Rapid microwave-assisted solution-phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2012.10.028

Rapid microwave-assisted solution-phase peptide synthesis protocol has been developed. The reaction temperature of 50 °C, 40 W and short coupling time of 28 min gave racemization-free synthesis of peptides in 50-80% yield. The method is applicable with eq

Full text of DOI:10.1016/j.tetlet.2012.10.028

Tetrahedron Letters 53 (2012) 6931–6935
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Tetrahedron Letters
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Rapid microwave-assisted solution-phase peptide synthesis
⇑
Amit Mahindra, Krishna K. Sharma, Rahul Jain
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Punjab 160 062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Rapid microwave-assisted solution-phase peptide synthesis protocol has been developed. The reaction
temperature of 50 °C, 40 W and short coupling time of 28 min gave racemization-free synthesis of pep-
tides in 50–80% yield. The method is applicable with equal ease in coupling both side-chain protected and
unprotected amino acids indicating reactive-functional group tolerance and high atom-economy. In the
case of dipeptides, DIC/HONB-mediated coupling rendered the best results. In the case of tri- and tetra-
peptides, HATU/HOAt/DIEA emerged as the best coupling combination.
Received 24 August 2012
Revised 4 October 2012
Accepted 6 October 2012
Available online 15 October 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Solution-phase peptide synthesis
Microwave-assisted synthesis
Activators
Boc chemistry
Fmoc chemistry
Peptides and peptidomimetics play a pivotal role in medicinal
and pharmaceutical research. Peptides have received meticulous
attention over the years as they possess a wide range of biological
properties and are assigned as privileged pharmacophores in drug
development.1,2 The myriad properties of peptides include, antimi-
crobial,3 antiplasmodial,4 antifungal,5 antitubercular,6 anti-
cancer,7 antidiabetic,8 neuro-protection,9 and hormonal activitiy.10
Two standard protocols are available for the synthesis of peptides—
one is through linkers attached to a solid support11 and the other
one is via solution-phase peptide synthesis.12 The standard proto-
col for the coupling of amino acids in solution-phase is cumber-
some and limited, requiring long coupling hours and low yield/
purity of crude peptides.13
duced reaction time, coupling of N-methyl amino acids, synthesis
of difficult sequences, and high purity/yield of crude peptides.17
However, the application of MW-irradiation in solution-phase pep-
tide synthesis is not well-explored,18 possibly due to high inci-
dences of enantiomerization in conventional solution-phase
peptide synthesis.
In this Letter, we report the utility of various coupling reagents
and activators to provide generalized synthetic protocol for pep-
tides in solution-phase under MW-irradiation. The amino acids
chosen for the study include, arginine (Arg), tryptophan (Trp),
and histidine (His). From the literature, it is evident that arginine
and histidine containing peptides are difficult to synthesize due
to the presence of participating side-chain groups resulting in
low yield.19 The new practical, rapid, and robust method could
be used to synthesize peptides containing up to four amino acids.
For higher peptides, purification of peptide rather than the ability
of the procedure becomes a limiting factor in the synthesis.
We first synthesized peptides using the standard solution-
phase protocol (Table 1). The synthesis of peptides was achieved
using DIC as coupling reagent and HONB as an auxiliary nucleo-
phile. The synthesis of Boc-His-Arg-OMe and Boc-Arg-Arg-OMe
(Table 1, entries 3 and 4) could only be achieved in 15–27% yield
In the past few years, the application of microwave (MW)-as-
sisted synthesis has been greatly used in the edifice of various
innovative organic moieties.14,15 The use of MW-irradiation in so-
lid-phase peptide synthesis (SPPS) has been well-documented.16
The benefits of MW-SPPS over conventional synthesis include, re-
Abbreviations: DIC, 1,3-diisopropylcarbodiimide; HONB, N-hydroxy-5-norborn-
ene-endo-2,3-dicarboximide; Ph, phenyl; An, anisolyl; Bom, benzyloxymethyl; Trt,
trityl; Mtr, 4-methoxy-2,3,6-trimethyl-benzenesulfonyl; Pbf, 2,2,4,6,7-pentame-
thyldihydrobenzofuran-5-sulfonyl; TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetram-
in 48 h. Similar pattern was observed when ring-arylated-L-histi-
dines20 were used to synthesize dipeptides (Table 1, entries 5–7,
yield = 18–34%). In the cases of tripeptides containing natural ami-
no acids, the yield was 15–26% (Table 1, entries 8 and 9), which
further decreased to 10–20%, when modified amino acids were
used (Table 1, entries 10 and 11). It is clear from these experiments
that conventional solution-phase synthesis of peptides results in
overall poor yield of 10–40% in 36–48 h.
ethyluronium tetrafluoroborate;
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; HATU, 2-
(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
HOBt, 1-hydroxy-benzotriazole; PyBOP,
HCTU, 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluor-
ophosphate; HOAt, 1-hydroxy-7-azabenzotriazole.
⇑
Corresponding author. Tel.: +91 172 2292024; fax: +91 172 2214692.
E-mail address: rahuljain@niper.ac.in (R. Jain).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.10.028

6932
A. Mahindra et al. / Tetrahedron Letters 53 (2012) 6931–6935
Table 1
Table 2
Solution-phase peptide synthesis at 0–4 °Ca
Comparison between 0–4 °C, conventional heating and microwave-assisted reactiona
Entry
Sequence
Reaction time (h)
Yieldb (%)
Entry
Sequence
Isolated yield (%)
1
2
3
4
5
6
7
8
Boc-Trp-His-OMe
Boc-His-His-OMe
Boc-His-Arg-OMe
Boc-Arg-Arg-OMe
Boc-Trp-His(2-Ph)-OMe
Boc-His(2-Ph)-His(2-Ph)-OMe
Boc-His(2-Ph)-Arg-OMe
Boc-His-Trp-His-OMe
Boc-Arg-His-Arg-OMe
Boc-His(2-Ph)-Trp-His(2-Ph)-OMe
Boc-Arg-His(2-Ph)-Arg-OMe
36
36
48
48
36
36
48
48
48
48
48
40
32
27
15
34
23
18
26
15
20
10
0–4 °C
Heating (50 °C)
MW
1
2
3
4
Boc-Trp-His-OMe
Boc-His-Arg-OMe
Boc-His-Trp-His-OMe
Boc-Arg-His-Arg-OMe
40
27
26
15
25
15
12
7
70
65
45
20
a
Reaction conditions: NH2-AA1-OMeÁ2HCl (1 mmol), Boc-AA2-OH (1.6 mmol),
DIC (1.6 mmol), HONB (1.6 mmol), and DMF (2 mL).
9
10
11
Table 3
a
Reaction conditions: NH2-AA1-OMeÁ2HCl (1 mmol), Boc-AA2-OH (1.6 mmol),
Screening of different activators for the synthesis of dipeptides and tripeptides under
microwave irradiationa
DIC (1.6 mmol), HONB (1.6 mmol), DMF (2 mL), and 0–4 °C.
b
Isolated yield after column chromatography.
Entry Activator Auxiliary
nucleophile
Sequence
Yieldb
(%)
1
2
3
4
5
6
DIC
HONB
HOBt
HOBt
HOAt
HOAt
HONB
Boc-His-Arg-OMe
Boc-His-Arg-OMe
Boc-His-Arg-OMe
Boc-His-Arg-OMe
Boc-His-Arg-OMe
Boc-Arg-His-Arg-
OMe
Boc-Arg-His-Arg-
OMe
Boc-Arg-His-Arg-
OMe
65
52
38
56
43
20
We then systematically investigated the application of
MW-irradiation in the solution-phase peptide synthesis. The MW
conditions for peptide synthesis were standardized by synthesiz-
ing Boc-His-Arg-OMe using DIC/HONB in DMF. Several reaction
conditions were used during the optimization process. From
Fig. 1 it is clear that the curve 2 (reaction conditions: 50 °C,
40 W) rendered the best results. It was observed that extending
the reaction time did not improve the yield and increasing the tem-
perature led to decreased yield, probably due to the formation of
side products. It was noted that the reaction temperature of
50 °C, 40 W and a short coupling time of 28 min were sufficient
to offer racemization-free synthesis of Boc-His-Arg-OMe in 65%
yield (Table 2, entry 2).
TBTU
PyBOP
HATU
HCTU
DIC
7
TBTU
HOBt
HOBt
HOAt
HOAt
45
23
50
37
8
PyBOP
HATU
HCTU
9
Boc-Arg-His-Arg-
OMe
Boc-Arg-His-Arg-
OMe
10
a
Reaction conditions: NH2-AA1-OMeÁ2HCl (1 mmol), Boc-AA2-OH (1.6 mmol),
To observe the advantages of MW-irradiation, peptides were
also synthesized by using conventional heating (oil bath) keeping
reaction conditions intact (Table 2). We found that under conven-
tional heating, the yields were even lower (7–25%) compared to the
0–4 °C reaction conditions (yield = 15–40%).
MW, 50 °C, 40 W, 28 min, DMF (2 mL). DIEA (3 mmol) was used in the synthesis of
tripeptides.
b
Isolated yield after column chromatography.
In the case of tripeptides, the use of DIC/HONB resulted in low
yield of 20–45%, even after employing MW-irradiation (Table 2,
entries 3 and 4). This prompted us to further investigate the MW-
assisted solution-phase peptide synthesis by screening various
well-known combinations of activators and auxiliary nucleophiles.
The combinations of DIC/HONB, TBTU/HOBt/DIEA, PyBOP/HOBt/
DIEA, HATU/HOAt/DIEA, and HCTU/HOAt/DIEA were investigated
for standardization of the coupling reaction. As observed earlier,
DIC/HONB proved to be the best combination for the synthesis of
dipeptides (Table 3, entry 1). The use of other combinations could
only produce dipeptides in 38–56% yield (Table 3, entries 2–5). The
combination of HATU/HOAt/DIEA in DMF produced best result in
the case of tripeptides (Table 3, entry 9, yield = 50%). It was evident
from the TLC chromatograms that the purity of the crude peptides
synthesized by using MW-irradiation was much higher as com-
pared to the standard protocol (0–4 °C). We believe that due to ra-
pid and focused nature of MW-based protocols, the formation of
several side products such as N-acylurea, diketopiperazine, and
c-lactam was possibly suppressed, resulting in enhanced yield
and purity of peptides.
With the optimized conditions in hand, the MW-assisted pep-
tide synthesis protocols were applied to synthesize several new
peptides (1a–l, 2a–h, and 3a–b, Scheme 1). All side-chain unpro-
tected amino acids were coupled satisfactorily using DIC/HONB
in DMF to produce dipeptides (Table 4, 1a–j, yield = 54–70%).21
The side-chain unprotected tripeptides (Table 4, 2a–f) were syn-
thesized in 50–59% yield using a combination of HATU/HOAt/DIEA
in DMF under MW-irradiation.22 The MW-assisted coupling proto-
cols were also investigated for peptides containing protected ami-
no acids. To our delight, Boc-His(Bom)-Arg-OMe (1k) and Boc-
Arg(Mtr)-His(Bom)-Arg-OMe (2g) were obtained in 77% and 61%
yields, respectively (Table 4).21,22 To broaden its usefulness, the
MW-assisted protocol was also investigated in Fmoc-based solu-
tion phase peptide synthesis. The desired peptides, Fmoc-
His(Trt)-Arg-OMe (1l) and Fmoc-Arg(Pbf)-His(Trt)-Arg-OMe (2h)
could be synthesized in 80% and 63% yields, respectively (Ta-
ble 4).21,22 Finally, the scope of MW-assisted method was further
expanded by synthesizing tetrapeptides, Boc-Trp-Arg(Mtr)-His(-
Bom)-Arg-OMe (3a) and Fmoc-Trp-Arg(Pbf)-His(Trt)-Arg-OMe
(3b) (Table 4, yield = 52–55%).22,23
It is well known that the SSPS requires expensive solid sup-
ported linker, 3- to 6-fold excess of coupling amino acids and acti-
Figure 1. Optimization of solution-phase peptide synthesis under microwave
irradiation.

A. Mahindra et al. / Tetrahedron Letters 53 (2012) 6931–6935
6933
Scheme 1.
Table 4
vators for driving the synthesis to completion. The MW-assisted
solution-phase peptide synthesis methodology reported herein
use about 1.6-fold excess of the coupling amino acids and activa-
tors making it attractive compared to SSPS, specifically for smaller
peptides.
The following analytical techniques were used to assign the
structure and purity of the isolated peptides: 1H, 13C, COSY, HSQC,
HRMS, and RP-HPLC analysis (peptides were analyzed on a Shima-
Microwave-assisted solution-phase peptide synthesis
S. No.
Sequence
Isolated yield (%)
1a
1b
1c
1d
1e
1f
1g
1h
1i
Boc-Trp-His-OMea
70
61
65
54
63
57
61
65
59
60
77
80
59
50
52
51
54
50
61
63
55
52
Boc-His-His-OMea
Boc-His-Arg-OMea
Boc-Arg-Arg-OMea
Boc-Trp-His(2-Ph)-OMea
Boc-His(2-Ph)-His(2-Ph)-OMea
Boc-His(2-Ph)-Arg-OMea
dzu HPLC system, equipped with a C-18, 300 Å, 5
l
m, 250 Â 4.6
Boc-Trp-His(2-An)-OMea
mm column, run for 60 min with a flow of 1 mL/min, using a
gradient of 95–5% where buffer A was 0.1% TFA in H2O and buffer
B was 0.1% TFA in CH3CN and detection at 220 nm).24–27 The
inspection of typical HPLC chromatograms (Figs. 2 and 3), indicates
that purity of peptides was more than 95%.
Boc-His(2-An)-His(2-An)-OMea
Boc-His(2-An)-Arg-OMea
Boc-His(Bom)-Arg-OMe
1j
1k
1l
Fmoc-His(Trt)-Arg-OMe
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
Boc-His-Trp-His-OMeb
Boc-Arg-His-Arg-OMeb
In conclusion, new robust and rapid synthetic protocols have
been established for peptides in solution-phase employing MW-
irradiation. The method allows rapid coupling of unprotected and
reactive groups containing amino acids resulting in high-functional
group tolerance and atom economy. As expected, comparatively
higher yields were obtained while coupling protected amino acids.
A combination of DIC/HONB in DMF was found to provide best
results for dipeptides. The HATU/HOAt/DIEA method of in situ acti-
vation has emerged as the best combination for the synthesis of tri-
and tetrapeptides. The issues related to solubility and purification
encountered during the synthesis of higher peptides render the
method unattractive beyond tetrapeptides. The MW-assisted
Boc-His(2-Ph)-Trp-His(2-Ph)-OMeb
Boc-Arg-His(2-Ph)-Arg-OMeb
Boc-His(2-An)-Trp-His(2-An)-OMeb
Boc-Arg-His(2-An)-Arg-OMeb
Boc-Arg(Mtr)-His(Bom)-Arg-OMeb
Fmoc-Arg(Pbf)-His(Trt)-Arg-OMeb
Boc-Trp-Arg(Mtr)-His(Bom)-Arg-OMeb
Fmoc-Trp-Arg(Pbf)-His(Trt)-Arg-OMeb
a
Reaction conditions: NH2-AA1-OMeÁ2HCl (1 mmol), Fmoc/Boc-AA2-OH
(1.6 mmol), DIC (1.6 mmol), HONB (1.6 mmol), and DMF (2 mL).
b
Reaction conditions: NH2-AA1-OMeÁ2HCl (1 mmol), Fmoc/Boc-AA2-OH
(1.6 mmol), HATU (1.6 mmol), HOAt (1.6 mmol), DIEA (3 mmol), and DMF (2 mL).
Figure 2. HPLC chromatogram (UV absorbance at 220 nm) of Boc-Trp-His(2-An)-OMe (1h).

6934
A. Mahindra et al. / Tetrahedron Letters 53 (2012) 6931–6935
Figure 3. HPLC chromatogram (UV absorbance at 220 nm) of Boc-His(2-An)-Trp-His(2-An)-OMe (2e).
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Acknowledgment
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AM thanks the Council of Scientific and Industrial Research
(CSIR), New Delhi for the award of Senior Research Fellowship.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.10.
028.
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21. General experimental procedure for dipeptides (1a–l): In
a 10 mL MW vial
equipped with a magnetic stir bar, amino acid (AA1) methyl ester (1 mmol) was
dissolved in DMF (2 mL). Fmoc/Boc-AA2-OH (1.6 mmol) was added followed by
DIC (1.6 mmol) and HONB (1.6 mmol). Mixture was subjected to MW-
irradiation (CEM DiscoverÒ microwave reactor) with gas cooling (pressure of
70 psi was maintained during irradiation) for 28 min at 40 W with magnetic
stirring, and a temperature limit of 50 °C (reaction time refers to the hold time
at the desired set temperature). DMF was evaporated and the reaction mixture
was purified on automated flash column chromatography system (BiotageÒ) to
give Fmoc/Boc-AA2-AA1-OMe.
22. General experimental procedure for N-a-Boc-protected peptides (2a–g, 3a): In a
10 mL MW vial equipped with a magnetic stir bar, Boc-AA2-AA1-OMe (1 mmol)
was subjected to 6 N HCl (5 mL) at 25 °C for 30 min to remove Boc group.
Dihydrochloride salt of dipeptide was neutralized with DIEA (3 mmol). The
resulting AA2-AA1-OMe (1 mmol) was dissolved in DMF (2 mL). Boc-AA3-OH
(1.6 mmol) was added followed by HATU (1.6 mmol) and HOAt (1.6 mmol).
Mixture was subjected to MW-irradiation (CEM DiscoverÒ microwave reactor)
with gas cooling (pressure of 70 psi was maintained during irradiation) for
28 min at 40 W with magnetic stirring, and a temperature limit of 50 °C
(reaction time refers to the hold time at the desired set temperature). DMF was
evaporated and the reaction mixture was purified on automated flash column
chromatography system (BiotageÒ
) to afford tripeptides (2a–g). The
deprotection and coupling cycle described above was repeated to obtain
tetrapeptide (3a).
23. General experimental procedure for N-
a-Fmoc-protected peptides (2h, 3b): In a
10 mL MW vial equipped with magnetic stir bar, Fmoc-AA2-AA1-OMe
a
(1 mmol) was subjected to 20% piperidine (2 mL) at 25 °C for 10 min
followed by the removal of excess piperidine. The resulting AA2-AA1-OMe
(1 mmol) was dissolved in DMF (2 mL). Fmoc-AA3-OH (1.6 mmol) was added
followed by HATU (1.6 mmol), HOAt (1.6 mmol), and DIEA (1.6 mmol). The
mixture was subjected to MW-irradiation (CEM DiscoverÒ microwave reactor)
10. (a) Rivier, J.; Vale, W.; Monahan, M.; Ling, N.; Burgus, R. J. Med. Chem. 1972, 15,
479–482; (b) Kaur, N.; Lu, X.; Gershengorn, M. C.; Jain, R. J. Med. Chem. 2005, 48,
6162–6165.

A. Mahindra et al. / Tetrahedron Letters 53 (2012) 6931–6935
6935
with gas cooling (pressure of 70 psi was maintained during irradiation) for
28 min at 40 W with magnetic stirring, and a temperature limit of 50 °C
(reaction time refers to the hold time at the desired set temperature). DMF was
evaporated and the reaction mixture was purified on automated flash column
chromatography system (BiotageÒ) to afford tripeptide (2h). The deprotection
and coupling cycle described above was repeated to obtain tetrapeptide (3b).
24. Spectral data of Boc-Trp-His(2-An)-OMe (1h): 1H NMR (400 MHz, CD3OD)
d = 7.78 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H),
7.12–7.06 (m, 2H), 7.04–6.94 (m, 3H), 6.84 (s, 1H), 4.77–4.70 (m, 1H), 4.38 (dd,
J = 5.9, 8.2 Hz, 1H), 3.84 (s, 3H), 3.68 (s, 3H), 3.21 (br s., 1H), 3.11 (d, J = 5.0 Hz,
1H), 3.08–2.98 (m, 2H), 1.37 (s, 9H); 13C NMR (100 MHz, CD3OD) d = 175.6,
174.8, 172.8, 161.7, 157.7, 148.1, 138.0, 135.5, 128.9, 128.0, 124.6, 123.9, 122.4,
120.2, 119.8, 119.4, 115.3, 112.3, 110.9, 80.7, 57.0, 55.8, 54.0, 52.8, 52.3, 45.7,
43.8, 29.9, 29.1, 28.7; HRMS (ESI-TOF) calcd for [M+H+]: 562.2665, found
562.2680; HPLC: tR = 47.08 min, 97.13%.
CD3OD) d = 174.5, 173.5, 162.0, 159.3, 158.6, 156.1, 148.6, 128.1, 127.1, 120.4,
115.4, 82.0, 55.9, 53.1, 52.9, 41.9, 30.5, 29.7, 28.7, 26.1; HRMS (ESI-TOF) calcd
for [M+H+]: 532.2880, found 532.2883; HPLC: tR = 28.38 min, 95.21%.
26. Spectral data of Boc-His(2-An)-Trp-His(2-An)-OMe (2e): 1H NMR (400 MHz,
CD3OD) d = 7.76 (d, J = 8.5 Hz, 4H), 7.33 (d, J = 8.0 Hz, 1H), 7.15 (s, 1H), 7.08 (d,
J = 8.0 Hz, 1H), 7.04–6.94 (m, 6H), 6.95 (br s., 1H), 6.92 (s, 1H), 4.65 (s, 2H),
4.36–4.23 (m, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.66 (s, 3H), 3.29–3.14 (m, 3H),
3.02 (br s., 2H), 2.94–2.81 (m, 1H), 1.39 (s, 9H); 13C NMR (100 MHz, CD3OD)
d = 174.1, 173.5, 172.3, 162.5, 147.4, 138.0, 128.8, 128.5, 124.8, 122.5, 119.9,
119.2, 115.6, 112.4, 110.3, 81.0, 56.0, 56.0, 55.5, 53.7, 53.0, 52.9, 30.8, 30.1,
29.1, 28.6, 27.9; HRMS (ESI-TOF) calcd for [M+H+]: 805.3673, found 805.3679;
HPLC: tR = 48.50 min, 93.36%.
27. Spectral data of Boc-Arg-His(2-An)-Arg-OMe (2f): 1H NMR (400 MHz, CD3OD)
d = 7.82 (d, J = 8.8 Hz, 2 H), 7.03–6.98 (m, 2H), 6.94 (br s., 1H), 4.70–4.65 (m,
1H), 4.43 (dd, J = 4.9, 9.2 Hz, 1H), 4.07–4.01 (m, 1H), 3.84 (s, 3H), 3.71 (s, 3H),
3.27–3.06 (m, 5H), 2.94 (br s., 1H), 1.99–1.87 (m, 1H), 1.82–1.74 (m, 2H), 1.70–
1.59 (m, 5H), 1.31 (s, 9H); 13C NMR (100 MHz, CD3OD) d = 174.8, 174.1, 173.5,
161.7, 158.6, 148.1, 127.9, 124.2, 115.3, 81.0, 55.9, 54.9, 53.3, 52.9, 42.0, 41.9,
40.4, 30.2, 29.5, 28.7, 26.2; HRMS (ESI-TOF) calcd for [M+H+]: 688.3894, found
688.3894; HPLC: tR = 33.12 min, 97.12%.
25. Spectral data of Boc-His(2-An)-Arg-OMe (1j): 1H NMR (400 MHz, CD3OD)
d = 7.83–7.77 (m, J = 8.8 Hz, 2H), 7.06–7.00 (m, J = 8.8 Hz, 2H), 6.98 (s, 1H),
4.47 (dd, J = 4.8, 9.0 Hz, 1H), 4.40 (dd, J = 5.6, 8.2 Hz, 1H), 3.86 (s, 3H), 3.69 (s,
3H), 3.20 (q, J = 6.7 Hz, 2H), 3.12 (dd, J = 5.1, 14.9 Hz, 1H), 2.94 (br s., 1H), 1.92
(br s., 1H), 1.74 ((br s., 1H), 1.70–1.61 (m, 2H),1.37 (s, 9H). 13C NMR (100 MHz,