Design, synthesis and biological evaluation of spiropyrazolopyridone derivatives as potent dengue virus inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127162

The effective treatment for dengue virus infection continues to be a challenge. We herein reported our continued SAR exploration on the spiropyrazolopyridone scaffold. Introducing different substituents at the 3?- or 5?-site of the pyrazolopyridone core or moving the benzyl chain to the adjacent nitrogen led to a significant loss of potency on DENV-2. While a narrow range of substitutions were tolerated at the para-position of the phenyl ring, di-substitution on the phenyl ring is beneficial for DENV-2 potency and has variable influences on DENV-3 potency depending on the exact compound. Among these molecules, compounds 22 (JMX0376) with 4-chloro-3-fluorobenzyl and 24 (JMX0395) with 2,4-bis(trifluoromethyl)benzyl showed the most potent and broadest inhibitory activities against DENV-1 to ?3 with nanomolar to low micromolar EC₅₀ values.

Full text of DOI:10.1016/j.bmcl.2020.127162

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of spiropyrazolopyridone
derivatives as potent dengue virus inhibitors
⁎
Jimin Xua,1, Xuping Xieb,1, Haiying Chena, Jing Zoub, Yu Xuea, Na Yea, Pei-Yong Shib,c,
,
⁎
Jia Zhoua,c,
a Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, United States
b Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, United States
c Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, United States
A R T I C L E I N F O
A B S T R A C T
The effective treatment for dengue virus infection continues to be a challenge. We herein reported our continued
Keywords:
́
́
Dengue virus
SAR exploration on the spiropyrazolopyridone scaffold. Introducing different substituents at the 3- or 5-site of
the pyrazolopyridone core or moving the benzyl chain to the adjacent nitrogen led to a significant loss of potency
on DENV-2. While a narrow range of substitutions were tolerated at the para-position of the phenyl ring, di-
substitution on the phenyl ring is beneficial for DENV-2 potency and has variable influences on DENV-3 potency
depending on the exact compound. Among these molecules, compounds 22 (JMX0376) with 4-chloro-3-fluor-
obenzyl and 24 (JMX0395) with 2,4-bis(trifluoromethyl)benzyl showed the most potent and broadest inhibitory
activities against DENV-1 to −3 with nanomolar to low micromolar EC50 values.
Spiropyrazolopyridone
NS4B inhibitors
Antiviral agents
Structure-activity relationship (SAR)
Dengue is a febrile disease that is caused by any one of the four
closely related serotypes of dengue viruses (DENV-1, -2, -3 and -4) with
asymptomatic, mild or severe symptoms. Common symptoms are flu-
like with patients experiencing high fever, headaches, retro-orbital pain,
joint/bone/muscle pain, vomiting, rash and fatigue. In most cases,
DENV infection is self-limiting, but sometimes it can develop into life-
threatening illnesses, dengue hemorrhagic fever (DHF) or dengue shock
syndrome (DSS).1 Dengue has become a major public health threat
throughout tropical and sub-tropical regions of the world, putting about
3.9 billion people in 128 countries at risk.2,3 Over the past 50 years,
there is an unprecedented increase in the incidence of dengue and 390
million people are estimated to get infected with dengue annually, 96
million of which experience clinical symptoms (with any severity of
disease), including 500,000 cases of severe dengue and 22,000 deaths
worldwide.4–6 Infection confers life-long immunity to that particular
serotype while cross-immunity against other serotypes is only partial
and temporary.7,8 However, a second infection with a different DENV
increases the probability of severe dengue through a process known as
antibody-dependent enhancement (ADE).9–12 As a result, an ideal
dengue antiviral or vaccine should be equally effective against all four
DENV serotypes. Recently, the first dengue vaccine (CYD-TDV from
Sanofi Pasteur) has been approved by regulatory authorities in about 20
countries with limited efficacy and safety issues.13–15 It requires a three-
dose regimen and is targeted for persons aged 9–45 years old who were
previously infected by DENV. Moreover, according to the re-
commendations of World Health Organization (WHO) regarding the use
of CYD-TDV in September 2018, seronegative vaccine recipients suffer
an excess risk of hospitalized and severe dengue compared to ser-
onegative unvaccinated individuals.16 While numerous efforts were
made towards anti-dengue drug discovery by different research groups,
there remains no specific therapy for dengue illness.17 Therefore, there
remains an urgent need to develop effective and safe antiviral agents for
the treatment of dengue infections.
Through a DENV-2 high-throughput phenotypic screening, spir-
opyrazolopyridone 1 (Fig. 1) was identified as a novel potent DENV
inhibitor.18–20 The R enantiomer 1a was then separated by chiral high-
performance liquid chromatography (HPLC) and confirmed to be far
more potent than the corresponding S enantiomer of racemate 1. Ge-
netic analysis showed that mutations in DENV-2 NS4B conferred re-
sistance to compound 1a inhibition, suggesting that this class of com-
pounds likely targets viral NS4B protein. To improve the
physicochemical properties, compound 2 with 3-pyridyl was discovered
with good in vivo pharmacokinetic profiles and efficacy in DENV-2 in-
fected AG129 mice. However, this class of compounds lacks potency
⁎ Corresponding authors at: Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, United States.
E-mail addresses: peshi@utmb.edu (P.-Y. Shi), jizhou@utmb.edu (J. Zhou).
1 These authors contributed equally to the manuscript.
https://doi.org/10.1016/j.bmcl.2020.127162
Received 25 February 2020; Received in revised form 27 March 2020; Accepted 28 March 2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:JiminXu,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2020.127162

J. Xu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 1
Antiviral
Activity
and
Cytotoxicity
of
Compounds
16-24a
Compd
R1
EC50 (μM)
CC50 (μM)
Fig. 1. The previously reported spiropyrazolopyridone derivatives as potent
D-1
2.4
D-2
D-3
D-4
dengue virus inhibitors.
1
0.11
0.006
> 10
> 10
NT
against DENV-1 and DNEV-4. Our group previously directed the
structure–activity relationship (SAR) exploration mainly on the amide
of the indolone moiety of this series and found that a wide range of
substitutions were well tolerated at this position. Compound 3 with an
isopentyl chain showed the most potent and broadest inhibitory activ-
ities, effective against DENV-1 to -3 with nanomolar to submicromolar
EC50 values, while exhibiting promising efficacy in the A129 mouse
models.21 These good in vivo results suggest that this spiropyr-
azolopyridone scaffold is worthy of further optimization efforts to
elucidate the chemical space for the potential to identify pan-serotype
DENV inhibitors. As part of our ongoing antiviral drug discovery and
development program,21–26 we herein report our continued SAR in-
vestigation of this series, mainly focused on the pyrazolopyridone
moiety.
16
NT
4.2
NT
NT
17
18
NT
NT
> 5
> 5
NT
NT
NT
NT
NT
NT
19
20
21
NT
NT
1.8
> 5
> 5
NT
NT
NT
NT
NT
NT
Previous studies showed a narrow range of para-substitutions were
tolerated on the phenyl ring while meta- or ortho-substitutions resulted
in a significant loss of potency on DENV-2.18 Based on these results, we
first attempted to introduce different functional groups at the para-
position and investigated the effect of di-substitutions on the phenyl
ring as well. Compounds 16–24 were prepared as outlined in Scheme 1.
Substitution of 3-aminopyrazole with different substituted benzyl bro-
mide or benzyl chloride afforded the final products as two isomers,
which were then successfully separated by column chromatography to
give 1-benzyl-5-aminopyrazole derivatives 5a-13a. The subsequent
0.062
> 5
> 5
> 10
22
1.8
0.050
0.019
> 5
> 10
23
24
2.3
1.6
0.14
0.39
> 5
> 5
> 10
> 10
0.038
0.017
a
EC50 values were determined on Huh7 cells stably expressing DENV-1 to
−4 replicon; CC50 values were measured using Huh7 cells stably expressing
DENV-2 replicon; D-1: DENV-1; D-2: DENV-2; D-3: DENV-3; D-4: DENV-4; NT:
not tested.
three component condensation of aminopyrazoles 5a-13a, 5-chlor-
oisatin and Meldrum’s acid provided a series of spiropyrazolopyridone
analogues 16–19 and 21–24.27 Acid analogue 20 was accessed from
ester 19 via hydrolysis. As shown in Table 1, 4-fluorobenzyl substitu-
tion (16) resulted in a significant loss of potency against DENV-2
(EC50 = 4.2 μM), while functional groups cyano (17), sulfonyl (18),
ester (19) and acid (20) on the para-position of the phenyl ring were all
unfavorable for potency, not effective against DENV-2 up to 5 μM.28
These results are consistent with the trend observed before. Interest-
ingly, di-substitutions analogues 21–24 displayed similar or improved
potency against DNEV-2 compared to mono-substitution analogue 1.
Compound 21 with 3,4-dichlorobenzyl exhibited improved potency
against DENV-1 and DENV-2 (EC50 = 1.8 μM and 0.062 μM, respec-
tively) meanwhile displaying a dramatic loss of potency against DENV-
3 (EC50 > 5 μM). Remarkably, replacing 3-Cl with 3-F (22) maintained
the same level of potency against DENV-1 (EC50 = 1.8 μM) and DENV-2
(EC50 = 0.019 μM), while it regained potent activity against DENV-3
with an EC50 value of 0.019 μM as compared with compound 21.
Moving the 3-F group to the ortho position (22) resulted in a 20-fold
loss of potency against DENV-3 (EC50 = 0.39 μM) and slightly de-
creased activities against DENV-1 (EC50 = 2.3 μM) and DENV-2
Scheme 1. Synthetic route of compounds 16–28. Reagents and conditions: (a)
NaH, various substituted benzyl bromide or benzyl chloride, DMF, 0 °C to r.t.,
12 h, 5a-14a, 5b and 9b. (b) K2CO3, 4-chlorobenzyl chloride, CH3CN, 60 °C,
12 h, 15a. (c) 5-chloroisatin, Meldrum’s acid, AcOH, 100 °C, 12 h. (d) NaOH,
MeOH/H2O, r.t., 2 h.
2

J. Xu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 2
Antiviral
Activity
and
Cytotoxicity
of
Compounds
25-32a
Scheme 2. Synthetic route of compound 29. Reagents and conditions: (a) i. t-
KOBu, THF, 0 °C to r.t., 30 min; ii·H2O, r.t.; iii. AcOH, 100 °C, 10 min; iv. NaOEt,
EtOH, r.t., 60 min. (b) 5-chloroisatin, Meldrum’s acid, NH4OAc, AcOH, 100 °C,
12 h.
Compd
R2
R3
EC50 (μM)
CC50 (μM)
D-1
D-2
D-3
D-4
(EC50 = 0.14 μM). Notably, compound 24 with 2,4-bis(trifluoromethyl)
benzyl showed the most potent and broad inhibitory activities against
DENV-1 to -3 with EC50 values of 1.6 μM, 0.038 μM and 0.017 μM,
respectively. Unfortunately, all these compounds did not show obvious
inhibitory activities against DENV-4 at the concentration up to 5 μM.
Next, we turned to explore the SAR around the pyrazolopyridone
core. These modifications, as shown in Scheme 1-3, yielded compounds
25–32 (Table 2). To this end, compounds 25–28 were prepared in a
similar manner to that described above for compounds 16–24. Benzy-
lation of aminopyrazole derivatives 4a-c was followed by a three-
component condensation to afford derivatives 25–28 (Scheme 1). Cy-
clization of succinonitrile, ethyl formate and 4-chlorobenzylamine
provided aminopyrrole 34 via multiple procedures, which was then
treated with 5-chloroisatin and Meldrum’s acid to produce spir-
opyrrolopyridone 29 (Scheme 2). Substitution of 4-chlorobenzyl
chloride with hydrazine provided substituted hydrazine 36, which was
condensed with (E)-ethyl 2-cyano-3-ethoxyacrylate to give amino-
carboxylate 37. Hydrolysis of ester 37 gave the sodium salt, which was
directly used for the subsequent three component reaction with Mel-
drum’s and ethyl acetoacetate or methyl cyanoacetate or methyl mal-
onamate to yield derivatives 30–32, respectively (Scheme 3). The
structures and purity of all synthesized compounds were confirmed by
1H and 13C NMR, HR-MS and HPLC analysis.29
25
26
27
28
NT
NT
2.5
NT
4.4
1.1
1.8
1.9
NT
NT
NT
> 5
NT
> 10
> 10
> 10
> 10
NT
CH3
H
H
H
0.025
NT
29
30
NT
NT
> 5
NT
NT
NT
NT
NT
> 10
> 20
31
32
H
H
CN
NT
NT
8.6
3.2
NT
NT
NT
NT
50
50
a
EC50 values were determined on Huh7 cells stably expressing DENV-1 to
−4 replicon; CC50 values were measured using Huh7 cells stably expressing
DENV-2 replicon; D-1: DENV-1; D-2: DENV-2; D-3: DENV-3; D-4: DENV-4; NT:
not tested.
DENV-2 (EC50 = 1.9 μM). Compound 29 with 3′-cyano pyrrolopyr-
idone core were inactive against DENV-2 up to 5 μM. The 5′-position of
the pyrazolopyridone core (α-carbonyl carbon) was also investigated
and introduction of different substituents yielded compounds 30–32.
These changes resulted in a dramatic loss in activities against DENV-2,
and only compound 32 with carbamoyl moiety exhibited low micro-
molar potency against DENV-2 with an EC50 value of 3.2 μM.
In summary, a series of spiropyrazolopyridone derivatives was
synthesized to explore the SAR around the pyrazolopyridone core.
Introducing different substituents at the 3′- or 5′-site or moving the
benzyl chain to the adjacent nitrogen led to a significant loss of potency
on DENV-2. While a narrow range of substitutions were tolerated at the
para-position of the phenyl ring, di-substitution on the phenyl ring is
beneficial for DENV-2 potency and has variable influences on DENV-3
potency depending on the exact compound. Among these molecules,
while compounds 22 (JMX0376) and 24 (JMX0395) displayed similar
potency profiles, compound 24 with 2,4-bis(trifluoromethyl)benzyl
showed the most potent and broadest inhibitory activities against
DENV-1 to -3 with EC50 values of 1.6 μM, 0.038 μM and 0.017 μM,
respectively.
As shown in Table 2, moving the benzyl chains to the adjacent ni-
trogen position (25 and 26) led to a sharp decrease in activity against
DENV-2 in comparison with compounds 1 and 21, respectively. Inter-
estingly, introduction of one methyl group at the 3′-site of the pyr-
azolopyridone core (27) retained the same level of potency against
DENV-1 (EC50 = 2.5 μM) and DENV-3 (EC50 = 0.025 μM) while di-
minishing the antiviral activity against DENV-2 (EC50 = 1.8 μM) in
contrast to compound 1. Substitution of 3′-methyl of compound 27 with
́
3-cyclopropyl produced compound 28 with similar potency against
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
Scheme 3. Synthetic route of compounds 30–32. Reagents and conditions: (a)
N2H4·H2O, EtOH, r.t., 48 h. (b) (E)-ethyl 2-cyano-3-ethoxyacrylate, EtOH, 80 °C,
8 h. (c) i. NaOH, EtOH/H2O, reflux, overnight; ii. 5-chloroisatin, ethyl acet-
oacetate or methyl cyanoacetate or methyl malonamate, AcOH, 100 °C, 6 h.
This work was supported by the John S. Dunn Foundation, the
Amon G. Carter Foundation, the Kleberg Foundation, the Gilson
Longenbaugh Foundation, the Summerfield Robert Foundation, CDC
3

J. Xu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
grant U01CK0000512, NIH grants U19AI142759, 1R41AI136126, and
R01AI127744, John Sealy Memorial Endowment Fund, John D. Stobo,
M.D. Distinguished Chair Endowment Fund, and Institute for
Translational Sciences (ITS) at UTMB.
and 4-(bromomethyl)-1,2-dichlorobenzene (1.43 g, 5.96 mmol) was added. The re-
sulting mixture was stirred at r.t. for 2 h. Then the mixture was diluted with 120 mL
of EtOAc, washed with water (2 × 30 mL) and brine (30 mL), dried (Na2SO4) and
concentrated. The residual was purified by column chromatography (Hex/EtOAc =
2/1 to 1/1) to give the amino intermediate 1-(3,4-dichlorobenzyl)-1H-pyrazol-5-
amine 9a (105 mg, 11%) as yellow oil. 1H NMR (300 MHz, CDCl3) δ 7.39 (d, J = 8.1,
1H), 7.33 (d, J = 1.8 Hz, 1H), 7.24 (d, J = 1.8 Hz, 1H), 6.99 (dd, J = 8.4, 2.1 Hz,
1H), 5.61 (d, J = 1.8 Hz, 1H), 5.15 (s, 2H), 3.38 (s, 2H). A solution of aminopyrazole
9a (105 mg, 0.43 mmol), 5-chloroisatin (86 mg, 0.48 mmol) and Meldrum’s acid (94
mg, 0.65 mmol) in 2 mL of AcOH was stirred at 100 °C for 12 h. The reaction mixture
was cooled to r.t. and concentrated in vacuo. The residue was stirred with water (10
mL) for 15 min. And the solid precipitate was filtered and further purified by column
chromatography (DCM/MeOH = 30/1 to 20/1) to give 5-chloro-1′-(3,4-di-
chlorobenzyl)-5′,7′-dihydrospiro[indoline-3,4′-pyrazolo[3,4-b]pyridine]-2,6′(1′H)-
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;
found, 431.0476. 5-Chloro-1′-(4-chloro-2-fluorobenzyl)-5′,7′-dihydrospiro[indoline-
3,4′-pyrazolo[3,4-b]pyridine]-2,6′(1′H)-dione (23). Yellow solid. HPLC purity 95.9%
(tR = 17.26 min). 1H NMR (300 MHz, DMSO-d6) δ 11.07 (s, 1H), 10.59 (s, 1H), 7.47
(dd, J = 9.9, 1.8 Hz, 1H), 7.37 – 7.24 (m, 3H), 7.07 (t, J = 8.1 Hz, 1H), 6.91 (d, J =
7.8 Hz, 1H), 6.80 (s, 1H), 5.38 (d, J = 15.9 Hz, 1H), 5.29 (d, J = 15.9 Hz, 1H), 3.12
(d, J = 15.9 Hz, 1H), 2.54 (d, J = 15.9 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ 178.
4, 168.7, 159.7 (d, J = 248.5 Hz), 140.8, 140.7, 134.5, 133.7, 133.2 (d, J = 10.3
Hz), 130.8 (d, J = 5.1 Hz), 128.6, 126.1, 124.8 (d, J = 3.6 Hz), 124.0, 123.2 (d, J =
14.9 Hz), 116.0 (d, J = 24.8 Hz), 111.3, 100.3, 45.8, 44.7. HRMS (ESI) calcd for
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C
20H14Cl2FN4O2, 431.0478 (M + H)+; found, 431.0474. 1′-(2,4-Bis(trifluoromethyl)
benzyl)-5-chloro-5′,7′-dihydrospiro[indoline-3,4′-pyrazolo[3,4-b]pyridine]-2,
6′(1′H)-dione (24). Yellow solid. HPLC purity 97.9% (tR = 18.63 min). 1H NMR (300
MHz, CD3OD + CDCl3) δ 7.94 (s, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.26 (dd, J = 8.4, 2.
1 Hz, 1H), 7.20 (d, J = 1.8 Hz, 1H), 6.99 (s, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.84 (d, J
= 8.4 Hz, 1H), 5.64 (d, J = 17.7 Hz, 1H), 5.56 (d, J = 17.7 Hz, 1H), 2.96 (d, J = 16.
2 Hz, 1H), 2.80 (d, J = 16.2 Hz, 1H). 13C NMR (75 MHz, CD3OD + CDCl3) δ 180.0,
170.3, 142.5, 140.6, 139.9, 136.5, 133.7, 130.9 (q, J = 33.5 Hz), 130.1 – 129.9 (m),
129.9, 129.0, 128.9, 128.6 (q, J = 32.0 Hz), 124.5, 124.1 (q, J = 272.2 Hz), 124.0 –
123.6 (m), 123.8 (q, J = 270.4 Hz), 112.2, 101.9, 48.5 (q, J = 3.9 Hz), 47.0, 41.1.
HRMS (ESI) calcd for C22H14ClF6N4O2, 515.0709 (M + H)+; found, 515.0707. 5-
Chloro-1′-(4-chlorobenzyl)-3′-methyl-5′,7′-dihydrospiro[indoline-3,4′-pyrazolo[3,4-
b]pyridine]-2,6′(1′H)-dione (27). Yellow solid. HPLC purity 96.7% (tR = 17.19
min). 1H NMR (300 MHz, DMSO-d6) δ 11.02 (s, 1H), 10.66 (s, 1H), 7.46 – 7.38 (m,
2H), 7.33 – 7.28 (m, 2H), 7.27 – 7.21 (m, 2H), 6.95 – 6.89 (m, 1H), 5.23 (d, J = 15.9
Hz, 1H), 5.16 (d, J = 15.9 Hz, 1H), 3.05 (d, J = 15.9 Hz, 1H), 2.59 (d, J = 15.9 Hz,
1H), 1.42 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 178.2, 168.6, 142.6, 140.6, 140.6,
136.1, 133.5, 132.1, 129.4 (2C), 128.6, 128.5 (2C), 126.0, 124.0, 111.3, 97.1, 49.6,
46.1, 11.8. HRMS (ESI) calcd for C21H17Cl2N4O2, 427.0729 (M + H)+; found, 427.
0725.
21. Xu J, Xie X, Ye N, et al. Design, synthesis, and biological evaluation of substituted
4,6-dihydrospiro[[1,2,3]triazolo[4,5-b]pyridine-7,3'-indoline]-2',5(3H)-dione analo-
gues as potent NS4B inhibitors for the treatment of dengue virus infection. J Med
Chem. 2019;62(17):7941.
22. Fan X, Xu J, Files M, et al. Dual activity of niclosamide to suppress replication of
integrated HIV-1 and Mycobacterium tuberculosis (Beijing). Tuberculosis (Edinb).
2019;116:28.
23. Niu Q, Liu Z, Alamer E, et al. Structure-guided drug design identifies a BRD4-se-
lective small molecule that suppresses HIV. J Clin Invest. 2019;129(8):3361.
24. Xu J, Berastegui-Cabrera J, Chen H, Pachón J, Zhou J, Sanchez-Cespedes J. Structure-
activity relationship studies on diversified salicylamide derivatives as potent in-
hibitors of human adenovirus infection. J Med Chem. 2020;63(6):3142.
25. Li Z, Brecher M, Deng Y-Q, et al. Existing drugs as broad-spectrum and potent in-
hibitors for Zika virus by targeting NS2B-NS3 interaction. Cell Res. 2017;27(8):1046.
26. J. Xu P.-Y. Shi H. Li J. Zhou Broad spectrum antiviral agent niclosamide and its
therapeutic potential. ACS Infect Dis. 2020: 10.1021/acsinfecdis.0c00052.
27. General procedures for synthesizing the final products (exemplified by compound
21). To a solution of 3-aminopyrazole (330 mg, 3.97 mmol) in 8 mL of DMF was
added NaH (238 mg, 5.96 mmol, 60% dispersion in mineral oil) at 0 °C. After ad-
dition, the mixture was stirred at 50 °C for 1 h. Then the mixture was cooled to r.t.
4