180 mg
N,N-diisopropylethylamine (1.39 mmol, 1.1 equiv.) were
introduced into a FalconÒ tube, dissolved in NMP, and
filled up to a volume of 12.7 cm3 with NMP. The solution
was reacted according to general procedure A at 160 °C
using an 8 cm3 coil at a flow rate of 0.5 cm3/min. To
improve the work-up process, NMP was removed under
high vacuum, the residue was dissolved in 25 cm3 chlo-
roform and extracted with a saturated solution of sodium
carbonate (3 9 25 cm3). Each aqueous phase was re-
extracted with 10 cm3 chloroform. The combined organic
phases were washed with brine (3 9 25 cm3). After drying
the organic phase with anhydrous sodium sulfate, filtration,
and evaporation of the solvent, crude product was obtained
as a brown oil. Further purification using chromatography
gave pure product 2 as a light brown solid in 60 % yield
(180 mg, 0.77 mmol). Spectral data were in accordance
with literature [25].
First step optimization in flow
As a starting point for the synthesis, a solution of 74 mg
4,6-dichloropyrimidine (0.50 mmol, 1.0 equiv.), 62 mg p-
anisidine (0.50 mmol, 1.0 equiv.), and 71 mg N,N-diiso-
propylethylamine (0.55 mmol, 1.1 equiv) in 50 cm3 NMP
was prepared. For each optimization step, 4 cm3 of starting
material solution was introduced into the system (4 cm3
coil) and reacted according to general procedure A at room
temperature and 150–200 °C in 10 °C increments at a flow
rate of 0.5 cm3/min. To improve the conversion rates,
starting material concentration was increased to 50 and
100 mM (based on 4,6-dichloropyrimidine), running at the
same molar ratio and reaction conditions. The progress of
the reaction optimization was monitored using UHPLC
measurements.
Second step optimization in flow
6-Chloro-N-(3-chlorophenyl)pyrimidine-4-amine (3)
4,6-Dichloropyrimidine (186 mg, 1.25 mmol, 1.0 equiv.),
160 mg m-chloroaniline (1.25 mmol, 1.0 equiv.), and
To tune the parameters of the second reaction step, a
starting material solution of 118 mg 1 (0.50 mmol, 1.0
equiv.), 149 mg cyclohexylamine (1.50 mmol, 3.0 equiv.),
and 194 mg N,N-diisopropylethylamine (1.50 mmol, 3.0
equiv.) were dissolved in 50 cm3 NMP. For each opti-
mization step, 4 cm3 of starting material solution was
introduced into the system (4 cm3 coil) and reacted
according to general procedure B at room temperature and
200–280 °C in 20 °C increments at a flow rate of 0.5 cm3/
min. In the optimization series, cyclohexylamine molar
ratio was increased to 5.0 and 10.0 equiv. and reaction
solution concentration was increased to 50 and 100 mM
(based on 1). Due to enhanced fluctuation in the system
pressure and increased number of system blockages, the
highest screening temperature was lowered stepwise from
280 to 220 °C. The progress of the reaction optimization
was monitored using UHPLC measurements.
178 mg N,N-diisopropylethylamine
(1.38 mmol,
1.1
equiv.) were introduced into a FalconÒ tube, dissolved in
NMP, and filled up to a volume of 12.5 cm3 with NMP.
The solution was reacted according to general procedure A
at 160 °C using an 8 cm3 coil at a flow rate 0.5 cm3/min.
After product purification described in the synthesis of 2,
pure product 3 was isolated as a white solid in 51 % yield
(153 mg, 0.64 mmol). Spectral data were in accordance
with literature [25].
6-Chloro-N-(2-methylphenyl)pyrimidine-4-amine
(4, C11H10ClN3)
4,6-Dichloropyrimidine (203 mg, 1.36 mmol, 1.0 equiv.),
145 mg o-toluidine (1.35 mmol, 1.0 equiv.), and
194 mg N,N-diisopropylethylamine
(1.50 mmol,
1.1
equiv.) were introduced into a FalconÒ tube, dissolved in
NMP, and filled up to a volume of 13.6 cm3 with NMP.
The solution was reacted according to general procedure A
at 160 °C using an 8 cm3 coil at a flow rate of 0.5 cm3/min.
After product purification described in the synthesis of 2,
pure product 4 was isolated as yellow crystals in 53 %
Synthesis of 4-substituted pyrimidines in flow
6-Chloro-N-(4-methoxyphenyl)pyrimidine-4-amine (1)
4,6-Dichloropyrimidine (190 mg, 1.28 mmol, 1.0 equiv.),
157 mg p-anisidine (1.27 mmol, 1.0 equiv.), and 180 mg
N,N-diisopropylethylamine (1.39 mmol, 1.1 equiv.) were
introduced into a FalconÒ tube, dissolved in NMP, and
1
yield (158 mg, 0.72 mmol). M.p.: 121–123 °C; H NMR
(DMSO-d6, 200 MHz): d = 2.18 (s, 3H), 6.53 (s, 1H),
123

Library synthesis of cardiomyogenesis inducing compounds using an efficient two-step-one-…
531
7.02–7.47 (m, 4H), 8.33 (s, 1H), 9.35 (br, 1H) ppm; 13C
NMR (DMSO-d6, 50 MHz): d = 17.8 (q), 103.0 (d), 125.9
(d), 126.1 (d), 126.5 (d), 130.8 (d), 133.3 (s), 136.1 (s),
158.3 (s), 158.6 (d), 162.5 (s) ppm; TLC (PE:E-
tOAc = 1:1): Rf = 0.59.
was isolated as a white solid in 70 % yield (113 mg,
0.38 mmol).
N4-Cyclohexyl-N6-(3-methoxyphenyl)pyrimidine-4,6-di-
amine (6)
4,6-Dichloropyrimidine (214 mg, 1.44 mmol, 1.0 equiv.),
177 mg m-anisidine (1.44 mmol, 1.0 equiv.), and 204 mg
N,N-diisopropylethylamine (1.58 mmol, 1.1 equiv.) were
introduced into a FalconÒ tube, dissolved in NMP, and
filled up to a volume of 12.0 cm3 with NMP. The solution
was reacted according to general procedure A at 160 °C
using an 8 cm3 coil at a flow rate of 0.5 cm3/min. Then, an
aliquot of 8.4 cm3 reacted solution (max. 1.01 mmol, 1.0
equiv.) was taken for the second reaction step and 997 mg
cyclohexylamine (10.05 mmol, 10.0 equiv.) and
Synthesis of 4,6-disubstituted pyrimidines in flow
using purified starting materials
N4-Cyclohexyl-N6-(4-methoxyphenyl)pyrimidine-4,6-di-
amine (5)
1 (237 mg, 1.01 mmol, 1.0 equiv.), 998 mg cyclohexy-
lamine (10.06 mmol, 10.0 equiv.), and 390 mg N,N-
diisopropylethylamine (3.02 mmol, 3.0 equiv.) were intro-
duced into a FalconÒ tube, dissolved in NMP, and filled up
to a volume of 10.1 cm3 with NMP. The solution was
reacted according to general procedure A at 200 °C using a
4 cm3 coil at a flow rate of 0.5 cm3/min. EtOAc (25 cm3)
was added to the reaction solution. By adding 25 cm3 of a
saturated solution of sodium carbonate, a precipitate
appeared in the organic phase, which could be identified
as product. EtOAc was added until the organic phase was
homogeneous again and the organic layer was extracted
390 mg N,N-diisopropylethylamine
(3.02 mmol,
3.0
equiv.) were added. The solution was reacted according
to general procedure B at 200 °C using an 8 cm3 coil at a
flow rate of 1.0 cm3/min. After evaporating NMP under
high vacuum, the residue was directly purified by MPLC
giving pure product 6 as a light brown solid in 48 % yield
(143 mg, 0.48 mmol). Spectral data were in accordance
with literature [25].
with
a
saturated solution of sodium carbonate
N4-Cyclohexyl-N6-(3-chlorophenyl)pyrimidine-4,6-diamine
(7)
(2 9 25 cm3). The combined aqueous phases were further
extracted with EtOAc (3 9 25 cm3) and the organic layers
were washed with brine (3 9 25 cm3). After drying the
organic phase with anhydrous sodium sulfate, filtration,
and evaporation of the solvent, crude product was obtained
as a brown oil. Further purification using chromatography
gave pure product 5 as a white, fluffy solid in 84 % yield
(251 mg, 0.84 mmol).
4,6-Dichloropyrimidine (214 mg, 1.44 mmol, 1.0 equiv.),
184 mg m-chloroaniline (1.44 mmol, 1.0 equiv.), and
204 mg N,N-diisopropylethylamine
(1.58 mmol,
1.1
equiv.) were introduced into a FalconÒ tube, dissolved in
NMP, and filled up to a volume of 12.0 cm3 with NMP.
The solution was reacted according to general procedure A
at 160 °C using an 8 cm3 coil at a flow rate of 0.5 cm3/min.
Then, an aliquot of 8.3 cm3 reacted solution (max.
0.99 mmol, 1.0 equiv.) was taken for the second reaction
step and 984 mg cyclohexylamine (9.92 mmol, 10.0
Synthesis of 4,6-disubstituted pyrimidines in flow
using a one-flow process
equiv.)
and
384 mg N,N-diisopropylethylamine
N4-Cyclohexyl-N6-(4-methoxyphenyl)pyrimidine-4,6-di-
amine (5)
(2.97 mmol, 3.0 equiv.) were added. The solution was
reacted according to general procedure B at 200 °C using
an 8 cm3 coil at a flow rate of 1.0 cm3/min. After product
purification described in the synthesis of 2, pure product 7
was isolated as a light brown solid in 42 % yield (126 mg,
0.42 mmol). Spectral data were in accordance with liter-
ature [25].
4,6-Dichloropyrimidine (214 mg, 1.44 mmol, 1.0 equiv.),
177 mg p-anisidine (1.44 mmol, 1.0 equiv.), and
204 mg N,N-diisopropylethylamine
(1.58 mmol,
1.1
equiv.) were introduced into a FalconÒ tube, dissolved in
NMP, and filled up to a volume of 12.0 cm3 with NMP.
The solution was reacted according to general procedure A
at 160 °C using a 4 cm3 coil at a flow rate of 0.5 cm3/min.
Then, an aliquot of 4.5 cm3 reacted solution (max.
0.54 mmol, 1.0 equiv.) was taken for the second reaction
step, and 534 mg cyclohexylamine (5.39 mmol,
10.0 equiv.) and 209 mg N,N-diisopropylethylamine
(1.62 mmol, 3.0 equiv.) were added. The solution was
reacted according to general procedure B at 200 °C using a
4 cm3 coil at a flow rate of 0.5 cm3/min. After product
purification described in the synthesis of 2, pure product 5
N4-Cyclohexyl-N6-(2-methylphenyl)pyrimidine-4,6-di-
amine (8, C17H22N4)
4,6-Dichloropyrimidine (214 mg, 1.44 mmol, 1.0 equiv.),
154 mg o-toluidine (1.44 mmol, 1.0 equiv.), and
204 mg N,N-diisopropylethylamine
(1.58 mmol,
1.1
equiv.) were introduced into a FalconÒ tube, dissolved in
NMP, and filled up to a volume of 12.0 cm3 with NMP.
The solution was reacted according to general procedure A
at 160 °C using an 8 cm3 coil at a flow rate of 0.5 cm3/min.
123

¨
M. Schon et al.
532
Then, an aliquot of 8.9 cm3 reacted solution (max.
1.07 mmol, 1.0 equiv.) was taken for the second reaction
step and 1.06 g cyclohexylamine (10.7 mmol, 10.0 equiv.)
and 413 mg N,N-diisopropylethylamine (3.20 mmol, 3.0
equiv.) were added. The solution was reacted according to
general procedure B at 200 °C using an 8 cm3 coil at a flow
rate of 1.0 cm3/min. After product purification described in
the synthesis of 2, pure product 8 was isolated as a white
solid in 51 % yield (153 mg, 0.54 mmol). M.p.:
219.5–221.5 °C; 1H NMR (CDCl3:CD3OD = 3:1,
200 MHz): d = 0.92–1.91 (m, 10H), 2.16 (s, 3H),
3.06–3.29 (m, 1H), 3.82 (br, 2H), 5.23 (s, 1H), 6.98–7.23
(m, 4H), 7.92 (s, 1H) ppm; 13C NMR (CDCl3:CD3-
OD = 3:1, 50 MHz): d = 17.6 (q), 24.5 (t), 25.4 (t), 32.6
(t), 49.6 (d), 80.9 (d), 125.4 (d), 126.1 (d), 126.7 (d), 131.1
(d), 133.5 (s), 136.4 (s), 157.5 (d), 161.3 (s), 161.8 (s) ppm;
TLC (PE:EtOAc = 1:1): Rf = 0.34.
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123