Oral Delivery of Propofol with Methoxymethylphosphonic Acid as the Delivery Vehicle

Article abstract of DOI:10.1021/acs.jmedchem.7b01133

Phosphonamidate 3a of methoxymethylphosphonic acid (MMPA) with propofol (1) and l-alanine ethyl ester was found to be an efficient scaffold for the oral delivery of compound 1. The synthesis and evaluation of MMPA based phosphonamidates of compound 1, HSK3486 (2), and other phenolic drugs revealed the general application of MMPA as the effective delivery vehicle for phenolic drugs. On the basis of plasma concentrations of compound 1 and SN38 (14), the oral bioavailability of compound 3a and 15 in beagle dogs was found to be 97.6% and 34.1%, respectively.

Full text of DOI:10.1021/acs.jmedchem.7b01133

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Article
Oral Delivery of Propofol with
Methoxymethylphosphonic Acid as the Delivery Vehicle
Yonggang Wei, Guanpeng Qiu, Bailin Lei, Linlin Qin, Hongzhu Chu, Yonghua Lu, Guozhi Zhu, Qiu Gao,
Qingping Huang, Guofei Qian, Pengfei Liao, Xinfeng Luo, Xiaowei Zhang, Chen Zhang, Yao Li, Suxin Zheng,
Yan Yu, Pingming Tang, Jia Ni, Pangke Yan, Yi Zhou, Pan Li, Xia Huang, Aisheng Gong, and Jianyu Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01133 • Publication Date (Web): 02 Oct 2017
Downloaded from http://pubs.acs.org on October 4, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Huang, Xia; Haisco Pharmaceuticals Group Co. Ltd, Haisco Pharmaceuticals
Group
Gong, Aisheng; Haisco Pharmaceuticals Group Co. Ltd, Haisco
Pharmaceuticals Group
Liu, Jianyu; Haisco Pharmaceuticals Group Co. Ltd.
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Oral Delivery of Propofol with Methoxymethylphosphonic
Acid as the Delivery Vehicle
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Yonggang Wei*, Guanpeng Qiu, Bailin Lei, Linlin Qin, Hongzhu Chu, Yonghua Lu,
Guozhi Zhu, Qiu Gao, Qingping Huang, Guofei Qian, Pengfei Liao, Xinfeng Luo,
Xiaowei Zhang, Chen Zhang, Yao Li, Suxin Zheng, Yan Yu, Pingming Tang, Jia Ni,
Pangke Yan, Yi Zhou, Pan Li, Xia Huang, Aisheng Gong, Jianyu Liu
Haisco Pharmaceuticals Group Co. Ltd., 136 Baili Road, Wenjiang District, Chengdu,
611130, China
KEYWORDS: Phenolic drugs, Propofol, Prodrug strategies, Phosphonamidate Prodrug
ABSTRACT Phosphonamidate 3a of methoxymethylphosphonic acid (MMPA) with
propofol (1) and Lꢀalanine ethyl ester was found to be an efficient scaffold for the oral
delivery of compound 1. The synthesis and evaluation of MMPA based
phosphonamidates of compound 1, HSK3486 (2) and other phenolic drugs revealed the
general application of MMPA as the effective delivery vehicle for phenolic drugs. Based
on plasma concentrations of compound 1 and SN38 (14), the oral bioavailability of
compound 3a and 15 in beagle dogs was found to be 97.6% and 34.1% respectively.
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INTRODUCTION
2
1
We have recently reported that HSK3486 (2, Figure 1), a close analog of propofol (1)
now in advanced phase II clinical trial in China, has shown equivalent potency of 1 at 1/5
of the dosage in similar lipid emulsion formulation. Given that compounds 1 and 2 may
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hold therapeutic potentials in insomnia, migraine, anxiety, and analgesia etc, we have
decided to explore the oral delivery of 1 and 2 through prodrug strategy, and evaluate the
potential clinical usages of these oral prodrugs. Herein we report the discovery of an
efficient oral delivery vehicle for phenolic drugs such as propofol (1) and HSK3486 (2).
Figure 1. Chemical structures of 1 and 2.
Previous attempts to make 1 orally bioavailable through formulation and prodrug
7
ꢀ11
modifications have met with limited success.
Our efforts turned to the
12ꢀ16
aryloxyphosphonamidate ProTide technology first reported by the McGuigan group,
which was used subsequently by Gilead Sciences for the marketed drug tenofovir
1
7
18
alafenamide. As illustrated with the prodrug of tenofovir (A, Figure 2), oral
absorption is facilitated by the neutral phosphonamidate A of tenofovir with an amino
acid ester and phenol. It was postulated that the release of the phosphonic acid target drug
happened first through hydrolysis of the amino acid ester, affording the release of the
alcohol fragment A1. This was followed by intramolecular lactam formation affording
the release of the phenol fragment A3. This transient cyclic lactam A4 was then
hydrolyzed through a two step sequence, first the rapid hydrolysis of the PꢀO bond,
1
9
followed by a phosphoramidase mediated cleavage of the PꢀN bond in A5, affording the
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release of the target drug 9ꢀRꢀ(2ꢀphosphonomethoxypropyl)adenine and the amino acid
A6.
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Figure 2. MMPA based oral delivery of 1.
We reasoned that if this prodrug system was orally bioavailable as a whole, each of its
fragments would also be delivered orally just as efficiently, i.e. the alcohol, amino acid
and phenol fragments. We now demonstrate for the first time that simple replacement of
the phenol (A3) with 1 (Figure 2), together with methoxymethylphosphonic acid (B5) as
the delivery vehicle (Figure 2), afforded the oral delivery of 1 with the release of MMPA
(B5), alcohol B1 and amino acid B4. Unlike the tenofovir prodrug, oral bioavailability of
MMPA prodrug is not dependent upon collapse of transient intermediate B3 to liberate
the target drug. With 1 as the model phenolic drug, we herein report preliminary results
of the proof of concept of these MMPA prodrugs with the syntheses and preliminary
pharmacological evaluation of a series of phosphonamidate prodrugs.
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RESULTS AND DISCUSSION
Chemistry. Given the pivotal role that amino acid ester hydrolysis plays in the MMPA
prodrugs as previously discussed, a series of phosphonamidates of MMPA for 1
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(
compounds 3 to 9) and 2 (compound 13) were designed and prepared (Scheme 1). The
synthesis started with readily available diethyl (hydroxymethyl)phosphonate 20 that was
first methylated using iodomethane and NaH in THF to give diethyl
(methoxymethyl)phosphonate 21. Reaction of diethyl (methoxymethyl)phosphonate 21
with 2 equivalents of bromotrimethylsilane in CH CN selectively removed the two ethyl
3
groups to give the crude bistrimethylsilylphosphonate 22 that readily underwent
chlorination using oxalyl chloride in CH Cl to provide the key intermediate
2
2
2
0
methoxymethylphosphonic acid dichloride 23, which could be purified by distillation.
Reaction of the dichloride with compound 1 and various amino acid esters in
Et N/CH Cl afforded prodrugs 3 to 9 in 30% to 51% yield. The phosphonamidate
3
2
2
prodrug 13 of 2 with Lꢀalanine ethyl ester was prepared in a similar manner.
Scheme 1. Synthesis of Phosphonamidates 3–9 and 13
Reagents and conditions: (a) CH I, NaH, THF, 0 ℃ to rt, 2 hrs; (b) TMSBr, CH CN,
3
3
5
0 ℃, 2 hrs; (c) oxalyl chloride, CH Cl , 0 ℃ to rt, 2 hrs; (d) 1, Et N, amino acid ester,
2 2 3
CH Cl , ꢀ10 ℃to rt; (e) 2, Et N, Lꢀalanine ethyl ester, CH Cl ꢀ10 ℃to rt.
2
2
3
2
2,
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The morpholinomethylphosphonic acid based prodrug of compound 1 containing Lꢀ
alanine ethyl ester was prepared (Scheme 2) first by converting diethyl
hydroxymethylphosphonate 20 into its mesylate 27. Dichloride 28 was prepared using the
previous two step sequence (TMSBr–oxalyl chloride), which was reacted with 1 and Lꢀ
alanine ethyl ester to give the mesylate intermediate 29. Displacement of the mesylate 29
with morpholine afforded the morpholinomethylphosphonamidate prodrug 11. Similarly
the benzyloxymethylphosphonamidate intermediate 26 containing 1 and Lꢀalanine ethyl
ester was obtained in a similar fashion through the benzyl ether 24 followed by the two
step chlorination sequence (TMSBr–oxalyl chloride) and phosphonamidate formation to
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Debenzylation
of
26
under
mild
conditions
afforded
the
hydroxymethylphosphonamidate prodrug 10. Starting with diethyl propylphosphonate 30,
the propylphosphonic acid dichloride 31 was prepared in the same two step sequence
(TMSBr–oxalyl chloride) and reacted with 1 and Lꢀalanine ethyl ester to afford the
propylphosphonamidate 12.
Scheme 2. Synthesis of Phosphonamidates 10–12
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Reagents and conditions: (a) BnBr, NaH, THF, 0 ℃ 12 hrs; (b) TMSBr, CH CN, 50 ℃,
3
2–3 hrs; (c) oxalyl chloride, CH Cl , 0 ℃to rt, 1–12 hrs; (d) 1, Et N, Lꢀalanine ethyl ester,
2
2
3
CH Cl ꢀ10 ℃to rt; (e) H , 10% Pd/C, CH Cl , rt, 8 hrs; (f) MsCl, Et N, CH Cl , 0 ℃to rt,
2
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hrs; (g) morpholine, Et N, THF, 80 ℃, 8 hrs.
3
All of the phosphonamidate prodrugs above were isolated as their diastereoisomeric
mixtures. Prodrugs 3 and 13 were further purified by SFC (Supercritical Fluid
Chromatography) to produce each pair of diastereoisomers 3a–3b and 13a–13b
respectively. Since all these compounds were either waxy solids or liquids, the absolute
configuration
of
3a
was
successfully
determined
through
the
hydroxymethylphosphonamidate analog 10a (Scheme 3). Crystallization of the
diastereoisomeric mixture of 10 from a mixed solvent (ethyl acetate /nꢀhexane = 1/10)
afforded 10a, whose absolute stereochemistry was determined by Xꢀray crystallography
to be (S, S ). Methylation of compound 10a under neutral conditions using iodomethane
P
and silver oxide in dichloromethane at room temperature produced compound 32 that has
been found to be identical in all aspects to compound 3a. Similarly, the absolute
stereochemistry of compound 13b was determined through silver oxide mediated
methylation of the corresponding hydroxymethylphosphonamidate compound 34b (S, SP)
to 35b (Scheme 3). For a given amino acid ester, the chemical shift of the phosphorus
atom of the Sꢀstereoisomer has so far been found to be always more upfield than the Rꢀ
31
stereoisomer in the P NMR (see supporting information), which is in accord with the
3
1
17
P NMR of tenofovir alafenamide.
Scheme 3. Determination of the Absolute Configuration of Compound 3a and 13b
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Reagents and conditions: (a) recrystallization (ethyl acetate/nꢀhexane = 1/10); (b) MeI,
Ag O, CH Cl , rt, overnight; (c) 2, Et N, Lꢀalanine ethyl ester, CH Cl , ꢀ10 ℃to rt, 2 hrs;
2
2
2
3
2
2
(
d) H , 10% Pd/C, MeOH, rt, 8 hrs.
2
Biological Evaluation Initially, the in vivo anesthetic effects of phosphonamidate
2
1
prodrugs were evaluated by loss of righting reflex (LORR) experiments in ICR mice
through oral administration. The onꢀset and duration of anesthesia of phosphonamidate
prodrugs allowed preliminary accessment of the impacts of amino acids and esters on the
oral delivery of 1 and 2. Delightfully, all compounds were found to produce moderate to
noticeable anesthetic effects, an indication of at least some oral bioavailability for all
compounds. Compound 3 produced the most pronounced anesthetic action with an onset
of anesthesia (4.50 ± 1.58 min) and duration time (60.31 ± 57.76 min) (Table 1). The
bulkier the ester moiety of the prodrug was, the slower was the onset of anesthesia
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(
compound 3 vs compound 4, 5 and 6), which might be the result of the relative rate of
hydrolysis of different ester groups (ethyl > isobutyl > benzyl > isopropyl). It is
interesting to note that the ester moiety could also affect the duration of anesthetic action,
as compound 6 with a benzyl ester exhibited a much longer duration than compound 3, 4
and 5.
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Furthermore, phosphonamidate prodrugs containing different amino acids were
investigated. With the incorporation of an unnatural amino acid, compound 7 showed
similar potency when compared with compound 3. However, it showed a slower onset of
anesthetic action than its Lꢀalanine analog. This may be due to the unnatural amino acid
used in 7 not being the natural substrate for the esterase responsible in the first step of
hydrolysis.
Compound 8 exhibited a 2ꢀfold longer anesthetic duration with a comparable LORR
effect to compound 3. This much longer duration of anesthetic action might be due to
steric hindrance of the bulkier dimethyl amino acid in the hydrolysis step. It is somewhat
surprising that compound 9 with a less bulky glycine ester produced a significantly
slower onset of anesthesia than 3, which has relatively more steric bulk at the alpha
2
2
position of the amino acid. This observation is in accord with a report that a substituted
amino acid such as alanine is the more preferable substrate than glycine for the esterase
responsible in the first step of hydrolysis.
The effect of substituents on the methoxymethylphosphonic acid (MMPA) moiety was
also studied. Compounds 10, 11 and 12 had slower onset of anesthetic action and shorter
anesthetic duration times compared with compound 3. This might indicate that the
electronic and relative steric effect of those substituents could also be rateꢀdetermining in
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the hydrolysisꢀcyclization sequence of phosphonamidate prodrugs (Figure 2). More inꢀ
depth investigation is required to delineate the scope and utilities for such substitutions.
It is noted that prodrug 13 showed a similar anesthetic action at a lower dose of 100
mg/kg, which could be attributed to the higher potency of 2 compared with compound 1.
Also, the higher ratio of mortality caused by 13 might be due to the lower LD of 2
0
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compared with 1 as reported in our previous work, and a more detailed dose response
study will have to be conducted.
With the results discussed above, we demonstrated that the rate of activation and the
duration time of these phosphonamidate prodrugs were tunable through modifications of
the ester moiety and the substituents on MMPA. These properties are critical for selecting
the right substitutions for the oral delivery of 1 and 2 based on the therapeutic demands.
These results have also built the foundation of expanding the scope of this MMPA based
prodrug technology onto other phenolic drugs with diverse indications and clinical
demands.
Table 1. Anesthetic Effect of Prodrugs in ICR Mice
Dose
Onset
(min)
Duration
(min)
LORR
(%)
mortality
(%)
Compound
Structure
(
mg/kg)
a
3
4
5
6
200
4.50 ± 1.58
12.70 ± 4.90
8.36 ± 3.31
9.34 ± 5.48
60.31 ± 57.76
59.38 ± 20.57
24.47 ± 15.56
94.61 ± 55.89
100
40
10
0
a
200
a
200
100
50
10
10
a
200
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a
7
8
200
200
200
200
9.48 ± 2.46
6.74 ± 1.11
14.37 ± 7.40
6.91 ± 0.42
44.80 ± 35.46
115.9 ± 63.56
32.48 ± 23.19
19.19 ± 3.59
100
100
70
10
20
0
a
a
a
a
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9
10
40
10
1
1
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2
200
200
7.65 ± 1.95
16.18 ± 7.49
36.11 ± 25.10
60
60
10
0
a
11.21 ± 3.43
b
1
3
100
4.49 ± 2.38
74.44 ± 6.57
100
50
a
b
2
00 mg ꢀ eq/kg parent drug, 100 mg ꢀ eq/kg parent drug
To further investigate the effect of the chirality on the phosphorus, the anesthetic
effects of each pair of enantiomeric pure compounds 3a–3b, 13a–13b were studied with
results shown in Table 2. Compound 3a (S, S ) and 3b (S, R ) produced a similar
P
P
anesthetic duration time, but a significant difference in the onset of anesthetic action and
LORR (3a, 4.38 ± 0.55, 100% vs 3b, 9.68 ± 1.19, 33%). These results indicated that the
chirality of the phosphorus had pronounced effect on the pharmacological behavior.
Either the esterase mediated hydrolysis or formation of the transient lactam intermediate
B3 or both could have been affected by the chirality on the phosphorus. Meanwhile,
compound 13a (S, S ) and 13b (S, R ) showed a slight difference in the onset of
P
P
anesthetic action and duration. The higher potency of 2 might have masked the difference.
The pharmacological difference could be more obvious at lower dose that will be one of
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4
4
5
5
5
5
5
5
5
5
5
5
6
the subjects for further studies.
Table 2. Anesthetic Effect of 3a, 3b, 13a and 13b in ICR Mice
Dose
Onset
(min)
Duration
(min)
LORR mortality
Compound
Structure
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
mg/kg)
(%)
(%)
a
3
200
4.50 ± 1.58
4.38 ± 0.55
9.68 ± 1.19
4.49 ± 2.38
60.31 ± 57.76
40.10 ± 32.51
47.24 ± 19.69
74.44 ± 6.57
100
10
a
3
a
200
100
33
0
0
3b
a
2
00
b
1
3
100
100
100
100
100
100
50
0
b
1
3a
3b
3.98 ± 0.84
4.35 ± 0.39
107.68 ± 53.44
85.23 ± 23.70
b
1
17
a
b
200 mg ꢀ eq/kg parent drug, 100 mg ꢀ eq/kg parent drug
The pharmacokinetic profiles of prodrugs 3a (S, S ) and 13a (S, S ) in beagle dogs
P
P
were investigated (Table 3). Oral bioavailability was calculated as a percentage of the
active compound quantitated in plasma after oral dosing of the prodrug versus
intravenously administered active compound by measuring plasma concentrations of the
active compound. Based on AUC(0ꢀt), the oral bioavailability (F) of 3a and 13a in beagle
dog was found to be 97.6 % and 29.5 % respectively.
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Table 3. Pharmacokinetic 3a and 13a in Beagle Dogs
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14
15
16
17
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Dose
t1/2
(h)
Cmax
AUC0ꢀt
F
Compound
Structure
(mg/kg)
ng/mL
(ng/mL·h)
(%)
IV
3.0
0.20 ± 0.08
0.64 ± 0.15
2050 ± 815
97 ± 29
616 ± 173
102 ± 34
/
1
PO
PO
6.9
7.3
3a
2
25.0
0.71 ± 0.16
1613 ± 889
2316 ± 1422
97.6
IV
2.0
0.20 ± 0.05
0.44 ± 0.21
1611 ± 123
233 ± 251
417 ± 27
/
PO
10.0
162 ± 192
7.7
PO
25.0
1.05 ± 0.18
452 ± 137
764 ± 327
29.5
13a
Based on the pharmacokinetic profile of 3a, we proposed that compound 3a might be
used for other indications such as insomnia. The hypnotic potency of 3a was evaluated
2
3
based on the prolongation of sleep induced by pentobarbital in ICR mice, at the dosages
where compound 3a alone caused no LORR. Compound 3a produced a dose dependent
prolongation of pentobarbital ꢀ induced sleep time at dosages of 55, 65 and 75 mg/kg
(Figure 3). At dosages of 65.0 and 75.0 mg/kg, compound 3a remarkably increased sleep
duration to 69 ± 20 min and 82 ± 53 min, respectively, compared to the vehicle group of
3
3 ± 20 min.
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0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Effects of 3a on sleep duration in pentobarbitalꢀtreated mice (* p<0.05, **
p<0.001), compared to vehicle group.
The generality of the MMPA platform to other phenolic drugs with more complicated
24
structures was investigated. Phosphonamidate prodrugs of phenolic drugs SN38 (14),
25
26
estradiol (16) and naloxone (18) (Figure 4) were synthesized by using the same
method as described for compound 3–9 (Scheme 1). The pharmacokinetic profiles of
compounds 15, 17 and 19, each as a mixture of its diastereoisomeric pair, were evaluated
27
in beagle dogs via oral administration (Table 4). As an active metabolite of irinotecan ,
14 was not orally active. To our delight, the oral bioavailability of 14 as its MMPA
prodrug 15 was found to be 34.1%. Even 16 was taken orally in clinic, its plasma
concentration was not measurable in our hands when given orally. However, as its
MMPA prodrug 17, the bioavailability of 16 was found to be 18.6%. The oral
bioavailability of 19 as its MMPA prodrug was found to be 15.7%, a moderate increase
from 9.6% for compound 18. These results provide support on the generality of MMPA
prodrug platform on phenolic drugs and warrant additional studies for other linking atoms
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and methods in this platform.
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45
46
47
48
49
50
51
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55
56
57
58
59
60
Figure 4. Chemical structures of 14–19.
Table 4. Pharmacokinetic Parameters of Selected Prodrugs (15, 17 and 19) in Beagle
Dogs
Dose
t1/2
(h)
Cmax
AUC0ꢀt
F
Compound
(
mg/kg)
ng/mL
(ng/mL·h)
(%)
IV
2.0 2.67 ± 0.22 983 ± 244
410 ± 63
NA
/
1
4
PO
PO
IV
16.0
NA
ND
NA
34.1
/
15
25.0 1.04 ± 0.11 1167 ± 27
1117 ± 182
28 ± 12
NA
0.2 0.27 ± 0.08
76 ± 24
ND
1
6
7
PO
PO
IV
4.4
NA
NA
18.6
/
1
8.0 0.84 ± 0.34 116 ± 17
1.0 0.38 ± 0.05 196 ± 18
115 ± 44
122 ± 16
117 ± 10
172 ± 7
1
8
PO
PO
10.0 8.99 ± 3.43
43 ± 4
9.6
15.7
1
9
16.4 1.23 ± 0.45 104 ± 26
ND: Not Detected; NA: Not Available
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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CONCLUSION
This work disclosed above has demonstrated how MMPA could be used as delivery
vehicle for oral administration of phenolic drugs such as 1 and 2. The oral bioavailability
of 1 will allow further studies for other indications. Prodrugs 3a and 13a were chosen for
further preclinical development. MMPA platform has shown promising results to other
phenolic drugs with more sophisticated structures including 14, 16 and 18. MMPA
platform may allow oral delivery of drug targets of interest through different linking
atoms and linkers. Efforts in exploring different linkers and linkages to different
functional groups in the MMPA platform are currently underway as well as the oral
delivery of the B1 alcohol and B4 amino acid (Figure 2) through this technology. The
ongoing mechanistic studies of MMPA platform and all related biological intermediates
and metabolites will be reported in due course.
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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7
8
9
0
1
2
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0
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2
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9
0
1
2
3
4
5
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7
8
9
0
EXPERIMENTAL SECTION
Anesthetic effects Loss of righting reflex (LORR), a validated rodent model of general
anesthesia, was applied to evaluate the anesthetic effects of the prodrugs of 1 and 2 in
ICR mice. Animals were fasted over night before experiment and administered with
prodrugs in 10% of dimethyl sulfoxide, 20% of Solutol HSꢀ15 and 70% of saline by
gavage (n = 10/dose group for diastereoisomeric mixtures, n = 6/dose group for
enantiopure compounds 3a, 3b, 13a and 13b). Then the mice were placed on their backs
continually until they stopped righting themselves. Anesthetic effects were assessed using
onset of LORR and anesthetic duration.
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Animals Male and female ICR mice (20
±
2 g) were purchased from Chengdu Dossy
Experimental Animals Co., Ltd. Animal facilities, animal care, and study programs were
conducted in conformity with inꢀhouse guidelines of Institute of Laboratory Animal
Resources, Commission on Life Science, National Research Council (National Academy
10
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14
15
16
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Press, Washington, DC. 2010). All animals were housed in cages at 21 ± 2
℃ and provided
free access to food and water. Rooms with constant temperature and humidity were in a cycle
of 12 hrs of light and 12 hrs of dark.
Pharmacokinetic profiles of prodrugs in beagle dogs were investigated. Parent drugs
which were formulated in 5% of dimethyl sulfoxide, 5% of Solutol HSꢀ15 (30%, w/v)
and 90% of saline were administered a single i.v. bolus injection to beagle dogs
(
n=3/group), and blood samples were collected into heparinized tubes at pre–dose and at
2
, 5, 10, 20, 30 min, 1, 2, 4, 6, 8, 10, 12, and 24 hrs postdose. After a week washout, the
prodrugs which were prepared in 5% of dimethyl sulfoxide, 5% of Solutol HSꢀ15 (30%,
w/v) and 90% of saline were administered to the same beagle dogs via i.g. and blood
samples were collected into heparinized tubes at preꢀdose and at 5, 10, 20, 30 min, 1, 2, 4,
6
, 8, 10, 12, and 24 hrs postdose after administration. All blood samples were centrifuged
at 5500 rpm for 10 min to obtain plasma at 2–8 ℃. Plasma samples were stored at ꢀ80 ℃
until analysis. Parent drugs concentrations in all plasma samples were determined by
liquid chromatographyꢀtandem mass spectrometry (LCꢀMS/MS). Pharmacokinetic
parameters were analyzed by the WinNonlin V6.3 software (Pharsight, Mountain View,
CA, USA) using nonꢀcompartmental analysis.
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Pentobarbital–Induced Sleep Hypnotic potency of compound 3a was evaluated based
on the prolongation of sleep induced by pentobarbital in ICR mice. Fasted male and
female mice were randomly divided into four groups, including vehicle group and three
dose groups (55, 65, 75 mg/kg, n=10/ group). Compound 3a was formulated in 10% of
dimethyl sulfoxide, 20% of Solutol HSꢀ15 (30%, w/v) and 70% of saline, and was
administrated orally 5 min before the pentobarbital injection. After the intraperitoneal
injection of 40 mg/kg pentobarbital, the mice were observed for measurement of sleep
duration. The sleep duration was defined as period from falling into sleep to recovering
the righting reflex. Data were expressed as mean ± SEM. Statistical analysis was
performed with Sigma Stat software (SPSS Inc., Chicago, USA).
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0
Synthetic Procedures in Chemistry All purchased starting materials were used
1
13
without further purification. H NMR and C NMR spectra were acquired at ambient
temperature on a Bruker Avance HD 400 spectrometer (400 MHz and 101 MHz) in
CDCl and CD OD with residual solvent signal (CHCl and CD HOD) as reference (7.26
3
3
3
2
3
1
and 77.16, 3.31 and 49.00 respectively). P NMR spectra were recorded at ambient
temperature on a Bruker Avance HD 400 spectrometer (162 MHz), with proton
decoupling. Chemical shifts were reported relative to external 85 % H PO . Chemical
3
4
shifts are expressed in parts per million (ppm, δ units), and coupling constants are
reported in hertz (Hz). Hight Resolution Mass Spectra (HRMS) were obtained on LTQ
FT Ultra of Thermo Fisher Scientific from the National Center for Organic Mass
Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academic
of Science. Mass spectra were obtained on FinniganLCQAd instrument (ESI). Most
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masses were reported as those of the protonated parent ions. Preparative column
chromatography was performed using 200–300 mesh silica. Purities of title compounds
were ≥95 % by HPLC.
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14
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Ethyl
(2S)-2-[[(2,6-diisopropylphenoxy)-(methoxymethyl)phosphoryl]amino]
propanoate (3) Under nitrogen atmosphere, to a stirred solution of 23 (4.5 g, 21 mmol)
in 50 mL of dichloromethane was added a solution of 1 (3.7 g, 21 mmol) and
triethylamine (8.7 g, 86 mmol) in dichloromethane (50 mL) at ꢀ10 ℃. The reaction
mixture was allowed to warm to room temperature, stirred for 1 hr, and Lꢀalanine ethyl
ester (5.0 g, 43 mmol) was then added. After 2 hrs, the reaction mixture was quenched
with a saturated solution of sodium dihydrogen phosphate (100 mL), the organic layer
was dried with anhydrous sodium sulfate and concentrated in vacuum. The residue was
purified by flash column chromatography (1:10–1:1 ethyl acetate/hexanes). The title
compound was obtained as yellow oil (2.5 g) in 30.9 % yield. R = 0.50 (1:2 ethyl
f
acetate/hexanes, TLC), major isomer : minor isomer = 1.18 : 1.00. HRMS (ESI): Calcd
+
for C H NO P [M+H] 386.2091; Found: 386.2090.
1
9
33
5
The diastereoisomeric mixture was separated by SFC to afford two optical isomers (see
supporting information for separation method). Major isomer: chiral HPLC retention time
1
=
18.2 min. H NMR (400 MHz, CD OD): δ 7.28–6.99 (m, 3H), 4.14–4.02(m, 2H), 3.98
3
(dq, J = 9.2, 7.2 Hz, 1H), 3.94 (dd, J = 13.2, 8.8 Hz, 1H), 3.87 (dd, J = 13.2, 8.0 Hz, 1H),
.54 (hept, J = 6.8 Hz, 2H), 3.45 (d, J = 1.2 Hz, 3H), 1.30 (d, J = 7.2 Hz, 3H), 1.22–1.18
3
1
3
(
m, 15H). C NMR (101 MHz, CD OD): δ 174.93 (d, J = 5.0 Hz), 146.60 (d, J = 11.4
3
Hz), 141.88 (d, J = 3.2 Hz), 126.66 (d, J = 1.7 Hz), 125.17 (d, J = 1.7 Hz), 69.99 (d, J =
1
57.9 Hz), 62.19, 61.57 (d, J = 15.2 Hz), 50.90, 28.21, 24.03, 23.80, 21.28 (d, J = 4.4
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1
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2
2
2
2
2
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3
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+
Hz), 14.46. P NMR (162 MHz, CD OD): δ 23.69. MS m/z (ESI): 386.2 [M+H] . Minor
3
1
isomer: chiral HPLC retention time = 19.1 min. H NMR (400 MHz, CD OD): δ 7.27–
3
7
.03 (m, 3H), 4.20–4.10 (m, 2H), 4.00 (dq, J = 7.6, 7.6 Hz, 1H), 4.02–3.90 (m, 2H) , 3.57
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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0
1
2
3
4
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0
1
2
3
4
5
6
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9
0
(
hept, J = 6.8 Hz, 2H), 3.48 (d, J = 1.2 Hz, 3H), 1.27–1.22 (m, 6H), 1.21 (d, J = 6.8 Hz,
1
3
12H). C NMR (101 MHz, CD OD): δ 175.30 (d, J = 4.0 Hz), 146.25 (d, J = 11.4 Hz),
3
1
42.03 (d, J = 3.1 Hz), 126.68 (d, J = 1.8 Hz), 125.15 (d, J = 1.8 Hz), 69.85 (d, J = 156.1
Hz), 62.29, 61.66 (d, J = 14.1 Hz), 50.91, 28.11, 24.03, 23.95, 20.75 (d, J = 5.4 Hz),
3
1
+
14.49. P NMR (162 MHz, CD OD): δ 24.96. MS m/z (ESI): 386.2 [M+H] .
3
Isopropyl
(2S)-2[[(2,6-diisopropylphenoxy)(methoxymethyl)phosphoryl]amino]
propanoate (4) Yellow oil (1.0 g), 40.9 % yield. R = 0.60 (1:2 ethyl acetate/hexanes,
f
TLC), major isomer : minor isomer = 1.63 : 1.00. HRMS (ESI): Calcd for C H NO P
2
0
35
5
+
[
M+H] 400.2247. Found: 400.2247. The diastereoisomeric mixture was separated by
1
SFC to afford two optical isomers. Major isomer: H NMR (400 MHz, CD OD): δ 7.15–
3
7
3
.08 (m, 3H), 4.95 (hept, J = 6.4 Hz, 1H), 4.04–3.74 (m, 3H), 3.58 (hept, J = 6.8 Hz, 2H),
13
.44 (d, J = 0.8 Hz, 3H), 1.29 (d, J = 7.2 Hz, 3H), 1.21–1.17 (m, 18H). C NMR (101
MHz, CD OD): δ 174.29 (d, J = 5.3 Hz), 146.50 (d, J = 11.3 Hz), 141.75 (d, J = 3.1 Hz),
3
1
26.60 (d, J = 1.7 Hz), 125.11 (d, J = 1.8 Hz), 69.97 (d, J = 157.8 Hz), 69.78, 61.57 (d, J
3
1
=
15.0 Hz), 50.88, 28.10, 24.14, 23.91, 22.04, 21.96, 21.44 (d, J = 4.1 Hz). P NMR (162
+
1
MHz, CD OD): δ 23.53. MS m/z (ESI): 400.3 [M+H] . Minor isomer: H NMR (400
3
MHz, CD OD): δ 7.33–6.95 (m, 3H), 5.00 (hept, J = 6.4 Hz, 1H), 4.06–3.90 (m, 3H),
3
1
3
3
.60 (hept, J = 6.8 Hz, 2H), 3.49 (d, J = 0.8 Hz, 3H), 1.33–1.12 (m, 21H). C NMR (101
MHz, CD OD): δ 174.77 (d, J = 4.0 Hz), 146.21 (d, J = 11.4 Hz), 141.99 (d, J = 3.1 Hz),
3
1
26.66 (d, J = 1.8 Hz), 125.13 (d, J = 1.8 Hz), 70.00, 69.84 (d, J = 156.1 Hz), 61.66 (d, J
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=
14.1 Hz), 50.96, 28.07, 24.08, 24.00, 22.01 (d, J = 3.0 Hz), 20.83 (d, J = 5.2 Hz). P
+
NMR (162 MHz, CD OD): δ 24.89. MS m/z (ESI): 400.3 [M+H] .
3
Isobutyl
(2S)-2-[[(2,6-diisopropylphenoxy)-(methoxymethyl)phosphoryl]amino]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
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propanoate (5). Yellow oil (5.8 g), 37.6 % yield. R = 0.70 and 0.6 (1:2 ethyl
f
acetate/hexanes, TLC), major isomer : minor isomer = 1.46 : 1.00. HRMS (ESI): Calcd
+
for C H NO P [M+H] 414.2404. Found: 414.2403. The diastereoisomeric mixture was
2
1
37
5
1
separated by chiral preparative HPLC to afford two optical isomers. Major isomer: H
NMR (400 MHz, CD OD): δ 7.15–7.09 (m, 3H), 4.01 (dq, J = 9.0, 7.2 Hz,1H), 3.97–3.84
3
(m, 2H), 3.86 (dd, J = 10.4, 6.8 Hz, 1H), 3.81 (dd, J = 10.4, 6.4 Hz, 1H), 3.56 (hept, J =
6
.8 Hz, 2H), 3.44 (d, J = 1.2 Hz, 3H), 1.98–1.75 (m, 1H), 1.31 (d, J = 7.2 Hz, 3H), 1.20
1
3
(d, J = 6.8 Hz, 12H), 0.90 (d, J = 6.8 Hz, 6H). C NMR (101 MHz, CD OD): δ 174.90
3
(d, J = 5.0 Hz), 146.57 (d, J = 11.3 Hz), 141.82 (d, J = 3.1 Hz), 126.65 (d, J = 1.7 Hz),
1
25.16 (d, J = 1.7 Hz), 72.16, 69.98 (d, J = 157.8 Hz), 61.59 (d, J = 15.3 Hz), 50.86,
3
1
2
8.85, 28.15, 24.09, 23.86, 21.44 (d, J = 4.4 Hz), 19.38 (d, J = 1.8 Hz). P NMR (162
+
1
MHz, CD OD): δ 23.56. MS m/z (ESI): 414.1 [M+H] . Minor isomer: H NMR (400
3
MHz, CD OD): δ 7.41–6.74 (m, 3H), 4.03 (dq, J = 7.2, 7.2 Hz,1H), 4.01–3.91 (m, 2H),
3
3
.93 (dd, J = 10.6, 6.6 Hz, 1H), 3.87 (dd, J = 10.6, 6.4 Hz, 1H), 3.58 (hept, J = 6.8 Hz,
H), 3.47 (d, J = 0.8 Hz, 3H), 2.07–1.80 (m, 1H), 1.26 (d, J = 7.2 Hz, 3H), 1.21 (d, J =
1
1
3
6.8 Hz, 12H), 0.93 (d, J = 6.8 Hz, 6H). C NMR (101 MHz, CD OD): δ 175.29 (d, J =
3
4
.1 Hz), 146.23 (d, J = 11.5 Hz), 142.01 (d, J = 3.1 Hz), 126.67 (d, J = 1.8 Hz), 125.14
(d, J = 1.7 Hz), 72.24, 69.83 (d, J = 156.1 Hz), 61.65 (d, J = 14.3 Hz), 50.89, 28.91,
3
1
2
8.08, 24.05, 23.98, 20.90 (d, J = 5.4 Hz), 19.37 (d, J = 2.4 Hz). P NMR (162 MHz,
+
CD OD): δ 24.88. MS m/z (ESI): 414.1 [M+H] .
3
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Benzyl
(2S)-2-(((2,6-diisopropylphenoxy)(methoxymethyl)phosphoryl)amino)
propanoate (6) Yellow oil (5.0 g), 36.4 % yield. R = 0.60 and 0.50 (1:2 ethyl
f
0
1
2
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4
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8
9
0
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acetate/hexanes, TLC), major isomer : minor isomer = 1.40 : 1.00. HRMS (ESI): Calcd
+
for C H NO P [M+H] 448.2247. Found: 448.2247. The diastereoisomeric mixture was
2
4
35
5
1
separated by chiral preparative HPLC to afford two optical isomers. Major isomer: H
NMR (400 MHz, CD OD): δ 7.45–7.23 (m, 5H), 7.14–7.08 (m, 3H), 5.19–5.08 (m, 2H),
3
4
.07 (dq, J = 9.0, 7.2 Hz, 1H), 3.92 (dd, J = 13.2, 8.8 Hz, 1H), 3.86 (dd, J = 13.2, 7.6 Hz,
H), 3.57 (hept, J = 6.8 Hz, 2H), 3.37 (d, J = 1.2 Hz, 3H), 1.32 (d, J = 7.2 Hz, 3H), 1.20
1
1
3
(d, J = 6.8 Hz, 6H), 1.19 (d, J = 6.8 Hz, 6H). C NMR (101 MHz, CD OD): δ 174.58 (d,
3
J = 4.8 Hz), 146.53 (d, J = 11.3 Hz), 141.78 (d, J = 3.1 Hz), 137.08, 129.49, 129.25,
1
29.20, 126.63 (d, J = 1.7 Hz), 125.14 (d, J = 1.7 Hz), 69.94 (d, J = 157.6 Hz), 67.73,
3
1
6
1.53 (d, J = 15.1 Hz), 50.91, 28.13, 24.07, 23.85, 21.24 (d, J = 4.4 Hz). P NMR (162
+
1
MHz, CD OD): δ 23.56. MS m/z (ESI): 448.3 [M+H] . Minor isomer: H NMR (400
3
MHz, CD OD) δ 7.43–7.24 (m, 5H), 7.13–7.11 (m, 3H), 5.19–5.08 (m, 2H), 4.09 (dq, J =
3
7
.2, 7.2 Hz, 1H), 3.92 (dd, J = 13.2, 8.0 Hz, 1H), 3.88 (dd, J = 13.2, 7.6 Hz, 1H), 3.58
(
hept, J = 6.8 Hz, 2H), 3.40 (d, J = 1.0 Hz, 3H), 1.27 (d, J = 7.2 Hz, 3H), 1.20 (d, J = 6.8
1
3
Hz, 12H). C NMR (101 MHz, CD OD): δ 175.00 (d, J = 3.7 Hz), 146.20 (d, J = 11.4
3
Hz), 141.98 (d, J = 3.1 Hz), 137.11, 129.53, 129.30, 129.27, 126.66 (d, J = 1.8 Hz),
1
25.13 (d, J = 1.8 Hz), 69.79 (d, J = 156.0 Hz), 67.93, 61.60 (d, J = 14.3 Hz), 50.92,
3
1
28.07, 24.03, 23.96, 20.64 (d, J = 5.5 Hz). P NMR (162 MHz, CD OD): δ 24.94. MS
3
+
m/z (ESI): 448.3 [M+H] .
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Ethyl
(2R)-2-[[(2,6-diisopropylphenoxy)-(methoxymethyl)phosphoryl]amino]
propanoate (7) Yellow oil (6.0 g), 51 % yield. R = 0.50 (1:2 ethyl acetate/hexanes,
f
10
11
12
13
14
15
16
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18
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TLC), major isomer : minor isomer = 1.32 : 1.00. HRMS (ESI): Calcd for C H NO P
19 33
5
+
[
M+H] 386.2091. Found: 386.2090. The diastereoisomeric mixture was separated by
1
chiral preparative HPLC to afford two optical isomers. Major isomer: H NMR (400
MHz, CD OD): δ 7.21–7.08 (m, 3H), 4.20–4.10 (m, 2H), 4.00 (dq, J = 7.2, 7.2 Hz, 1H),
3
3
2
.98 (dd, J = 13.2, 8.0 Hz, 1H), 3.94 (dd, J = 13.2, 8.0 Hz, 1H), 3.57 (hept, J = 6.8 Hz,
1
3
H), 3.48 (d, J = 1.2 Hz, 3H), 1.27–1.23 (m, 6H), 1.21 (d, J = 6.8 Hz, 12H). C NMR
(101 MHz, CD OD): δ 175.31 (d, J = 3.8 Hz), 146.25 (d, J = 11.4 Hz), 142.03 (d, J = 3.2
3
Hz), 126.69 (d, J = 1.8 Hz), 125.16 (d, J = 1.8 Hz), 69.85 (d, J = 156.1 Hz), 62.29, 61.66
3
1
(
d, J = 14.1 Hz), 50.90, 28.11, 24.03, 23.95, 20.74 (d, J = 5.5 Hz), 14.49. P NMR (162
+
1
MHz, CD OD): δ 24.96. MS m/z (ESI): 386.2 [M+H] . Minor isomer: H NMR (400
3
MHz, CD OD): δ 7.15–7.09 (m, 3H), 4.15–4.05 (m, 2H), 3.99 (dq, J = 9.0, 7.2 Hz, 1H),
3
3
2
.94 (dd, J = 13.2, 8.8 Hz, 1H) , 3.87 (dd, J = 13.2, 8.0 Hz, 1H), 3.55 (hept, J = 6.8 Hz,
13
H), 3.44 (d, J = 0.8 Hz, 3H), 1.31 (d, J = 7.2 Hz, 2H), 1.21–1.17 (m, 15H). C NMR
(101 MHz, CD OD): δ 174.84 (d, J = 5.1 Hz), 146.55 (d, J = 11.4 Hz), 141.82 (d, J = 3.0
3
Hz), 126.63 (d, J = 1.8 Hz), 125.13 (d, J = 1.8 Hz), 69.97 (d, J = 158.0 Hz), 62.12, 61.58
3
1
(
d, J = 15.1 Hz), 50.83, 28.15, 24.08, 23.84, 21.32 (d, J= 4.3 Hz), 14.49. P NMR (162
+
MHz, CD OD): δ 23.62. MS m/z (ESI): 386.2 [M+H] .
3
Ethyl 2-[[(2,6-diisopropylphenoxy)-(methoxymethyl)phosphoryl]amino]-2-methyl-
propanoate (8) Yellow oil (5.0 g), 50.1 % yield. R = 0.70 (1:2 ethyl acetate/hexanes,
f
+
TLC), isomer 1 : isomer 2 = 1.00 : 1.00. HRMS (ESI): Calcd for C H NO P [M+H]
2
0
34
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00.2247. Found: 400.2246. The diastereoisomeric mixture was separated by chiral
1
preparative HPLC to afford two optical isomers. Isomer 1: H NMR (400 MHz, CD OD):
3
δ 7.33–6.88 (m, 3H), 4.20–4.01 (m, 2H), 3.94 (dd, J = 13.2, 8.0 Hz, 1H), 3.90 (dd, J =
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3.2, 7.6 Hz, 1H), 3.63 (hept, J = 6.8 Hz, 2H), 3.49 (d, J = 1.2 Hz, 3H), 1.41(s, 3H), 1.37
1
3
(s, 3H), 1.22–1.17 (m, 15H). C NMR (101 MHz, CD OD): δ 176.84 (d, J = 5.4 Hz),
3
1
46.17 (d, J = 11.6 Hz), 142.14 (d, J = 3.1 Hz), 126.63 (d, J = 1.8 Hz), 125.08 (d, J = 1.8
Hz), 70.27 (d, J = 156.9 Hz), 62.58, 61.60 (d, J = 14.0 Hz), 58.46, 28.72 (d, J = 4.8 Hz),
31
28.10, 27.90 (d, J = 2.2 Hz), 24.06, 23.86, 14.36. P NMR (162 MHz, CD OD): δ 23.69.
3
+
1
MS m/z (ESI): 400.1 [M+H] . Isomer 2: H NMR (400 MHz, CD OD): δ 7.18–7.10 (m,
3
3
H), 4.17–4.03 (m, 2H), 3.94 (dd, J = 13.2, 8.0 Hz, 1H), 3.90 (dd, J = 13.2, 7.6 Hz, 1H),
3.63 (hept, J = 6.8 Hz, 2H), 3.49 (d, J = 1.2 Hz, 3H), 1.41 (s, 3H), 1.38 (s, 3H), 1.24–1.16
13
(m, 15H). C NMR (101 MHz, CD OD): δ 176.81 (d, J = 5.5 Hz), 146.15 (d, J = 11.7
3
Hz), 142.11 (d, J = 3.1 Hz), 126.62 (d, J = 1.8 Hz), 125.07 (d, J = 1.8 Hz), 70.26 (d, J =
1
56.9 Hz), 62.56, 61.60 (d, J = 14.1 Hz), 58.44, 28.72 (d, J = 4.9 Hz), 28.08, 27.90 (d, J
31
=
2.0 Hz), 24.07, 23.87, 14.37. P NMR (162 MHz, CD OD): δ 23.67. MS m/z (ESI):
3
+
400.1 [M+H]
Ethyl 2-[[(2,6-diisopropylphenoxy)-(methoxymethyl)phosphoryl]amino]acetate (9)
Yellow oil (5.0 g), 43.9 % yield. R = 0.50 (1:2 ethyl acetate/hexanes, TLC), isomer 1 :
f
+
isomer 2 = 1.00 : 1.00. HRMS (ESI): Calcd for C H NO P [M+H] . 372.1934. Found:
18
31
5
3
72.1932. The diastereoisomeric mixture was separated by chiral preparative HPLC to
1
afford two optical isomers. Isomer 1: H NMR (400 MHz, CD OD): δ 7.18–7.10 (m, 3H),
3
4
.16 (q, J = 6.8 Hz, 2H), 4.02 (dd, J = 13.2, 8.0 Hz, 1H), 3.98 (dd, J = 13.2, 8.0 Hz, 1H),
.81 (dd, J = 18.0, 10.0 Hz, 1H), 3.68 (dd, J = 18.0, 13.6 Hz, 1H), 3.57 (hept, J = 6.8 Hz,
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H), 3.48 (d, J = 0.8 Hz, 3H), 1.28–1.18 (m, 15H). C NMR (101 MHz, CD OD): δ
3
1
72.63 (d, J = 3.8 Hz), 146.31 (d, J = 11.4 Hz), 141.94 (d, J = 3.0 Hz), 126.64 (d, J = 1.8
Hz), 125.14 (d, J = 1.8 Hz), 69.93 (d, J = 156.7 Hz), 62.12, 61.65 (d, J = 14.3 Hz), 43.44,
10
11
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59
60
31
2
8.17, 24.04, 23.98, 14.52. P NMR (162 MHz, CD OD): δ 25.68. MS m/z (ESI): 372.2
3
+
1
[M+H] . Isomer 2: H NMR (400 MHz, CD OD): δ 7.18–7.10 (m, 3H), 4.14 (q, J = 7.2
3
Hz, 2H), 4.02 (dd, J = 13.2, 8.0 Hz, 1H), 3.98 (dd, J = 13.2, 8.0 Hz, 1H), 3.81 (dd, J =
1
8.0, 10.0 Hz, 1H), 3.70 (dd, J = 18.0, 13.6 Hz, 1H), 3.57 (hept, J = 6.8 Hz, 2H), 3.48 (d,
1
3
J = 0.8 Hz, 3H), 1.27–1.20 (m, 15H). C NMR (101 MHz, CD OD): δ 172.62 (d, J = 3.9
3
Hz), 146.30 (d, J = 11.4 Hz), 141.92 (d, J = 2.9 Hz), 126.64 (d, J = 1.8 Hz), 125.14 (d, J
=
1.8 Hz), 69.92 (d, J = 156.8 Hz), 62.12, 61.64 (d, J = 14.3 Hz), 43.44, 28.17, 24.04,
3
1
+
23.99, 14.52. P NMR (162 MHz, CD OD): δ 25.67. MS m/z (ESI): 372.2 [M+H] .
3
Ethyl
(2S)-2-[[(2,6-diisopropylphenoxy)-
(
hydroxymethyl)phosphoryl]amino]propanoate (10) To a stirred solution of 26 (15.0
g, 32.5 mmol) in 100 mL of dichloromethane was added Pd/C (10.0 g, 10 wt %). The
mixture was stirred under hydrogen atmosphere at room temperature for 8 hrs. The
resulting solution was filtered. The solvent was removed under reduced pressure. The
title compound was obtained as yellow oil (8.0 g) in 66.3 % yield. R = 0.40 (1:1 ethyl
f
acetate/hexanes, TLC), major isomer : minor isomer = 1.75 : 1.00. HRMS (ESI): Calcd
+
for C H NO P [M+H] 371.1856. Found: 371.1858. The diastereoisomeric mixture was
1
8
30
5
separated by chiral preparative HPLC to afford two optical isomers. Major isomer: chiral
1
retention time = 7.81 min, H NMR (400 MHz, CD OD): δ 7.16–7.09 (m, 3H), 4.19–4.11
3
(
m, 2H), 4.10–4.05 (m, 2H), 4.03 (dq, J = 7.2, 7.2 Hz, 1H), 3.61 (hept, J = 6.8 Hz, 2H),
1
.27 (d, J = 7.2 Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H), 1.23 (d, J = 6.8 Hz, 6H), 1.22 (d, J =
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.8 Hz, 6H). C NMR (101 MHz, CD OD): δ 175.85 (d, J = 3.9 Hz), 146.17 (d, J = 11.7
3
Hz), 142.04 (d, J = 3.0 Hz), 126.58 (d, J = 1.8 Hz), 125.11 (d, J = 1.8 Hz), 62.43, 59.71
3
1
(
d, J = 152.9 Hz), 50.88, 28.06, 24.07, 23.95, 20.71 (d, J = 5.3 Hz), 14.44. P NMR (162
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
MHz, CD OD): δ 28.02. MS m/z (ESI): 394.2 [M+Na] . Minor isomer: chiral HPLC
3
retention time = 12.41 min. This isomer can also be isolated by crystallization method. 10
(
3.0 g) was dissolved in 10 mL ethyl acetate/nꢀhexane (v : v = 1:10) at 70 ℃, cooled
down to room temperature and stayed overnight. After filtration, 10a was obtained as
white crystal (0.70 g) in 23.3 % yield. The optical purity of the isolated diastereoisomer
1
was determined by chiral HPLC to be 100 %. H NMR (400 MHz, CD OD): δ 7.16–7.07
3
(m, 3H), 4.10–3.98 (m, 5H), 3.57 (hept, J = 6.8 Hz, 2H), 1.36 (d, J = 6.8 Hz, 3H), 1.21(d,
13
J = 6.8 Hz, 12H), 1.19 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CD OD): δ 175.02 (d, J
3
=
5.3 Hz), 146.25 (d, J = 11.6 Hz), 141.61 (d, J = 3.1 Hz), 126.52 (d, J = 1.8 Hz), 125.10
(
d, J = 1.8 Hz), 62.19, 59.74 (d, J = 156.2 Hz), 50.82, 28.14, 24.08, 23.82, 21.49 (d, J =
31
+
4
.0 Hz), 14.42. P NMR (162 MHz, CD OD): δ 27.01. MS m/z (ESI): 394.2 [M+Na] .
3
Ethyl (2S)-2-[[(2,6-diisopropylphenoxy)-(morpholinomethyl)phosphoryl]amino]
propanoate (11) To a stirred solution of 29 (4.94 g, 11.0 mmol) in 20 mL of
tetrahydrofuran was added morpholine (2.87 g, 33.0 mmol) and triethylamine (5.67 g,
56.0 mmol). The reaction mixture was heated to 80 ℃ and stirred for 8 hrs. The resulting
mixture was concentrated in vacuum. The residue was purified by flash column
chromatography (1:10–1:1 ethyl acetate/hexanes). The title compound was obtained as
yellow oil (4.0 g) in 82.6 % yield. R = 0.60 and 0.5 (1:2 ethyl acetate/hexanes, TLC),
f
major isomer : minor isomer = 1.41 : 1.00. HRMS (ESI): Calcd for C H N O P
2
2
38
2
5
+
441.2513 [M+H] . Found: 441.2509. The diastereoisomeric mixture was separated by
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chiral preparative HPLC to afford two optical isomers. Major isomer: H NMR (400
MHz, CD OD): δ 7.18–7.06 (m, 3H), 4.15–3.95 (m, 2H), 4.07–3.95 (m, 1H), 3.69 (m,
3
4
H), 3.57 (hept, J = 6.8 Hz, 2H), 3.04 (dd, J = 15.4, 11.2 Hz, 1H), 2.98 (dd, J = 15.4, 10.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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42
43
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48
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57
58
59
60
Hz, 1H), 2.73 (m, 2H), 2.63 (m 2H), 1.40 (d, J = 7.2 Hz, 3H), 1.21 (d, J = 6.8 Hz, 12H),
1
3
1.19 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CD OD): δ 175.54 (d, J = 4.4 Hz), 146.15
3
(d, J = 11.8 Hz), 142.16 (d, J = 3.0 Hz), 126.65, 12 5.18, 68.07, 62.28, 57.54 (d, J =
31
1
55.1 Hz), 56.60 (d, J = 10.0 Hz), 51.09, 28.10, 24.12, 23.99, 21.12, 14.52. P NMR
+
1
(162 MHz, CD OD): δ 26.58. MS m/z (ESI)
:
441.3 [M+H] . Minor isomer: H NMR (400
3
MHz, CD OD): δ 7.21–7.08 (m, 3H), 4.27–4.08 (m, 2H), 4.00 (dq, J = 7.2, 7.2 Hz, 1H),
3
3
.74–3.66 (m, 4H), 3.55 (hept, J = 6.8 Hz, 2H), 3.09 (dd, J = 15.4, 10.2 Hz, 1H), 3.01
1
3
(dd, J = 15.4, 10.8 Hz, 1H), 2.78–2.60 (m, 4H), 1.30–1.17 (m, 18H). C NMR (101
MHz, CD OD): δ 175.53 (d, J = 4.4 Hz), 146.14 (d, J = 11.8 Hz), 142.15 (d, J = 3.0 Hz),
3
1
26.65 (d, J = 1.6 Hz), 125.18 (d, J = 1.8 Hz), 68.07, 62.28, 57.54 (d, J = 155.0 Hz),
31
5
6.60 (d, J = 9.9 Hz), 51.09, 28.10, 24.13, 24.00, 21.15 (d, J = 4.9 Hz), 14.52. P NMR
+
(
162 MHz, CD OD): δ 27.56. MS m/z (ESI)
:
441.4 [M+H] .
3
Isopropyl
(2S)-2-[[(2,6-diisopropylphenoxy)-propyl-
phosphoryl]amino]propanoate (12) Under nitrogen atmosphere, to a stirred solution of
1 (4.00 g，25.0 mmol) in 25 mL of dichloromethane was added a solution of 1 (4.45
3
g，25.0 mmol) and triethylamine (10.0 g，99.0 mmol) in dichloromethane (20 mL) at ꢀ
20 ℃, then stirred for 2 hrs. The Lꢀalanine ethyl ester (5.03 g, 43.0 mmol) was then
added. The reaction mixture was allowed to warm to room temperature and stirred for 3
hrs, quenched with water (20 mL) and extracted with ethyl acetate (50 mL). The organic
layer was washed with sodium dihydrogen phosphate (15 wt %, 20 mL × 3), dried with
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
5
5
5
5
5
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6
anhydrous sodium sulfate and concentrated in vacuum. The residue was purified by flash
column chromatography (1:9–3:7 ethyl acetate/hexanes). The title compound was
obtained as yellow oil (2.0 g) in 20.9 % yield. R = 0.60 (1:5 ethyl acetate/hexanes, TLC),
f
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
major isomer : minor isomer = 1.15 : 1.00. HRMS (ESI): Calcd for C H NO P [M+H]
2
0
35
4
384.2298. Found: 384.2297. The diastereoisomeric mixture was separated by chiral
1
preparative HPLC to afford two optical isomers. Major isomer: H NMR (400 MHz,
CD OD): δ 7.20–7.07 (m, 3H), 4..22–4.07 (m, 2H), 3.89 (dq, J = 7.6, 7.6 Hz, 1H), 3.53
3
(hept, J = 6.8 Hz, 2H), 2.12–1.89 (m, 2H), 1.89–1.66 (m, 2H), 1.30–1.17 (m, 15H), 1.13–
13
1
1
.05 (m, 6H). C NMR (101 MHz, CD OD): δ 175.63 (d, J = 4.1 Hz), 146.76 (d, J =
3
1.5 Hz), 142.10 (d, J = 3.1 Hz), 126.45 (d, J = 1.9 Hz), 125.06 (d, J = 1.8 Hz), 62.16,
50.69, 31.73 (d, J = 130.0 Hz), 28.46, 23.96, 23.76, 20.58 (d, J = 5.5 Hz), 17.13 (d, J =
31
4
.8 Hz), 15.80 (d, J = 18.4 Hz), 14.48. P NMR (162 MHz, CD OD): δ 34.56. MS m/z
3
+
1
(
ESI): 384.2 [M+H] . Minor isomer: H NMR (400 MHz, CD OD): δ 7.17–7.05 (m, 3H),
3
4
.04–3.97 (m, 2H), 3.94 (dq, J = 9.6, 7.0 Hz, 1H), 3.49 (hept, J = 6.8 Hz, 2H), 2.04–1.87
(m, 2H), 1.76–1.70 (m, 2H), 1.29 (d, J = 7.0 Hz, 3H), 1.21 (d, J = 6.8 Hz, 6H), 1.20 (d, J
1
3
= 6.8 Hz, 6H), 1.15 (t, J = 7.2 Hz, 3H), 1.07 (td, J = 7.2, 1.6 Hz, 3H). C NMR (101
MHz, CD OD): δ 174.82 (d, J = 4.8 Hz), 147.06 (d, J = 11.4 Hz), 141.88 (d, J = 3.0 Hz),
3
1
26.38 (d, J = 1.8 Hz), 125.07 (d, J = 1.8 Hz), 62.06, 50.49, 31.45 (d, J = 131.3 Hz),
28.50, 23.96, 23.74, 21.53 (d, J = 4.6 Hz), 17.25 (d, J = 4.5 Hz), 15.71 (d, J = 18.2 Hz),
31
+
1
4.42. P NMR (162 MHz, CD OD): δ 33.57. MS m/z (ESI)
:
384.2 [M+H] .
3
Ethyl (2S)-2-[[[2-[(1R)-1-cyclopropylethyl]-6-isopropyl-phenoxy](methoxymethyl)
phosphoryl]amino]propanoate (13) Yellow oil (2.5 g), 28.0 % yield. R = 0.60 (1:2
f
ethyl acetate/hexanes, TLC), major isomer : minor isomer = 1.10 : 1.00. HRMS (ESI):
ACS Paragon Plus Environment

Page 29 of 38
Journal of Medicinal Chemistry
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2
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+
Calcd for C H NO P [M+H] 412.2247. Found: 412.2245. The diastereoisomeric
2
1
35
5
mixture was separated by chiral preparative HPLC to afford two optical isomers. Major
1
isomer: H NMR (400 MHz, CD OD): δ 7.28 (dd, J = 6.2, 3.0 Hz, 1H), 7.20–7.10 (m,
3
10
11
12
13
14
15
16
17
18
19
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60
2
H), 4.20–4.04 (m, 2H), 3.97 (dq, J = 9.0, 7.0 Hz,1H), 3.90 (dd, J = 13.4, 8.8 Hz, 1H),
3.84 (dd, J = 13.4, 7.8 Hz, 1H), 3.56 (hept, J = 6.8 Hz, 1H), 3.44 (d, J = 1.0 Hz, 3H), 2.80
(dq, J = 9.0, 7.0 Hz, 1H), 1.30 (d, J = 7.0 Hz, 3H), 1.28 (d, J = 6.8 Hz, 3H), 1.25–1.17
13
(m, 9H), 1.00–0.88 (m, 1H), 0.58–0.50 (m, 1H), 0.37–0.18 (m, 3H). C NMR (101 MHz,
CD OD): δ 174.81 (d, J = 5.2 Hz), 146.76 (d, J = 11.4 Hz), 141.69 (d, J = 3.0 Hz),
3
1
40.77 (d, J = 3.2 Hz), 126.54 (d, J = 1.7 Hz), 126.17 (d, J = 1.7 Hz), 125.15 (d, J = 1.7
Hz), 70.03 (d, J = 157.3 Hz), 62.16, 61.53 (d, J = 15.4 Hz), 50.82, 38.27, 28.15, 24.00,
31
23.91, 21.47, 21.34 (d, J = 4.3 Hz), 19.23, 14.50, 5.42, 4.22. P NMR (162 MHz,
+
1
CD OD): δ 23.42. MS m/z (ESI): 412.3 [M+H] . Minor isomer: H NMR (400 MHz,
3
CD OD): δ 7.27 (dd, J = 6.4, 3.2 Hz, 1H), 7.15–7.11 (m, 2H), 4.22–4.10 (m, 2H), 3.99
3
(
dq, J = 7.2, 7.2 Hz, 1H), 3.94 (dd, J = 13.2, 8.0 Hz, 1H), 3.90 (dd, J = 13.2, 8.0 Hz, 1H),
.57 (hept, J = 6.8 Hz, 1H), 3.47 (d, J = 1.0 Hz, 1H), 2.81 (dq, J = 9.0, 7.0 Hz, 1H), 1.30
(d, J = 7.0 Hz, 3H), 1.27–1.21 (m, 9H), 1.20 (d, J = 6.8 Hz, 3H), 0.98–0.88 (m, 1H),
3
1
3
0
.59–0.51 (m, 1H), 0.37–0.17 (m, 3H). C NMR (101 MHz, CD OD): δ 175.24 (d, J =
3
3
.8 Hz), 146.39 (d, J = 11.4 Hz), 141.81 (d, J = 3.1 Hz), 141.08 (d, J = 3.1 Hz), 126.57
(d, J = 1.8 Hz), 126.17 (d, J = 1.7 Hz), 125.09 (d, J = 1.8 Hz), 69.81 (d, J = 156.0 Hz),
2.27, 61.61 (d, J = 14.6 Hz), 50.85, 38.32, 28.12, 24.32, 23.69, 21.53, 20.76 (d, J = 5.5
6
3
1
Hz), 19.38, 14.50, 5.46, 4.29. P NMR (162 MHz, CD OD): δ 24.77. MS m/z (ESI):
3
+
4
34.3 [M+Na] .
ACS Paragon Plus Environment