3-Hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxylates-A new class of HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.02.055

A new class of inhibitors of HIV-1 integrase has been optimized to provide selective and highly efficient strand transfer inhibition.

Full text of DOI:10.1016/j.bmcl.2009.02.055

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1930–1934
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxylates—A new class
of HIV-1 integrase inhibitors
*
Monica Donghi , Olaf D. Kinzel, Vincenzo Summa
Department of Medicinal Chemistry, IRBM, Merck Research Laboratories Rome, Via Pontina km 30,600, 00040 Pomezia, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of inhibitors of HIV-1 integrase has been optimized to provide selective and highly efficient
strand transfer inhibition.
Received 19 December 2008
Revised 12 February 2009
Accepted 13 February 2009
Available online 20 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HIV-1 integrase
Inhibitor
Spread assay
Pyridopyrimidine
Oxalamide
The Acquired Immunodeficiency Syndrome (AIDS) is a major
epidemic with more than 33 million infected people worldwide.1
Its etiological agent has been identified as human immunodefi-
ciency virus type 1 (HIV-1). Current approved therapies target
four steps of the HIV life cycle (fusion, reverse transcription,
integration and proteolytic maturation).2 Triple therapy, com-
monly referred to as HAART (highly active antiretroviral therapy)
is the standard for HIV treatment. Unfortunately, despite the
great efficacy of HAART long term treatment has many side ef-
fects and the selection of mutated viruses leads to the failure
of the therapy. Therefore there is a continuous need for new
agents with improved properties.
Integrase is an enzyme encoded by the HIV genome and repre-
sents a well established target. It catalyses the insertion and inte-
gration of the proviral DNA into the genome of the host cell in two
steps: 30-processing (endonucleolytic sequence-specific hydrolysis
of the 30-ends of the viral cDNA) and strand transfer (ligation of the
viral 30-OH cDNA ends to the phosphate backbone of the host DNA
acceptor).3
inhibitor (raltegravir) was approved in the U.S. and in Europe for
treatment of HIV infection.6
In a previous article, we reported the discovery of bicyclic
pyrimidinones which were created by linking the N1-Methyl
group of the pyrimidinone scaffold 17 into a saturated cycle as
in 2 (Fig. 1).8
Herein we describe further studies evolving the 6,7,8,9-tetrahy-
dropyrido[1,2-a]pyrimidin-4-one scaffold
2 into a pyrido[1,2-
a]pyrimidin-4-one scaffold 3 with the aim of removing the ben-
zylic stereocenter.
In order to evaluate the validity of the scaffold, the unsubstitut-
ed template 4 was first prepared and tested in the enzymatic assay
QUICKIN (QI) (Table 1).
The compound resulted to be a very potent inhibitor of the
strand transfer showing an IC50 of 22 nM.
Efforts were then directed towards the evaluation of different
linker atoms like N and C for the substituents at position 9 of the
bicyclic scaffold.
Amines and amides were tolerated by the enzyme. N-methyl
amino derivative 5 lost sixfold compared to the unsubstituted scaf-
fold 4 while the N-ethyl amino derivative 6 and the benzamide
derivative 8 maintained high activity. The N,N-dimethylamino
substituted compound 7 lost threefold.
The substitution of 4 with a methyl group in position 9 led to a
twofold gain in enzymatic activity (9, IC50 = 9 nM). In the light of
this result we investigated the homologation of amine and amide
derivatives. With the introduction of basic substituents (e.g., 10
and 11) a nearly 20-fold loss in intrinsic potency was observed.
While first generation integrase inhibitors attempted to block
the whole assembly process, recent series have been designed to
specifically inhibit the strand transfer reaction.4 Selective inhibi-
tion of the integrase activity causes an interruption of the HIV-1
replication cycle and represents an answer to the unmet medical
need for new improved treatments.5 Recently the first integrase
* Corresponding author.
E-mail address: monica_donghi@merck.com (M. Donghi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.055

M. Donghi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1930–1934
1931
O
N
O
O
N
OH
NH
F
OH
NH
F
OH
NH
F
N
N
N
O
N
N
O
N
O
N
O
R1 R2
R1 R2
1
2
3
Figure 1. From bicyclic pyrimidinones to fused pyridopyrimidones.
On the other hand, the homologated benzamide 12 retained
activity.
Unfortunately, inhibition in the cell based assay (Spread) was
lower than desired even under low serum condition (10% fetal bo-
vine serum). This effect could be due to the combination of subop-
timal physical chemical properties, including high lipophilicity.
Table 1
In vitro activity
O
N
OH
F
N
H
N
9
R1
O
Only 6 exhibited activity (Table 2) less than 1.5 lM.
Compound
R1
QUICKIN IC50 (nM)
a
Therefore 6 was chosen as starting point for further SAR and
was acylated giving compounds 13–19 (Table 2). Based on our pre-
vious experience the introduction of amines or small polar hetero-
cycles was beneficial to modulate physico-chemical properties and
to improve activity in the cell based assay. Following this strategy
some interesting results were obtained.
4
5
6
7
8
9
H
22
130
36
70
18
9
NHMe
NHEt
NMe2
NHCOPh
Me
Upon introduction of N,N-dimethylglycine a nearly fivefold loss
in intrinsic potency was observed (compound 13). However, the
intrinsic activity of 6 could be maintained by substitution with
morpholin-4-yl acetamide (14). Unfortunately, no improvement
in the cell-based assay was achieved.
O
N
10
170
N
O
11
185
24
N
All compounds 15–19 containing
a heterocycle fragment
12
CH2NHCOPh
Strand transfer inhibition assay, see Ref. 9.
showed activity in the strand transfer assay with IC50 between
20 and 50 nM and improved activity in the spread assay. The intro-
a
Table 2
Amide substitution
O
N
OH
F
H
N
N
N
O
R3
R2
Compound*
R2
R3
H
QUICKIN IC50 (nM)
Spread CIC95
(nM)
50% NHS
a
b,c
10% FBS
1250
6
Et
Et
36
>1000
O
13
185
>1000
>1000
>1000
N
N
O
O
O
14
15
16
Et
Et
Et
23
50
21
>1000
>1000
1000
>1000
250
N
O
O
O
O
N
O
N
17
18
Et
50
500
>1000
N
N
S
Et
Et
21
18
250
500
>1000
>1000
19
O
N
a
Strand transfer inhibition assay, see Ref. 9.
HIV-1 infection spread inhibition in cell culture, see Ref. 9.
All compounds did not show any toxicity up to 50 M.
b
c
l

1932
M. Donghi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1930–1934
duction of pyridine-2-carboxamide 16 and thiazolecarboxamide 18
resulted in a fivefold improvement in cell-based activity. Interest-
ingly, the position of the pyridine nitrogen does seem to play a role
in activity, since the para-derivative 15 has a comparatively low
activity in the spread assay. This might have to do with the change
in basicity of the pyridine nitrogen.
The introduction of an oxadiazole 17 as precedented by ralte-
gravir or of an isoxazole 19 led to a twofold gain in cell-based
activity under low-serum conditions.
In other series of HIV integrase inhibitors the presence of an
oxalamide had been found to have a positive effect on activity
and pharmacological profile.6a,8 Hence, we decided to study this
substitution as well in our new template. All compounds prepared
showed comparatively high activity in the enzymatic assay and im-
proved activity in the cell based spread assay in the presence of 10%
fetal bovine serum and 50% human serum with respect to 6. Substi-
tutions were carried out on the two extremities of the oxalamide
fragment (Table 3). First N,N-dimethyl oxalamides differing on the
amino group attached to the scaffold (R2 = Me, Et, nPr, iPr, 20–23)
were prepared and the Et derivative 21 was identified as the most
potent compound in the cell-based spread assay at high serum
condition, (CIC95: 10% FBS = 62 nM; 50% NHS = 250 nM).
The synthesis of the compounds described in Tables 1–3 was
based on our recent report on the new pyridopyrimidone scaf-
fold.10 The methyl esters 28–29 were transformed into the corre-
sponding amides 4 and 9 by standard aminolysis using para-
fluorobenzyl amine (Scheme 1).
Compound 29 was further elaborated by radical bromination
of the methyl group in the benzylic position of the scaffold, fur-
O
N
O
N
4F-BnNH2
MeOH, reflux
OBz
OH
N
N
O
CO2Me
R
R
HN
4 (R = H)
28 (R = H)
9 (R = Me)
29 (R = Me)
F
N
Scheme 1. Benzylamide formation.
NBS
AIBN
O
N
O
R1
R2
For further evaluation, R2 = Et was kept fixed and R4 was mod-
ified. Compounds 24–27 maintained a good activity in the enzy-
matic assay, which for compounds 25–26 translated without a
significant shift into the low-serum spread assay. However, this
was paired with a pronounced shift (>4-fold) under high serum
conditions.
The most potent compound identified was the N,N-diethyl-
amide 24 which exhibited nanomolar potency in the spread assay
at low serum conditions and a twofold shift under high serum con-
ditions (CIC95: 10% FBS = 62 nM; 50% NHS = 125 nM).
OH
OBz
N
H
CCl4, 80 ºC
N
29
O
MeOH,
then
pFBnNH2,
reflux
N
CO2Me
50%
R1
NH
N
Br
30
R2
1) NaN3
2) P(nBu)3
THF/H2O
F
10 (R1R2NH = morpholine)
3) pFBnNH2,
11 (R1R2NH = N-acetyl piperazine)
reflux
O
OH
N
In order to evaluate the PK properties of the series, 21 was fur-
ther investigated as a representative compound. It was dosed at
3 mg/kg both IV and PO and the rat PK profile was obtained. The
O
N
F
HN
NHBz
compound showed 40% oral bioavailability (F%), plasma exposure
*
of 2.9 lM h (AUC), plasma half life of 4.5 h (t ) and after IV dosing
½
12
a plasma clearance of 24 ml/min/kg.
Scheme 2. Synthesis of aminomethyl pyrido pyrimidines derivatives.
Table 3
Oxalamide substitution
O
N
OH
F
N
H
N
O
N
O
O
R2
R4
R2
R4
QUICKIN IC50 (nM)
Spread CIC95
(nM)
a
b,c
Compound
10% FBS
50%NHS
20
21
22
23
24
Me
Et
nPr
iPr
Et
NMe2
NMe2
NMe2
NMe2
NEt2
26
36
18
21
19
125
62
31
62
62
500
250
500
>1000
125
25
26
Et
Et
Et
25
21
21
31
31
250
1000
500
N
N
N
O
27
125
N
a
Strand transfer inhibition assay, see Ref. 9.
HIV-1 infection spread inhibition in cell culture, see Ref. 9.
All compounds did not show any toxicity up to 50 M.
b
c
l

M. Donghi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1930–1934
1933
nishing the bromo derivative 30 in 50% yield. The benzylic bro-
mide 30 was displaced with a variety of secondary amines at
room temperature. The corresponding amides 10–11 were ob-
tained by one pot aminolysis with para-fluorobenzyl amine
(30–40%) (Scheme 2).
characterized by a pyrido[1,2-a]pyrimidin-4-one scaffold thus
eliminating the presence of any stereocenters in the core of the
inhibitor. Various substituents were tolerated at position 9 of the
bicyclic scaffold. The presence of an oxalamide was very beneficial
for both intrinsic and cellular activity. A number of very potent
compounds with nanomolar activity both in the enzymatic and
in the cellular spread assay under high serum conditions were
identified. The PK profile of the representative compound 21
showed good parameters.
Compound 30 was also reacted under Staudinger conditions
with sodium azide to yield, after reduction of the azide, the amino
methyl derivative. During this reaction, migration of the benzoate
from the 50-OH to the newly formed amino functionality was ob-
served (14%, compound 12).
The methyl ester 3110 was transformed with para-fluorobenzyl-
amine into the corresponding amide 32a (Scheme 3).
Acknowledgements
Treatment of 32a with 37% hydrogen bromide in acetic acid led
to the cleavage of the Cbz group liberating the amine 32b as its
hydrobromide salt in quantitative yield. This material was sub-
jected to reductive amination under standard conditions leading
to the alkylated amines 5–7 and 33a–b (30–60%). These amines
were then acylated with a series of acid chlorides leading to com-
The authors thank Silvia Pesci for NMR studies, William B.
Schleif and Peter J. Felock for biological testing and Fabrizio Fiore
and Ralph Laufer for PK studies.
References and notes
1. Source:
WHO
Factsheet
HIV.
http://www.who.int/hiv/data/
pounds 8 and 15–19 (46–56%). For the synthesis of the
a-amino
2008_global_summary_AIDS_ep.png, accessed September 15th, 2008.
2. (a) Mushawar, I. K. Perspect. Med. Virol. 2007, 13, 75; (b) Murphy, E.-M.;
Jimenez, H. R.; Smith, S. M. Adv. Pharmacol. 2008, 56, 27.
acid derivatives 13–14, the acylation was carried out with chloro-
acetyl chloride, followed by substitution of the resulting alkyl chlo-
ride with the corresponding amine (21–25%).
Alternatively, acylation of the monoamines (5, 7, 34) was car-
ried out by heating them with oxalic acid methylester chloride
(Scheme 4). The resulting intermediary oxalic acid methylester
amides were transformed in one pot into unsymmetric bis-amides
by treatment with different amines, yielding compounds 20–27
(29–56%).11
3. (a) Nair, V. Rev. Med. Virol. 2002, 12, 179; (b) Young, S. D. Ann. Rep. Med. Chem.
2003, 38, 173; (c) De Clercq, E. Exp. Opin. Emerging Drugs 2005, 10, 241.
4. (a) Zhuang, L.; Wai, J. S.; Embrey, M. W.; Fisher, T. S.; Egbertson, M. S.; Payne, L.
P., ; Guare, J. P., Jr.; Vacca, J. P.; Hazuda, D. J.; Felock, P. J.; Wolfe, A. L.; Stillmock,
K. A.; Witmer, M. V.; Moyer, G.; Schleif, W. A.; Gabryelski, L. J.; Leonard, Y. M.;
Lynch, J. J., Jr.; Michelson, S. R.; Young, S. D. J. Med. Chem. 2003, 46, 453; (b)
Hazuda, D. J.; Anthony, N. J.; Gomez, R. P.; Jolly, S. M.; Wai, J. S.; Zhuang, L.;
Fisher, T. E.; Embrey, M. W.; Guare, J. P., Jr.; Egbertson, M. S.; Vacca, J. P.; Huff, J.
R.; Felock, P. J.; Witmer, M. V.; Stillmock, K. A.; Danovich, R.; Grobler, J.; Miller,
M. D.; Espeseth, A. S.; Jin, L.; Chen, I.-W.; Lin, J. H.; Kassahun, K.; Ellis, J. D.;
Wong, B. K.; Xu, W.; Pearson, P. G.; Schleif, W. A.; Cortese, R.; Emini, E.; Summa,
V.; Holloway, M. K.; Young, S. D. Proc. Natl. Acad. Sci. 2004, 101, 11233; (c)
Petrocchi, A.; Koch, U.; Matassa, V. G.; Pacini, B.; Stillmock, K. A.; Summa, V.
Bioorg. Med. Chem. Lett. 2007, 17, 350; (d) Egbertson, M. S. Curr. Top. Med. Chem.
2007, 7, 1251.
In conclusion, we have designed and studied a new viable alter-
native bicyclic template of HIV-1-integrase inhibitors, which is
O
N
O
N
5. Pommier, Y.; Johnson, A. A.; Marchand, C. Nat. Rev. Drug Discovery 2005, 4,
236.
4F-BnNH2
OH
OH
N
N
MeOH, reflux
6. (a) Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi, M.; Ferrara, M.;
Fiore, F.; Gardelli, C.; Gonzalez Paz, O.; Hazuda, D. J.; Jones, P.; Kinzel, O.; Laufer,
R.; Monteagudo, E.; Muraglia, E.; Nizi, E.; Orvieto, F.; Pace, P.; Pescatore, G.;
Scarpelli, R.; Stillmock, K.; Witmer, M. V.; Rowley, M. J. Med. Chem. 2008, 51,
5843; (b) Pace, P.; Rowley, M. Curr. Opin. Drug Disc. Dev. 2008, 11, 471; (c)
Deeks, S. G.; Kar, S.; Gubernick, S. I.; Kirkpatrick, P. Nature Rev. Drug. Disc. 2008,
7, 117; (d) Sayana, S.; Khanlou, H. Expert Rev. Anti-Inf. Ther. 2008, 6, 419.
7. Gardelli, C.; Nizi, E.; Muraglia, E.; Crescenzi, B.; Ferrara, M.; Orvieto, F.; Pace, P.;
Pescatore, G.; Poma, M.; Rico Ferreira, M. d. R.; Scarpelli, R.; Homnick, C. F.;
Ikemoto, N.; Alfieri, A.; Verdirame, M.; Bonelli, F.; Gonzales Paz, O.; Taliani, M.;
Monteagudo, E.; Pesci, S.; Laufer, R.; Felock, P.; Stilmock, K. A.; Hazuda, D.;
Rowley, M.; Summa, V. J. Med. Chem. 2007, 50, 4953.
8. (a) Guare, J. P.; Wai, J. S.; Gomez, R. P.; Anthony, N. J.; Jolly, S. M.; Cortes, A. R.;
Vacca, J. P.; Felock, P. J.; Stillmock, K. A.; Schleif, W. A.; Moyer, G.; Gabryelski, L.
J.; Chen, I.; Hazuda, D. J.; Young, S. D. Biorg. Med. Chem. Lett. 2006, 16, 2900; (b)
Hazuda, D. J.; Young, S. D.; Guare, J. P.; Anthony, N. J.; Gomez, R. P.; Wai, J. S.;
Vacca, J. P.; Handt, L.; Motzel, S. L.; Klein, H. J.; Dornadula, G.; Danovich, R. M.;
Witmer, M. V.; Wilson, K. A. A.; Tussey, L.; Schleif, W. A.; Gabryelski, L. S.; Jin, L.;
Miller, M. D.; Casimiro, D. R.; Emini, E. A.; Shiver, J. W. Science 2004, 305, 528;
(c) Muraglia, E.; Kinzel, O.; Gardelli, C.; Crescenzi, B.; Donghi, M.; Ferrara, M.;
Nizi, E.; Orvieto, F.; Pescatore, G.; Laufer, R.; Gonzalez-Paz, O.; Di Marco, A.;
Fiore, F.; Monteagudo, E.; Fonsi, M.; Felock, P. J.; Rowley, M.; Summa, V. J. Med.
Chem. 2008, 51, 861.
O
CO2Me
NHR
HN
NHCbz
31
32a (R=Cbz)
32b (R=H)
HBr
AcOH
F
O
N
O
R3COCl,
RCHO
NaCNBH3
OH
OH
N
N
DCE, 80 ºC
O
O
N
R3
N
HN
N
HN
R2
R3
R2
O
5 (R2 = Me)
8
6 (R2 = Et)
and
F
7 (R2 = R3 = Me)
33a (R2 = nPr)
33b (R2 = iPr)
F
15-19
9. Zhuang, L.; Wai, J. S.; Embrey, M. W.; Fisher, T. S.; Egbertson, M. S.; Payne, L. P.;
Guare, J. P., Jr.; Vacca, J. P.; Hazuda, D. J.; Felock, P. J.; Wolfe, A. L.; Stillmock, K.
A.; Witmer, M. V.; Moyer, G.; Schleif, W. A.; Gabryelski, L. J.; Leonard, Y. M.;
Lynch, J. J., Jr.; Michelson, S. R.; Young, S. D. J. Med. Chem. 2003, 46, 453.
10. Kinzel, O. D.; Donghi, M.; Maguire, C. K.; Muraglia, E.; Pesci, S.; Rowley, M.;
Summa, V. Tetrahedron Lett. 2008, 49, 6556.
Scheme 3. Synthesis of amino pyrido pyrimidines derivatives.
O
1)
O
N
O
N
Cl
11. Synthetic and brief spectroscopic data on compound 24: Step 1: Compound 3110
(1.1 mmol) was dissolved in MeOH (0.08 M) and p-fluorobenzylamine
(2.16 mmol, 2 equiv) was added. The resulting suspension was stirred for
16 h at 80 °C. The solvent was evaporated and the residue washed with HCl
2 M in Et2O.1HNMR (400 MHz, DMSO-d6) d 12.45 (s, 1H), 10.44 (s br, 1H), 10.02
(s, 1H), 8.45 (d, J = 7.1 Hz, 1H), 8.24 (d, J = 7.5 Hz, 1H), 7.48–7.38 (m, 6H), 7.18
(m, 3H), 5.29 (s, 2H), 4.61 (s, br, 2H); MS (ES+) m/z 463 (M+H)+.Step 2: benzyl
MeO
OH
OH
N
N
O
O
O
O
DCE, 50-80 ºC
HN
HN
R5
N
HN
2) R5 R5
R2
N
R2
N
R5
O
H
5 (R2 = Me)
7 (R2 = Et)
20-27
(2-{[(4-fluorobenzyl)amino]
carbonyl}-3-hydroxy-4-oxo-4H-pyrido[1,2-
a]pyrimidin-9-yl)carbamate (1.1 mmol) was dissolved in acetic acid (0.07 M)
and HBr (30% in acetic acid) was added. The resulting solution was stirred at rt
for 2 h. The solvent was evaporated, the residue dissolved several times in
toluene and the solvent evaporated. The resulting solid was dissolved in 1,2-
dichloroethane-MeOH (1:1, 0.02 M) acetaldehyde (1.1 mmol, 1 equiv) and
34a (R2 = nPr)
34b (R2 = iPr)
F
F
Scheme 4. Oxalamide synthesis.

1934
M. Donghi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1930–1934
sodium cyanoborohydride (1.1 mmol, 1 equiv) were added and the mixture
heated to 50 °C and stirred for 1 h. The solvent was evaporated and the residue
was stirred at rt. After 30 min. the reaction mixture was quenched by the
addition of water. The solvent was evaporated and the residue washed with
water, (95% yield). 1HNMR (400 MHz, DMSO-d6) 12.19 (s, 1H), 10.05 (s br, 1H),
8.03 (d, J = 6.9 Hz, 1H), 7.41(m, 2H), 7.20 (m, 2H), 7.04 (m, 1H), 6.5 (d, J = 7.4 Hz,
1H), 4.62 (d, J = 6.0 Hz, 2H), 3.3 (m, 2H), 1.24 (t, J = 6.8 Hz, 3H); MS (ES+) m/z
356 (M+H)+.Step 3: Compound N-(4fluorobenzyl)-3-hydroxy 9(ethylamino)-4-
oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide (0.17 mmol) was dissolved in
1,2-dichloroethane (0.02 M) and methyl chloro(oxo)acetate (0.34 mmol,
2 equiv) was added. The resulting solution was heated to 80 °C and stirred
for 1 h. The solvent was evaporated; the residue dissolved in MeOH (0.02 M)
and diethylamine (0.17 mmol, 1 equiv) was added. The resulting solution was
dissolved in acetonitrile and purified by RP-HPLC (stationary phase: Symmetry
C18, 5
lm, 19 ꢀ 300 mm. Mobile phase: acetonitrile/H2O buffered with 0.1%
TFA). The fractions were combined and lyophilised to afford the title
compounds as yellow powder (23% yield). The 1H NMR spectrum shows the
presence of two conformers in a ratio of 3:2.Compound 24: 1H NMR (400 MHz,
CD3CN) d 12.48 (s, br, 0.6H), 12.08 (s, br, 0.4H), 9.63 (s, br, 0.4H), 8.74 (d,
J = 7.41 Hz, 0.4H), 8.59 (s, br, 0.6H), 7.64 (d, J = 6.9 Hz, 0.4H), 7.59 (d, J = 6.9 Hz,
0.6H), 7.48 (t, br, 0.8H), 7.42 (t, br, 1.2H), 7.13–7.06 (m, 3H), 4.68–4.56 (m, 2H),
4.43 (m, 0.6H), 3.5–3.42 (m, 4H), 3.23 (m, 0.4H), 2.91–2.83 (m, 1H), 1.20 (t,
J = 6.9 Hz, 1.5H), 1.11 (t, J = 6.5 Hz, 1.5H), 0.9 (t, J = 6.8 Hz, 1.5H), 0.58 (t,
J = 6.9 Hz, 1.5H). MS (ES+) m/z 484 (M+H)+.