Substituted 4-morpholine N-arylsulfonamides as γ-secretase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.08.024

The design, synthesis, SAR, and biological profile of a substituted 4-morpholine sulfonamide series of γ-secretase inhibitors (GSIs) were described. In several cases, the resulting series of GSIs reduced CYP liabilities and improved γ-secretase inhibition activity compared to our previous research series. Selected compounds demonstrated significant reduction of amyloid-β (Aβ) after acute oral dosing in a transgenic animal model of Alzheimer's disease (AD).

Full text of DOI:10.1016/j.ejmech.2016.08.024

Accepted Manuscript
Substituted 4-morpholine N-arylsulfonamides as γ-secretase inhibitors
Zhiqiang Zhao, Dmitri A. Pissarnitski, Hubert B. Josien, Thomas A. Bara, John
W. Clader, Hongmei Li, Mark D. McBriar, Murali Rajagopalan, Ruo Xu, Giuseppe
Terracina, Lynn Hyde, Lixin Song, Lili Zhang, Eric M. Parker, Rebecca Osterman,
Alexei V. Buevich
PII:
S0223-5234(16)30667-5
10.1016/j.ejmech.2016.08.024
EJMECH 8820
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 March 2016
Revised Date: 11 August 2016
Accepted Date: 12 August 2016
Please cite this article as: Z. Zhao, D.A. Pissarnitski, H.B. Josien, T.A. Bara, J.W. Clader, H. Li, M.D.
McBriar, M. Rajagopalan, R. Xu, G. Terracina, L. Hyde, L. Song, L. Zhang, E.M. Parker, R. Osterman,
A.V. Buevich, Substituted 4-morpholine N-arylsulfonamides as γ-secretase inhibitors, European Journal
of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.024.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Substituted 4-morpholine N-arylsulfonamides as γ-
secretase inhibitors
Zhiqiang Zhao,a,* Dmitri A. Pissarnitski,a Hubert B. Josien,a Thomas A. Bara,a John W. Clader,a
Hongmei Li,a Mark D. McBriar,a Murali Rajagopalan,a Ruo Xu,a Giuseppe Terracina,b Lynn
Hyde,b Lixin Song,b Lili Zhang,b Eric M. Parker,b Rebecca Osterman,c and Alexei V. Buevichc
a Department of Medicinal Chemistry, Merck Research Laboratories, 2000 Galloping Hill Road,
Kenilworth, NJ 07033, USA
b Department of Neurobiology, Merck Research Laboratories, 2000 Galloping Hill Road,
Kenilworth, NJ 07033, USA
c NMR Structure Elucidation, Merck Research Laboratories, 2000 Galloping Hill Road,
Kenilworth, NJ 07033, USA
*Zhiqiang.Zhao@Merck.com
ABSTRACT: The design, synthesis, SAR, and biological profile of a substituted 4-morpholine
sulfonamide series of γ-secretase inhibitors (GSIs) were described. In several cases, the resulting
series of GSIs reduced CYP liabilities and improved γ-secretase inhibition activity compared to
our previous research series. Selected compounds demonstrated significant reduction of amyloid-
β (Aβ) after acute oral dosing in a transgenic animal model of Alzheimer’s disease (AD).
1

ACCEPTED MANUSCRIPT
Kew words: γ-secretase inhibitor; Alzheimer's disease; substituted 4-morpholine sulfonamide
1. Introduction
Alzheimer’s disease (AD) is the most common form of dementia that causes problems with
memory, thinking and behavior in the elderly. These problems can become so severe over time
that the patients will not be able to participate in conversation and to respond to their
environment. AD is the sixth leading cause of death across all ages in the United States. An
estimated 5.3 million Americans have AD and causing $226 billion in cost to the nation in 2015
[1]. As the elderly population continues to grow, the prevalence of AD will increasingly be a
major burden to the nation’s health care system.
It is known that γ-secretase is one of the critical enzymes required for the generation of Aβ
peptides. Accumulation and deposition of Aβ plaques leads to the damage and death of neurons,
which is correlated with severity of AD [2]. Thus, inhibition of γ-secretase is considered to be a
disease-modifying approach for this disorder [3]. Small-molecule GSIs have been shown to
lower levels of plasma, cerebrospinal fluid (CSF), and cortical Aβ peptides in the animal
models[4]. A review of recent advances in the identification of GSIs was published in a Journal
of Medicinal Chemistry perspective [5]. The same review also discussed the application of GSIs
beyond AD, such as for the treatment of leukemia [6] and breast cancer [7].
The previously published research from our group identified novel series of arylsulfonamides
as γ-secretase inhibitors. Josien and Pissarnitski et. al. reported N-arylsulfonamides as orally
active GSIs (Structure I, Fig. 1) [8,9]. Li et. al. demonstrated that adding a hydroxyl group on
piperidine ring can further reduce the CYP inhibition while retain the GSI activity (Structure II,
Fig. 1) [10].
2

ACCEPTED MANUSCRIPT
Fig. 1. Design of morpholine sulfonamide series of GSIs (III)
Two intermediate compounds (A and B in Fig. 1) were tested for both Aβ40 and CYP3A4
inhibition activity. Potential hydrolysis of structures I and II in vivo may produce A which is a
strong CYP3A4 inhibitor. Cyclopropanol group is a suspected liability, because compound B
without it has a clean CYP3A4 profile. However, cyclopropyl is important for γ-secretase
activity. Based on these known results, herein we describe the design, synthesis, SAR, and
biological profile of a novel substituted morpholine sulfonamide series of GSIs III [11].
Borrowing SAR elements of known sulfonamide GSIs, one carbon on the piperidine ring was
replaced by oxygen; carbamate right hand side was changed to amide to avoid the potential
generation of cyclopropanol group from in vivo hydrolysis. An optional hydroxyl group was also
added at R5 position of III to expand SAR by analogy to structure II. Selected compounds of
type III demonstrated a significant reduction of amyloid-β (Aβ) after acute oral dosing in a
transgenic animal model of AD.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of compounds 15
3

ACCEPTED MANUSCRIPT
Our synthesis started with an inexpensive commercially available chemical 1, diethyl
cyclopropane-1,1-dicarboxylate [12] purchased from Sigma-Aldrich (Scheme 1). Compound 1
was reduced by lithium aluminum hydride (LAH) to cyclopropane-1,1-diyldimethanol [13],
which was mono protected with p-methoxybenzyl (PMB) group to give (1-((4-
methoxybenzyloxy)methyl)-cyclopropyl)methanol 2. The mono-protection was moderately
selective, but the bis-protected by-product could be selectively converted to the desired mono
protected product 2 by treatment with one equivalent of 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ). Compound 2 was then oxidized to an aldehyde 3 by Swern oxidation.
Through the Bucherer-Bergs reaction, compound 3 was converted to a hydantoin 4. Hydrolysis
of 4, followed by protection of the resulting amine group with tert-butoxycarbonyl (Boc) gave
the carboxylic acid 5. Alkylation of 5 with 2-bromo-1-cyclopropylethanone under basic
condition gave compound 6. PMB protecting group was removed by treatment of 6 with DDQ,
and the hydroxyl group was re-protected with tert-butyldiphenylsilyl (TBDPS) group to give
compound 7. Treatment of compound 7 with trifluoroacetic acid (TFA) to deprotect Boc from
amine was followed by an intramolecular reaction to form a cyclic imine which was then
reduced to a cyclic amine 8 by sodium triacetoxyborohydride. Sulfonylation of the amine 8
furnished sulfonamide 9. The lactone group in compound 9 was reduced by sodium borohydride
to a diol and followed by Mitsunobu reaction to give the morpholine core compound 10. The
TBDPS protecting group was removed by using tetra-n-butylammonium fluoride (TBAF) to give
alcohol 11. The hydroxyl group was then transformed to iodide 12 which was further
transformed to a nitrile group 13. Compound 13 was reduced to an aldehyde by
diisobutylaluminum hydride (DIBAL-H), followed by oxidation by sodium chlorite to give the
carboxylic acid 14. The carboxylic acid 14 was then converted to a carbonyl chloride
4

ACCEPTED MANUSCRIPT
intermediate which was used to prepare a series of final amide compounds 15a-i by reacting with
a variety of amines.
Scheme 1.a Synthesis of compounds 15
a Reagents and conditions. (a) LAH, THF, 0°C, 87%; (b) PMBCl, NaH, DMF, 0 °C, 72%; (c)
oxalyl chloride, DMSO, Et3N, CH2Cl2, -78 °C – RT, 92%; (d) KCN, (NH4)2CO3, Et3N,
water/EtOH, 60 °C, 89%; (e) Ba(OH)2, water, reflux, 98%; (f) (Boc)2O, Et3N/dioxane, RT, 85%;
(g) 2-bromo-1-cyclopropylethanone, KOH, KI, MeOH/CH2Cl2, RT, 91%; (h) DDQ, CH2Cl2; (i)
TBDPSCl, Et3N, imidazole, THF, 71% (two steps); (j) TFA, CH2Cl2; (k) NaBH(OAc)3, CH2Cl2,
88% (two steps); (l) 4-chlorobenzene-1-sulfonyl chloride, pyridine, 51%; (m) NaBH4, CaCl2,
THF/EtOH, 69%; (n) PPh3, DEAD, toluene, 60%; (o) TBAF, THF, 95%; (p) I2, Ph3P, imidazole,
CH3CN/Toluene, 89%; (q) n-Bu4NCN, CH3CN, 91%; (r) DIBAL, CH2Cl2, -78 °C, 82%; (s)
NaClO2, CH3CHC(CH3)2, t-BuOH/water, 0 °C, 97%; (t) oxalyl chloride, Et3N, HNR1R2.
2.1.2. Synthesis of compound 25
Following the same chemical route as described in Scheme 1, we used 2-chlorocyclopentanone
for reaction with 5. The intermediate compound 5 reacts with 2-chlorocyclopentanone to give a
mixture of two diastereomers of compound 16 (Scheme 2). The two diastereomers of 16 were
5

ACCEPTED MANUSCRIPT
separated by flash chromatography purification on silica gel, followed by crystallization from
diethyl either. One single diastereomer 16a (white crystal form) was carried over to compound
17. In the cyclization step of 17 to 18, we obtained the fused ring compound 18 which was
sulfonylated to provide diastereomerically pure compound 19a which was used to assign
stereochemistry for the whole series. In order to gain additional confidence in the assignment,
the alternative diastereomer 19b was also secured by carrying a diastereomeric mixture 16a/b
through the same steps, followed by separation of two diastereomers 19a and 19b by flash
chromatography purification on silica gel.
O
O
Boc
NH
O
O
O
O
+
a
OH
PMBO
HN
OPMB
HN
OPMB
O
Boc
Boc
5
16a
16b
O
O
O
O
O
O
O
OTBDPS
d, e
b, c
f
OTBDPS
16a
N
OTBDPS
N
H
18
O
HN
O
S
Boc
Ar
17
19a
O
O
1. b ꢀ f
2. Chromatography
19a
16a/b
+
N
OTBDPS
O
O
i
S
Ar
19b
O
O
O
j
g, h
19a
I
OH
N
N
OTBDPS
N
O
S O
O
S O
Ar
O
S O
20
21
22
Ar
Ar
O
O
N
O
NR1R2
N
k
n
l, m
OH
O
S O
O
N
CN
O
O
S
O
S O
Ar
O
Ar
23
24
25
Cl
Scheme 2. a Synthesis of fused ring morpholine compounds 25
6

ACCEPTED MANUSCRIPT
a Reagents and conditions. (a) 2-Chlorocyclopentanone, KOH, KI, MeOH, RT, 70%;
chromatography, crystallization; (b) DDQ, CH2Cl2; (c) TBDPSCl, Et3N, imidazole, THF, 92%
(two steps); (d) TFA, CH2Cl2, 87%; (e) NaBH(OAc)3, CH2Cl2, 73% (two steps); (f) 4-
chlorobenzene-1-sulfonyl chloride, pyridine, 71%; (g) NaBH4, CaCl2, THF/EtOH, 62%; (h)
PPh3, DEAD, THF, 80%; (i) TBAF, THF, 76%; (j) I2, Ph3P, imidazole, CH3CN/Toluene, 90%;
(k) n-Bu4NCN, CH3CN, 89%; (l) DIBAL, CH2Cl2, -78 °C, 94%; (m) NaClO2, CH3CHC(CH3)2,
t-BuOH/water, 0 °C, 55%; (n) oxalyl chloride, Et3N, HNR1R2
For the determination of relative stereochemistry of compounds 19a and 19b, the NMR signals
for protons 2, 5,6,7, 11, and 13 (Fig. 2) were assigned on the basis of gCOSY, gHSQC, and
NOESY (Supporting info). Compound 19a reveals a strong NOE H5-H6, consistent with the
cis- configuration of these protons. Furthermore, protons H7a and H7b are differentiated based
on the magnitude of their NOE signal with H6 (2.4% and 1.2% respectively). Consistent with the
overall shape of the molecule 19a, H7b has a significant NOE with H13b, and H2 has a weak,
but measurable NOE with H5. On the other hand, the structure of diastereomer 19b is
consistent with trans- configuration of protons H5 and H6, as evident by the NOE signals H5-
H7b and H6-H7a respectively (Fig. 2). The assignment is re-enforced by observation of NOEs
between the protons of cyclopropyl group (H11a, H12a) with proton H7b [14].
Figure 2. Assignment of structure for compounds 19a and 19b. 4-Clorophenylsulfonamide
moiety is not shown for clarity.
7

ACCEPTED MANUSCRIPT
Compound 19a was transformed to the compound 21 following the same procedures in
Scheme 1. Compound 21 was resolved by chiral OJ column to give the single enantiomer 21
which was carried to a series of final compounds 25a-e (Scheme 2).
2.1.3. Synthesis of compound 29
The intermediate compound 11 was oxidized to a corresponding aldehyde through Dess-
Martin reaction (Scheme 3). Reaction of the aldehyde with KCN and TBSCl in the presence of a
catalytic amount of ZnI2 produced TBS-protected cyanohydrine 26. The CN group in 26 was
reduced by DIBAL-H to aldehyde to give compound 27. The aldehyde group in 27 was oxidized
to an acid by NaClO2 and the TBS protecting group was removed by TBAF to give compound
28. The alpha-hydroxy acid intermediate 28 was converted to the final compound 29 through the
amide coupling reaction with an amine.
Scheme 3. a Synthesis of alpha-hydroxy amide sulfonyl morpholine
a Reagents and conditions. (a) DMP, CH2Cl2, 59%; (b) TBSCl, KCN, ZnI2 (cat.), CH3CN, 55
°C; (c) separation of diastereomers (d) DIBAL-H, CH2Cl2, -78 °C, 35%; (e) NaClO2, NaH2PO4,
CH3CHC(CH3)2, t-BuOH/water, 87%; (f) TBAF/THF, 71%; (g) BOP, NMM, HNR1R2, CH2Cl2,
68-76%.
8

ACCEPTED MANUSCRIPT
2.2.Biological Evaluation
2.2.1. Rational and design
Herein, we disclose the design of a series of substituted 4-morpholine sulfonamide γ-secretase
inhibitors, III (Fig. 3), utilizing the cyclohexane framework C as a reference compound (Fig. 3).
Although molecule C itself is a potent γ-secretase inhibitor in vitro [15], it experienced
extensive
Fig. 3. Conformational comparisons for the design of GSIs.
metabolism at the methylene carbons of the cyclohexyl ring in in vitro metabolic stability assays
[16]. In addition, its poor aqueous solubility may have negatively impacted oral absorption. As
discussed in our recent paper [17] the arylsulfone group must maintain the equatorial position for
the potency of GSIs. Our newly substituted morpholine arylsulfonamide series maintained the
active chair conformation with arylsulfonamide at equatorial position. The added substituents at
C3 and C5 may help to stabilize the active chair conformation. The morpholine ring will not only
increase the polarity of the molecule but may also block the in vitro metabolite of the methylene
carbons of the cyclohexyl ring (Fig. 3).
2.2.2. Biological test results
The biological test results are summarized in Table 1.
Table 1. Biological Data for compound Шa and Шb
9

ACCEPTED MANUSCRIPT
Entry
Structure
NR1R2
X
R5 Compound
Cell
CYP3A4IC50
AUCc
Шa/Шb
Aβ40 Co-inc; Pre- (h•ng/mL)
a
IC50
inc b
(nM)
(µM)
N
1
2
3
Cl
Cl
Cl
H
H
H
15a (IIIa)
15b (IIIa)
15c (IIIa)
5.9
12
>30, 1.9
>30, 2.0
>30, <0.4
464
1292
2728
N
OH
6.0
4
5
6
7
8
Cl
Cl
H
H
15d (IIIa)
15e (IIIa)
3.5
18
10
24
30
9.9, <0.3
30, 4.3
1160
N
N
O
Cl OH 29a (IIIa)
Cl OH 29b (IIIa)
>30, 9.0
>30, >30
>30, 1.5
254
267
575
N
N
N
N
F
H
15f (IIIa)
N
N
9
F
F
F
H
H
H
15g (IIIa)
15h (IIIa)
15i (IIIa)
67
32
14
30, 17
>30, 6.9
>30, 2.2
3158
1973
O
10
11
12
13
Cl
Cl
H
H
25a (IIIb)
25b (IIIb)
1.8
1.7
30, 30
36
21, 8.0
10

ACCEPTED MANUSCRIPT
14
Cl
H
25c (IIIb)
4.8
30, 15
15
16
Cl
Cl
H
H
25d (IIIb)
25e (IIIb)
16
18
>30, >30
30, 30
a Values are means of two experiments; b Pre-incubation values were determined 30 min after combining the
compound with the enzyme; c Measured in rat (n = 2) over 0-6 h period after a 30 mg/kg oral dosing.
The test results show that substituted 4-morpholine sulfonamide compounds were very active γ-
secretase inhibitors. The IC50 for the inhibition of Aβ40 are in the range of 1.7 to 67 nM (Table
1). A direct comparison of compound 15a (entry 1, Table 1) with our previously published
compound Іa [8] shows that 15a is six times more potent than Іa in the γ-secretase assay and
also has a lessened CYP3A4 liability by more than four folds (Fig. 4). Compound 15a was
evaluated in a transgenic CRND8 mouse model of AD and demonstrated 89% inhibition of total
Aβ in the plasma after a single oral dose of 30 mg/kg (Fig. 5).
Fig. 4. Direct comparisons of 15a and Іa
11

ACCEPTED MANUSCRIPT
Fig. 5. Reduction of plasma Aβ40 of transgenic CRND8 mice dosed with 15a, 30 mg/kg, PO,
measured 3 h after the dosing, compared to the group treated with vehicle only (Veh).
As expected in our design, adding a hydroxy group in molecule IIIa at R5 position
significantly reduced CYP3A4 activity. Comparison of entries 4 and 6 shows that when R5
changed from hydrogen to hydroxyl group, CYP3A4 activity changed from 9.9 µM to greater
than 30 µM for co-incubation, and from less than 0.3 µM to 9.0 µM for pre-incubation
respectively. Comparison of entry 2 and 7 demonstrates that when R5 changed from hydrogen to
hydroxyl group, CYP3A4 activity changed from 2 µM to greater than 30 µM for pre-incubation.
As evident from entries 5 and 9, 4-chlorophenyl sulfonamide compound 15e is 3 fold more
potent than 4-fluorophenyl sulfonamide compound 15g; but 15e is also more active than 15g for
CYP3A4 inhibition. The fused ring morpholine series (IIIb) not only reduced the CYP3A4
activity, but also improved the γ-secretase activity. Comparison of entries 5 and 14 demonstrates
that, when left side of the molecule is changed from cyclopropyl to fused cyclopentyl ring,
CYP3A4 activity was ameliorated from 4.3 µM to greater than 15 µM for pre-incubation; the γ-
secretase IC50 values improved from 18 nM to 4.8 nM. Likewise, comparison of entry 8 and 13
shows that CYP3A4 activity changed from 1.5 µM to 8.0 µM for pre-incubation, and the γ-
secretase IC50 improved from 30 nM to 1.7 nM.
12

ACCEPTED MANUSCRIPT
3. Conclusion
In conclusion, based on the SAR elements of known sulfonamide GSIs from our previous
research results, we have designed a novel series of morpholine sulfonamide core series of GSIs.
The new substituted morpholine series did not only improved the in vitro activity significantly,
but also reduced the CYP3A4 liability. Selected compound 15a demonstrated significant
reduction of total Aβ after acute oral dosing in a transgenic animal model of AD.
4. EXPERIMENTAL
4.1. General for chemistry
Purity of all final compounds was determined by HPLC and exceeded 95% [18]. LC/MS data
analyses were performed using an Applied Biosystems API-100 mass spectrometer and
Shimadzu SCL-10A, LC column: Alltech platinum C18, 3 micron, 33mm x 7mm ID; gradient
flow: 0 min – 10% CH3CN, 5 min – 95% CH3CN, 7 min – 95% CH3CN, 7.5 min – 10% CH3CN,
9 min – stop. The observed parent ions are given. Preparative chromatography was carried out on
Analogix flash chromatography instrument, chiral separations were conducted on chiral OJ
1
columns (Daicel Corp) using isopropyl alcohol and hexanes as the mobile phase. H NMR and
13C NMR spectra were obtained on either a Varian Gemini-300 (300 MHz) or XL-400 (400
MHz) and are reported as ppm down field from Me4Si with number of protons, multiplicities,
and coupling constants in Hertz indicated parenthetically. Chemical names were generated from
ChemDraw. Unless noted otherwise, compounds were isolated as amorphous solids.
4.2. General for biological tests
4.2.1. In vitro cell based γ-secretase assay
In vitro γ-secretase assays was conducted in whole cells. HEK293 cells stably transfected with
a human APP cDNA with both the Swedish and London familial AD mutations in the pcDNA3.1
13

ACCEPTED MANUSCRIPT
vector (Invitrogen, HEK293Swe‑Lon cells) were treated with γ-secretase inhibitors for 5 h. Aβ
in conditioned medium was measured using MesoScale Discovery (MSD) technology-based
sandwich immunoassays. Aβ40 was measured using the antibody pair of TAG-G2-10 and biotin-
4G8. IC50 values for the compounds were determined as described earlier for the membrane-
based assay [19]. IC50 values were calculated from a non-linear fit of initial reaction velocities
versus compound concentration using inhibition dose-response equation (four-parameter;
variable slope) using PRISM® software (GraphPad). The data presented in this study were the
means of the duplicate or triplicate measurements in each experiment.
4.2.2. In vivo studies
All animal procedures were performed within an Association for Assessment and Accreditation
of Laboratory Animal Care (AAALAC) International-accredited facility and approved by the
Merck Institutional Animal Care and Use Committee (IACUC).
Transgenic 5–7 weeks old CRND8 mice carrying the Swedish and Indiana familial Alzheimer’s
disease APP mutations under the control of the Syrian hamster prion protein [20] used in these
studies were bred at Merck Research Laboratories in Kenilworth, NJ, or Taconic in
Germantown, NY, as described previously [21]. Male and female mice, counterbalanced across
groups, were singly housed in a plastic igloo with nesting material before the dosing began and
for the duration of the study. γ-Secretase inhibitor 15a was formulated in 20% hydroxypropyl-
ꢀ-cyclodextrin and dosed via oral gavage at a dose of 20 mg/kg. After a period of 3 hours, mice
were sacrificed by CO2 asphyxiation, total blood was collected from the posterior vena cava in
EDTA Microtainer® tubes (BD Biosciences), and plasma was isolated. Plasma Aꢀ40 was
quantified using biotin-4G8 and S-tag G2-10 using MesoScale Discovery (MSD) technology-
based sandwich immunoassay.
14

ACCEPTED MANUSCRIPT
4.2.3. Determination of rat plasma AUCs after an oral dosing
Two male Sprague-Dawley rats (Charles River, Co.) were used per each compound. The rats
were pre-cannulated (femoral artery) in order to facilitate precise blood sampling times, to
increase throughput, and to reduce the stress on the animals caused by serial bleedings. Rats
were fasted overnight prior to oral dosing at a dose of 10 mg/kg in a 5-mL/kg dose (compounds
were formulated in 0.5% methylcellulose). Blood was collected into heparin-containing tubes
serially from each animal at 0.5, 1, 2, 3, 4 and 6 h post-dosing (100 ꢀL per time point) and
centrifuged to generate plasma. The plasma samples were stored at -20 °C until analysis. The
concentrations of compounds in plasma was determined by a selective, but non-validated LC-
MS/MS assay using selected reaction monitoring (SRM) methods developed for each compound
prior to analysis of the plasma samples. The individual SRM transitions were based on a
fragmentation from the protonated molecule ([MH]+) to a characteristic product ion [22].
4.2.4. CYP inhibition assay
Assays for CYP inhibition were conducted using commercial recombinant 3A4, 2D6, and 2C9
enzymes (Thermo Fisher) by measuring appearance of fluorescent products from non-fluorescent
substrates:
3-cyano-7-ethoxy-coumarin,
dibenzyl
fluorescein,
and
7-methoxy-4-
trifluoromethylcoumarin,
7-methoxy-4-(aminomethyl)-coumarine,
7-benzyloxy-4-
trifluoromethylcoumarin. Data for the pre-incubation experiment was collected 30 min after
addition of the compound to the assay system. Known CYP inhibitors, sulfaphenazole and
ketoconazole were used as the positive controls. All compounds were inactive in 2D6 and 2C9
assays up to the maximal measured concentrations of 30 ꢀM.
4.3. Procedures for synthesis of 3,5-cis disubstituted morpholine compounds 15a-i
4.3.1. Synthesis of (1-((4-methoxybenzyloxy)methyl)cyclopropyl)methanol 2
15

ACCEPTED MANUSCRIPT
To solid LAH (16.6 g, 0.44 mol) in a 2 L flask at 0
°C was added dry THF (350 mL) slowly,
and then starting material compound 1 (32 g, 0.202 mol) in dry THF (50 mL) was added
dropwise through addition funnel at 0 ºC. Addition of the compound 1 was interrupted when too
much bubbling occurred. After addition was complete, the mixture was stirred at 0 ºC for 30
minutes. The reaction was then quenched by adding water dropwise at 0 ºC with reflux
condenser. The precipitate was aged for 3-4 h, filtered through celite. The precipitate was
washed with 1 L of THF. The filtrate was dried (MgSO4) and concentrated in vacuo to obtain a
diol product (17.9 g, 87%).
To NaH (7.71 g, 193 mmol, 60% in mineral oil) in DMF (300 mL) under nitrogen at 0 ºC, was
added the diol compound (17.9 g, 175 mmol) obtained from the above in DMF (200 mL)
dropwise. After 30 minutes, para-methoxybenzyl chloride (PMBCl) (30.2 g, 193 mmol) in DMF
(100 mL) was added dropwise over 30 minutes. The reaction was stirred at 0 ºC for 2 h. The
mixture was allowed to warm up to room temperature and stirred for another 4 h. TLC analysis
(30% EtOAc/Hexane) showed that the reaction was complete and had two sports. The reaction
mixture was quenched with ice, extracted with diethyl ether (3×150 mL). The combined organic
phases were washed with water and brine, dried (MgSO4) and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel, eluting with 0 – 30% EtOAc/Hexane in 1h
1
to obtain the desired mono PMB protected product (22.8 g, 58.6%). H NMR: 7.23 (2H, d, J =
8.60), 6.86 (2H, d, J = 8.62), 4.44 (2H, s), 3.79 (3H, s), 3.55 (2H, s), 3.42 (2H, s), 0.45 – 0.55
(4H, m). Mass Calcd: 222.13. m/z found: 223.3. The reaction also produce a bis PMB protected
by-product (9.9 g, 16.5%). However, the bis-PMB protected by-product can be converted to the
desired mono PMB protected product by the following procedure. To the by-product (9.9 g, 28.9
mmol) dissolved in a mixture of CH2Cl2/ water (200 mL/4 mL), was added DDQ (6.56 g, 28.9
16

ACCEPTED MANUSCRIPT
mmol) in CH2Cl2 (25 mL) dropwise over a period of 30 minutes. The reaction was stirred
overnight. TLC analysis (100% CH2Cl2) showed that the reaction was complete. The solid was
filtered off and was washed with CH2Cl2 (3 times). The organic phases from filtrate were washed
with water and brine, dried (MgSO4) and concentrated in vacuo. The residue was purified by
flash chromatography on silica gel, eluting with 0 – 30% EtOAc/Hexane to obtain the desired
product (5.3 g, 82.5%).
4.3.2. Synthesis of compound 1-((4-methoxybenzyloxy) methyl)cyclopropanecarbaldehyde 3
To oxalyl chloride (4.36 mL, 50 mmol) in CH2Cl2 (140 mL) at -78 ºC, was added DMSO (4.43
mL, 62.5 mmol) in CH2Cl2 (20 mL) dropwise. The mixture was stirred for 10 minutes, and then
compound 2 (5.56 g, 25 mmol) in CH2Cl2 (70 mL) was added slowly and stirred for another 10
minutes, followed by addition of Et3N (34.9 mL, 250 mmol, neat). Cooling bath was removed
and the reaction mixture was stirred for 30 minutes. The reaction was quenched with water,
extracted with CH2Cl2 (3 times). The combined organic phases were washed with water and
brine, dried (MgSO4) and concentrated in vacuo. The crude product was purified by flash
chromatography on silica gel, eluting with 0 - 20% EtOAc/Hexane in 1 h to afford compound 3
1
(5.06g, 92%). H NMR: 9.0 (1H, s), 7.25 (2H, d, J = 8.59), 6.87 (2H, d, J = 8.43), 4.48 (2H, s),
3.80 (3H, s), 3.66 (2H, s), 1.22 (2H, m), 1.10 (2H, m). Mass Calcd: 220.11. m/z found: 221.2.
4.3.3. Synthesis of 5-(1-((4-methoxybenzyloxy)methyl)cyclopropyl)imidazolidine-2,4-dione 4
To compound 3 (18.2 g, 0.083 mol) in 50% aqueous alcohol (100 mL) in a pressure flask, was
added KCN (8.1 g, 0.124 mol) and ammonium carbonate (23.9 g, 0.25 mol) and triethylamine
(50 mL, 0.35 mol). The flask was placed on the ultrasonicator for 2 minutes, and then stirred at
60 ºC overnight. TLC analysis (25%) EtOAc/Hexane showed that the reaction was complete.
The reaction mixture was extracted with CH2Cl2 (3 times). The combined organic phases were
17

ACCEPTED MANUSCRIPT
washed with water and brine, dried (MgSO4) and concentrated. The crude product was
crystallized from MeOH/CH2Cl2/Hexane to yield compound 4 (21.42g, 89%). 1H NMR: 7.21
(2H, d, J = 8.53), 6.87 (2H, d, J = 8.66), 4.42 (2H, d, J = 2.1), 3.92 (1H, s), 3.80 (3H, s), 3.77
(1H, d, J = 9.07), 2.96 (1H, d, J = 10.3), 0.44 – 0.84 (4H, m). Mass Calcd: 290.13. m/z found:
291.3.
4.3.4.
Synthesis
of
2-(tert-butoxycarbonylamino)-2-(1-((4-
methoxybenzyloxy)methyl)cyclopropyl) acetic acid 5
To a solution of hydantoin compound 4 (15.56 g, 53.67 mmol) in water (200 mL) was added
Ba(OH)2 (18.4 g, 107.3 mmol). The flask was fitted with a reflux condenser and the mixture was
refluxed overnight. TLC analysis (0.5% MeOH/ CH2Cl2) showed that the reaction was complete.
The hot reaction mixture was filtered, then the filtrate was boiled with (NH4)2CO3 (12.8 g) to
remove barium as insoluble BaCO3. Boiling was continued to decompose excess (NH4)2CO3.
1
The resulting filtrate was concentrated to give an amino acid intermediate (14.0g, 98%). H
NMR: 7.29 (2H, d, J = 8.75), 6.89 (2H, d, J = 8.75), 4.50 (1H, d, J = 11.37), 4.36 (1H, d, J =
11.36), 3.80 (1H, d, J = 9.83), 3.77 (3H, s), 3.15 (1H, s), 2.97 (1H, d, J = 9.83), 0.55 - 0.95 (4H,
m). Mass Calcd: 265.13. m/z found:266.2.
To a mixture of amino acid intermediate (18.0 g, 0.0679 mol) in dioxane (50 mL) and water
(50 mL) was added Boc2O (26.68g, 0.122mol) and TEA (19 mL, 0.136 mol). The reaction was
stirred at RT for overnight. TLC analysis (5% MeOH/ CH2Cl2) showed that the reaction was
complete. Dioxane was removed in vacuo. The mixture was extracted with EtOAc (3 times), and
organic phase was washed with citric acid (20%). The aqueous phase was back extracted with
EtOAc. The combined organic phases were dried (MgSO4) and concentrated. The crude product
was purified by flash chromatography on silica gel, eluting with 0-15% MeOH/CH2Cl2 to yield
18

ACCEPTED MANUSCRIPT
the compound 5 (21.0g, 85%). 1H NMR: 7.25 (2H, d, J = 8.68), 6.87 (2H, d, J = 8.64), 6.06 (1H,
bs), 4.55 (1H, d, J = 11.16), 4.43 (1H, d, J = 11.32), 3.95 (1H, m), 3.80 (3H, s), 2.89 (1H, d, J =
9.94), 0.805 (1H, m), 0.64 (2H, t, J = 6.64), 0.55 (1H, m). Mass Calcd: 365.18. m/z found: 366.4.
4.3.5.
Synthesis
of
2-cyclopropyl-2-oxoethyl
2-(tert-butoxycarbonylamino)-2-(1-((4-
methoxybenzyloxy) methyl)cyclopropyl)acetate 6
To a solution of compound 5 (21.38g, 0.0737mol) in MeOH (150 mL) and CH2Cl2 (50 mL)
was added slowly a solution of KOH (4.14g, 0.0737 mol) in MeOH (150 mL). The reaction is
concentrated after 2 minutes. The mixture was concentrated for several times with CH2Cl2 to
remove any trace amount of MeOH as much as possible. The resulting solid and KI (1.22g,
7.37mmol) are taken up in DMF (50 mL) and a solution of 2-bromo-1-cyclopropylethanone
(12.0 g, 0.0737 mol) in DMF (25 mL) was slowly added (dark brown color to yellow with
precipitate). The reaction mixture was stirred at RT for 4 h. TLC analysis (30% EtOAc/Hexane)
showed that the reaction was complete. The reaction mixture was extracted with ether (3 times).
The combined organic phases were washed with water and brine, dried (MgSO4) and
concentrated in vacuo. The crude product was purified by flash chromatography on silica gel,
eluting with 0 to 30% EtOAc/Hexane to give compound 6 (29.9g, 91%). 1H NMR: 7.27 (2H, d, J
= 8.66), 6.86 (2H, d, J = 8.66), 6.37 (1H, d, J = 7.92), 4.95 (1H, d, J = 16.6), 4.72 (1H, d, J =
16.6), 4.49 (1H, d, J = 11.2), 4.35 (1H, d, J = 11.2), 3.95 (1H, d, J = 11.4), 3.80 (3H, s), 3.67 (1H,
d, J = 7.84), 2.88 (1H, d, J = 10.34), 1.98 (1H, m), 1.58 (1H, s), 1.43 (9H, s), 1.09 (1H, m), 0.96
(2H, m), 0.89 (1H, m), 0.79 (1H, m), 0.64 (1H, m), 0.57 (1H, m). Mass Calcd: 447.23. m/z
found: 448.3.
4.3.6. Synthesis of 2-cyclopropyl-2-oxoethyl 2-(tert-butoxycarbonylamino)-2-(1-((tert-
butyldiphenylsilyloxy) methyl) cyclopropyl)acetate 7
19

ACCEPTED MANUSCRIPT
To compound 6 (30.2 g, 0.0675 mol) in CH2Cl2 (400 mL) and water (8 mL), was added DDQ
(16.85 g, 0.0742 mol) as solid in portions over a period of 30 minutes. The reaction was stirred
overnight. TLC analysis (100% CH2Cl2, use stain) showed that the reaction was complete. The
solid was filtered off and washed with CH2Cl2 (3 times). The combined organic phases were
washed with water and brine, dried (MgSO4) and concentrated. The residue was purified by
flash chromatography on silica gel to collect all fractions, eluting with 0 to 35% EtOAc/Hexane
to obtain product (22.1 g, 100%, not pure, taken as is).
To a solution of the above product (22.1 g, 67.5 mmol) in THF (160 mL), was added imidazole
(9.19 g, 135 mmol), followed by TBDPSCl (21 mL, 8 1mmol). The reaction was stirred
overnight. TLC analysis (25% EtOAc/Hexane) showed that the reaction was complete (less
polar, UV active product). The mixture was extracted with ether (3 times). The combined
organic phases were washed with water, saturated NaHCO3 and brine, dried MgSO4 and
concentrated. The crude product was purified by flash chromatography on silica gel, eluting with
1
(30%-35% EtOAc/Hexane) to afford compound 7 (27.0g, 71%). H NMR: 7.67 (4H, m), 7.39
(6H, m), 6.81 (1H, d, J = 7.78), 4.98 (1H, d, J = 16.57), 4.70 (1H, d, J = 16.56), 4.28 (1H, d, J =
11.18), 3.69 (1H, d, J = 7.72), 2.83 (1H, d, J = 10.59), 1.99 (1H, m), 1.44 (9H, s), 1.08 (9H, s),
0.94 (3H, m), 0.81 (3H, m), 0.53 (1H, m), 0.41 (1H, m). Mass Calcd: 565.29. m/z found: 566.5.
4.3.7.
Synthesis
of
(3S,5R)-3-(1-((tert-butyldiphenylsilyloxy)methyl)cyclopropyl)-5-
cyclopropylmorpholin-2-one 8
To a solution of 7 (27.0 g, 47.76 mmol) in CH2Cl2 (270 mL) was added TFA (90 mL) and
stirred for 2 h. TLC analysis (10% EtOAc/Hexane) showed that the reaction was complete.
Solvent was removed. The residue was redissolved in CH2Cl2 and toluene, solvent was removed
in vacuo. This step was repeated two more times to remove TFA. The residue was dissolved in
20

ACCEPTED MANUSCRIPT
CH2Cl2 (250 mL), washed with aqueous NaHCO3, water and brine, dried (MgSO4) and
concentrated to yield the cyclized imine product (22.1 g, 100%, crude).
To a solution of the cyclized imine product (12.6 g, 28.17 mmol) in CH2Cl2 (280 mL) was
added NaBH(OAc)3 (11.94 g, 56.34 mmol). The reaction was stirred at RT overnight. The
reaction mixture was diluted with saturated NaHCO3, extracted with CH2Cl2 (3 times). The
combined organic phases were washed with water and brine, dried (MgSO4) and concentrated.
The crude product was purified by flash chromatography on silica gel, eluting with 0 to 35%
EtOAc/Hexane to afford compound 8 (11.2 g, 88%). 1H NMR: 7.70 (4H, m), 7.40 (6H, m), 4.31
(1H, dd, J = 3.38, 10.52), 4.21 (1H, t, J = 10.30), 3.88 (1H, d, J = 10.55), 3.48 (1H, d, J = 10.56),
3.07 (1H, s), 2.27 (1H, m), 1.05 (9H, s), 0.87 (1H, m), 0.27 – 0.69 (8H, m). 13C NMR
(Chloroform-d) δ (ppm): 169.30, 135.69, 135.64, 133.44, 133.28, 129.67, 129.64, 127.65,
127.64, 77.25, 76.75, 73.42, 67.52, 63.57, 57.25, 26.78, 24.85, 19.19, 12.25, 10.54, 7.99, 2.75,
1.99. Mass Calcd: 449.24. m/z found: 450.4.
4.3.8.
Synthesis
of
(3S,5R)-3-(1-((tert-butyldiphenylsilyloxy)methyl)cyclopropyl)-4-(4-
chlorophenylsulfonyl)-5-cyclopropylmorpholin-2-one 9
To a solution of starting compound 8 (4.20 g, 9.35 mmol) in pyridine (80.0 mL) was added 4-
chlorobenzene-1-sulfonyl chloride as solid. The reaction was stirred at 85 ºC overnight. The
reaction was diluted with aqueous NaHCO3, extracted with CH2Cl2 (3 times), the combined
organic phases were washed with water and brine, dried (MgSO4) and concentrated. The residue
was purified by flash chromatography on silica gel, eluting with 0 to 30% EtOAc/Hexane in 1 h
1
to afford compound 9 (7.39g, 51.0%). H NMR: 7.63 (6H, m), 7.41 (8H, m), 4.68 (1H, s), 4.54
(1H, dd, J = 10.11, 12.48), 4.11 (1H, dd, J = 5.75, 12.07), 3.74 (1H, d, J = 10.99), 3.55 (1H, d, J
= 10.95), 2.91 (1H, m), 1.03 (9H, s), 0.90 (1H, ,m), 0.82 (1H, m), 0.5 – 0.65 (6H, m), 0.21 (1H,
m). Mass Calcd: 623.19. m/z found: 624.1.
21

ACCEPTED MANUSCRIPT
4.3.9.
Synthesis
of
(3S,5R)-3-(1-((tert-butyldiphenylsilyloxy)methyl)cyclopropyl)-4-(4-
chlorophenylsulfonyl)-5-cyclopropylmorpholine 10
To starting compound 9 (10.35 g, 16.58 mmol) and CaCl2 (11.0 g, 99.0 mmol) in THF (85.0
mL) and EtOH (127 mL) was added NaBH4 (3.14 g, 83.0 mmol) as solid. The reaction was
stirred at RT for 2 h. TLC analysis (25% EtOAc/Hexane) showed that the reaction was
incomplete. Stirring was continued until the less polar product was converted completely to the
more polar product. At 0 ºC, the reaction was quenched (exothermic) with citric acid (20%),
extracted with CH2Cl2 (3 times). The combined organic phases were washed with water and
brine, dried (MgSO4) and concentrated. The crude product was purified by flash chromatography
on silica gel, eluting with 0 to 30% EtOAc/Hexane in 1 h to afford the desired diol product
(7.14g, 69.1%). 1H NMR: 7.77 (2H, d, J = 8.84), 7.62 – 7.55 (4H, m), 7.46 – 7.36 (8H, m), 4.10
(1H, d, J = 10.26, 3.86 (1H, t, J = 10.11), 3.80 (2H, d, J = 6.01), 3.60 (1H, s), 3.40 (1H, t, 7.08),
2.86 (1H, d, J = 11.70), 2.75 (1H, q, J = 9.87), 0.95 (1H, m), 0.87 (1H, m), 0.67 (3H, m), 0.13 –
0.29 (3H, m), 0.04 (1H, m). Mass Calcd:627.22. m/z found:628.3.
To the diol starting material (7.14 g, 11.38 mmol) obtained from above and PPh3 (6.57 g,
25.04 mmol) in toluene (75.0 mL) was added DEAD (3.96 mL, 25.04 mmol) at RT. The
reaction was stirred at RT overnight. TLC analysis (25% EtOAc/Hexane) showed that the
reaction was complete with formation of a less polar product. The reaction mixture was diluted
with water, extracted with CH2Cl2 (3 times). The combined organic phases were washed with
water and brine, dried (MgSO4) and concentrated. The crude product was purified by flash
chromatography on silica gel, eluting with 0 to 35% EtOAc/Hexane to afford the product 10 (4.2
1
g, 59.7%). H NMR: 7.63 (2H, d, J = 8.81), 7.61 – 7.66 (4H, m), 7.40 (6H, m), 7.32 (2H, d, J =
8.87), 4.19 (1H, d, J = 4.29), 4.01 (1H, d, J = 10.16), 3.88 (1H, d, J = 10.16), 3.70 (1H, d, J =
22

ACCEPTED MANUSCRIPT
11.38), 3.55 (1H, d, J = 12.41), 3.11 (2H, m), 2.61 (1H, m), 1.05 (9H, s), 0.83 (3H, m), 0.45 (3H,
m), 0.11 (3H, m). Mass Calcd: 609.21. m/z found: 610.3.
4.3.10. Synthesis of (1-((3S,5R)-4-(4-chlorophenylsulfonyl)-5-cyclopropylmorpholin-3-
yl)cyclopropyl)methanol 11
To a starting compound 10 (4.2 g, 6.88 mmol) in THF (50 mL) was added TBAF (1 M in THF,
13.76 mmol) at RT. The reaction was stirred at RT for 2 h. TLC analysis (80% CH2Cl2/Hexane)
showed that the reaction was complete. The reaction mixture was quenched with water, extracted
with EtOAc (3 times). The combined organic phases were washed with water and brine, dried
(MgSO4) and concentrated. The crude product was purified by flash chromatography on silica
gel, eluting with 0 to 30% EtOAc/Hexane to obtain desired product 11 (2.43 g, 95%).
Compound 11 was further resolved by HPLC Chiral OJ column to obtain a single enantiomer.
[α]D20 = -125.84 (EtOH). 1H NMR: 7.75 (2H, d, J = 8.52), 7.52 (2H, d, J = 8.52), 4.68 (1H, dq, J
= 12.44, 2.2), 4.17 (1H, d, J = 4.28), 3.74 (1H, d, J = 11.50), 3.67 (1H, dd, J = 4.59, 10.43), 3.32
(1H, d, J = 12.61), 3.01 (1H, dd, J = 3.75, 11.46), 2.85 (1H, dd, J = 3.75, 10.54), 2.78 (1H, dd, J
= 4.44, 12.64), 2.64 (1H, dd, J = 10.49, 12.34), 1.65 (1H, m), 1.25 (1H, m), 0.51 – 0.81 (6H, m),
0.28 (1H, m). Mass Calcd: 371.10. m/z found: 372.3.
4.3.11.
Synthesis
of
(3R,5S)-4-(4-chlorophenylsulfonyl)-3-cyclopropyl-5-(1-
(iodomethyl)cyclopropyl)morpholine 12
The starting hydroxyl compound 11 (0.478 g, 1.29 mmol) in CH3CN (4 mL) and toluene (8
mL) was treated with Ph3P (406 mg, 1.55 mmol), I2 (393 mg, 1.55 mmol) and imidazole (263.5
mg, 3.87 mmol) at RT. The reaction was stirred at RT for 1 h. TLC analysis (30%
EtOAc/Hexane) showed that the reaction was complete. The reaction mixture was quenched with
NH4Cl, extracted with EtOEt (3 times). The combined organic phases were washed with
saturated NaHCO3 and brine, dried (MgSO4) and concentrated. The residue was purified by flash
23

ACCEPTED MANUSCRIPT
chromatography on silica gel, eluting with 0 to 80% CH2Cl2/Hexane in 1h to afford the desired
1
product (0.55g, 89%). H NMR: 7.82 (2H, d, J = 8.83), 7.51 (2H, d, J = 8.83), 4.33 (1H, d, J =
3.95), 4.12 (1H, d, J = 10.25), 3.74 (1H, d, J = 11.45), 3.46 (1H, d, J = 12.79), 3.17 (1H, dd, J =
3.88, 11.45), 3.13 (1H, d, J = 10.10), 3.09 (1H, dd, 4.30, 12.73), 2.73 (1H, dd, J = 3.67, 10.27),
1.97 (1H, m), 1.61 (1H, m), 0.99 (1H, m), 0.84 (1H, m), 0.72 (1H, m), 0.62 (1H, m), 0.52 (1H,
m), 0.38 (1H, m), 0.22 (1H, m). Mass Calcd: 481.00. m/z found: 482.1.
4.3.12. Synthesis of 2-(1-((3S,5R)-4-(4-chlorophenylsulfonyl)-5-cyclopropylmorpholin-3-
yl)cyclopropyl)acetonitrile 13
To a suspension of iodide 12 (0.55 g, 1.14 mmol) in CH3CN (10 mL, not soluble) was added
n-Bu4NCN (371 mg, 1.38 mmol) at RT. The reaction mixture was stirred at RT for 1.5 h. TLC
analysis (70% CH2Cl2/Hexane) showed that the reaction was complete. The reaction mixture was
diluted with water, and extracted with EtOAc (3 times). The combined organic phases were
washed with water and brine, dried (MgSO4) and concentrated. The crude product was purified
by flash chromatography, eluting with 0 to 30% EtOAc/Hexane in 40 minutes to give the
1
compound 13 (0.393g, 91%). H NMR: 7.71 (1H, d, J = 8.77), 7.51 (1H, d, J = 8.77), 4.06 (1H,
d, J = 4.17), 3.72 (1H, d, J = 11.47), 3.34 (1H, d, J = 12.81), 3.14 (2H, q, J = 17.46), 2.95 (1H,
dd, J = 3.74, 11.49), 2.86 (1H, dd, J = 4.35, 12.81), 2.78 (1H, dd, J = 3.43, 10.39), 1.63 (1H, m),
1.36 (1H, m), 0.91 (2H, m), 0.62 – 0.75 (4H, m), 0.30 (1H, m). Mass Calcd: 380.1. m/z found:
381.4.
4.3.13. Synthesis of 2-(1-((3S,5R)-4-(4-chlorophenylsulfonyl)-5-cyclopropylmorpholin-3-
yl)cyclopropyl)acetic acid 14
To the nitrile starting material 13 (0.393 g, 1.03 mmol) in CH2Cl2 (7 mL) was added DIBAL-H
(1.55 mL, 1.55 mmol) at -78 ºC dropwise. The reaction was stirred at -78 ºC for 4 h. TLC
analysis (10% EtOAc/Hexane) showed that the reaction was complete. The reaction mixture was
24

ACCEPTED MANUSCRIPT
quenched by slow addition of MeOH (2 mL) and stirred for 10 minutes. H2SO4 (1N, 2 mL) was
added and stirring continued for 45 minutes. Reaction mixture was extracted with CH2Cl2 (3
times), the combined organic phases were washed with water, brine, dried (MgSO4) and
concentrated. The crude product was purified by flash chromatography on silica gel, eluting with
1
0 to 20% EtOAc/Hexane to afford the aldehyde intermediate product (0.325g, 82%). H NMR:
9.83 (1H, s), 7.69 (2H, d, J = 8.81), 7.48 (2H, d, J = 8.81), 4.12 (1H, d, J = 4.40), 3.75 (1H, dq, J
= 18.43, 0.80), 3.73 (1H, d, J = 11.30), 3.36 (1H, d, J = 12.72), 3.01 (1H, dd, J = 3.78, 11.45),
2.84 (1H, dd, J = 3.60, 10.39), 2.78 (1H, dd, J = 4.43, 12.72), 1.83 (1H, d, J = 17.77), 1.72 (1H,
m), 1.29 (1H, m), 0.72 – 0.83 (2H, m), 0.65 (3H, m), 0.47 (1H, m), 0.28 (1H, m). Mass Calcd:
383.10. m/z found: 384.2.
To the aldehyde intermediate (0.325g, 0.85mmol) obtained above in t-BuOH (12 mL) and
water (3 mL) at 0 ºC, was added 2-methyl-2-butene (361ul, 3.4 mmol), followed by sodium
chlorite (246 mg, 2.72 mmol). The reaction was stirred at RT for 2 h. TLC analysis (30%
EtOAc/Hexane) showed that the reaction was complete. The reaction was quenched by saturated
NH4Cl (10 mL) and extracted with EtOAc (3×25 mL). The combined organic phases were
washed with water, brine, dried (MgSO4) and concentrated. The crude product was purified by
flash chromatography on silica gel, eluting with 0 to 30% EtOAc/Hexane to afford 14 (330 mg,
97%). 1H NMR: 7.72 (2H, d, J = 8.63), 7.46 (2H, d, J = 8.39), 4.24 (1H, d, J = 4.29), 3.73 (1H, d,
J = 11.74), 3.56 (1H, dd, J = 1.52, 17.72), 3.36 (1H, d, J = 12.74), 3.04 (1H, dd, J = 3.67, 11.32),
2.82 (2H, td, J = 12.55, 4.34), 1.92 (1H, d, J = 17.75), 1.72 (1H, m), 1.25 (1H, m), 0.63 – 0.79
(5H, m), 0.55 (1H, m), 0.27 (1H, m). Mass Calcd: 399.09. m/z found: 400.2.
4.3.14. General procedure for synthesis of final compounds 15a-i
25

ACCEPTED MANUSCRIPT
To the carboxylic acid 14 (60 mg, 0.15 mmol) in CH2Cl2 (2 mL) was added oxalyl chloride
(105 µl, 1.2 mmol), stirred for 20 min. Solvent was removed and the residue was placed on the
high vacuum for 1h. The crude product was dissolved in 1 mL of CH2Cl2 and added to a vial
contains amine (0.30 mmol) and Et3N (126 µl, 0.9 mmol) in CH2Cl2 (1 mL). The reaction was
stirred overnight. The reaction was diluted with aqueous NaHCO3, extracted with CH2Cl2 (3
times). The combined organic phases were washed with water and brine, dried (MgSO4) and
concentrated. The crude was purified by preparative TLC (7 % MeOH/ CH2Cl2) to obtain the
final product.
Compound 15a. Yield: 96%. 1H NMR: 7.70 (2H, d, J = 8.74), 7.45 (2H, d, J = 8.76), 4.31 (1H,
s), 3.68 (1H, d, J = 11.38), 3.35 – 3.65 (6H, m), 2.91 (1H, dd, J = 3.80, 11.41), 3.34 (2H, s), 2.74
(4H, m), 2.63 (1H, m), 2.50 (2H, m), 1.77 (1H, m), 1.70 (1H, d, J = 16.45), 1.27 (1H, m), 1.02
(6H, s), 0.63 – 0.83 (5H, m), 0.48 (1H, m), 0.28 (1H, m). 13C NMR (Chloroform-d) δ (ppm):
169.07, 139.60, 139.31, 129.88, 128.09, 68.51, 67.71, 66.12, 63.67, 58.46, 52.97, 46.12, 42.69,
38.85, 38.59, 20.32, 18.14, 15.04, 12.56, 11.16, 6.36, 4.80. Mass Calcd: 539.22. m/z found:
540.3.
1
Compound 15b. Yield: 71 %. H NMR: 7.71 (2H, m), 7.45 (2H, m), 4.70 (0.5 H, t, J = 6.60),
4.63 (0.5 H, t, J = 5.88), 4.46 (0.5H, d, J = 4.31), 4.29 (0.5 H, d, J = 4.23), 4.18 (0.5 H, t, J =
6.98), 4.05 (0.5 H, t, J = 5.37), 3.67 (1H, dd, J = 3.38, 11.42), 3.43 (2H, m), 2.80 (4H, m), 2.48
(4H, m), 1.51 – 2.15 (13H, m), 1.39 (2H, m), 1.25 (1H, m), 0.71 (6H, m), 0.52 (1H, m), 0.26
(1H, m). Mass Calcd: 575.26. m/z found: 576.1.
Compound 15c. Yield: 40%. 1H NMR: 7.72 (1H, d, J = 8.66), 7.69 (1H, d, J = 8.67), 7.45 (1H,
d, J = 8.71), 7.44 (1H, d, J = 8.67), 4.44 (0.5H, d, J = 4.21), 4.23 (1H, m), 4.08 (0.5 H, d, J =
12.92), 3.66 (2H, m), 3.58 (2H m), 3.49 (1H, m), 3.38 (1H, m), 3.23 (3H, m), 2.72 – 2.96 (4H,
26

ACCEPTED MANUSCRIPT
m), 2.53 (3H, m), 1.46 – 1.96 (5H, m), 1.27 (1H, m), 0.62 – 0.85 (6H, m), 0.45 (1H, m,), 0.28
(1H, m). Mass Calcd: 537.21. m/z found: 538.1.
1
Compound 15d. Yield: 64 %. H NMR: 7.71 (2H, m), 7.46 (2H, m), 4.44 (0.5H, d, J = 4.30),
4.14 – 4.29 (1.5 H, m), 3.35 – 3.70 (5H, m), 2.74 – 2.94 (3H, m), 1.61 – 2.06 (9H, m), 1.28 (1H,
m), 0.62 – 0.86 (6H, m), 0.47 (1H, m), 0.28 (1H, m). Mass Calcd: 493.18. m/z found: 494.3.
Compound 15e. Yield: 55 %. 1H NMR: 7.68 (2H, m), 7.46 (2H, m), 4.75 (1H, d, 15.45), 4.45
(1H, t, J = 4.6), 4.17 (2H, m), 3.68 (2H, m), 3.2 – 3.59 (3H, m), 2.72 – 2.96 (5H, m), 2.11 (3H,
s), 2.13 (2H, d, J = 1.4), 2.04 (1H, d, J = 6.60), 1.76 (1H, m), 1.30 (1H, m), 0.60 – 0.90 (6H, m),
0.45 (1H, m), 0.29 (1H, m). Mass Calcd: 535.19. m/z found: 536.3.
Compound 15 f. Yield: 72%. 1H NMR: 7.78 (2H, m), 7.16 (2H, m), 4.74 (0.5 H, d, J = 13.67),
4.67 (0.5 H, d, J = 13.63), 4.37 (0.5 H, d, J = 3.98), 4.27 (0.5 H, d, J = 4.22), 3.89 (1H, t, J =
14.31), 3.56 – 3.71 (2H, m), 3.37 (1H, d, J = 12.53), 3.07 (0.5H, t, J = 12.80), 2.91 (1.5 H, m),
2.77 (2H, m), 2.49 (5H, m), 1.78 (6H, m), 1.56 (4H, m), 1.41 (2H, m), 1.26 (1H, m), 0.64 – 0.81
(6H, m), 0.47 (1H, m), 0.27 (1H, m). Mass Calcd: 533.27. m/z found: 534.1.
1
Compound 15g. Yield: 74%. H NMR: 7.76 (2H, m), 7.16 (2H, m), 4.30 (1H, m), 3.92 (1H,
m), 3.34 – 3.80 (11H, m), 2.90 (1H, m), 2.76 (2H, m), 2.09 (3H, s), 1.78 (2H, s), 1.60 (1H, dd, J
= 6.90, 16.73), 1.29 (1H, m), 0.85 (1H, m), 0.75 (2H, m), 0.65 (2H, d, J = 7.85). Mass Calcd:
493.20. m/z found: 494.1.
1
Compound 15h. Yield: 87%. H NMR: 7.78 (2H, dd, J = 5.05, 8.82), 7.16 (2H, t, J = 8.50),
4.31 (1H, d, J = 4.19), 3.54 – 3.76 (7H, m), 3.44 (1H, m), 3.38 (1H, d, J = 12.69), 2.90 (1H, dd, J
= 3.81, 11.44), 2.75 – 2.79 (2H, m), 2.67 (1H, m), 2.57 (4H, t, J = 5.25), 2.47 (2H, m), 1.77 (1H,
m), 1.71 (1H, d, J = 16.45), 1.28 (1H, m), 0.64 – 0.83 (5H, m), 0.47 (1H, m). Mass Calcd:
495.22. m/z found: 496.3.
27

ACCEPTED MANUSCRIPT
1
Compound 15i. Yield: 63%. H NMR: 7.77 (2H, m), 7.15 (2H, t, J = 8.48), 4.00 – 4.84 (2H,
m), 3.30 – 3.71 (6H, m), 2.60 – 2.97 (6H, m), 2.15 – 2.51 (2H, m), 1.74 (3H, m), 1.22 – 1.48
(4H, m), 1.03 (3H, s), 0.97 (3H, s), 0.77 (2H, m), 0.64 (3H, m), 0.47 (1H, m), 0.27 (1H, m). Mass
Calcd: 537.27. m/z found: 538.3.
4.4. Procedures for synthesis of fused ring morpholine compounds 25a-e
4.4.1. Synthesis of 2-oxocyclopentyl 2-(tert-butoxycarbonylamino)-2-(1-((4-methoxybenzyloxy)
methyl)cyclopropyl) acetate 16
The same procedure as in the synthesis of compound 6 was used for compound 16. The
product was purified by flash chromatography, eluting with 0 – 30% EtOAc/Hexane to give a
mixture of two diastereomers (Total yield 71.0%). The mixture was separated by crystallization
from hot diethyl ether to give a white crystal and a yellow oil compound with a 1:1 ratio of two
diastereomers. Diastereomer one 16a.(35%, white solid, mp. 63-66 °C). 1H NMR: 7.27 (2H, d, J
= 8.66), 6.86 (2H, d, J = 8.66), 6.25 (1H, d, J = 7.69), 5.02 (1H, t, J = 8.80), 4.79 (1H, s), 4.47
(1H, d, J = 11.27), 4.35 (1H, d, J = 11.25), 3.79 (3H, s), 3.64 (1H, d, J = 7.84), 2.93 (1H, d, J =
10.38), 2.29 (2H, m), 2.08 (1H, m), 1.84 (2H, m), 1.43 (9H, s), 0.87 (1H, m), 0.78 (1H, m), 0.60
(1H, m), 0.54 (1H, m). Mass Calcd: 477.23. m/z found: 478.3. Diastereomer two 16b (36%,
yellow oil). 1H NMR: 7.29 (2H, d, J = 8.56), 6.87 (2H, d, J = 8.36), 6.35 (1H, d, J = 8.06), 5.21
(1H, t, J = 10.03), 4.53 (1H, d, J = 11.22), 4.35 (1H, d, J = 11.24), 4.01 (1H, d, J = 10.28), 3.80
(3H, s), 3.64 (1H, d, J = 8.13), 3.47 (1H, q, J = 7.55, 14.03), 2.84 (1H, d, J = 10.39), 2.21 – 2.41
(3H, m), 2.09 (1H, m), 1.87 (2H, m), 1.44 (9H, s), 1.21 (1H, t, J = 7.02), 0.79 (2H, m), 0.63 (1H,
m), 0.53 (1H, m). Mass Calcd: 477.23. m/z found: 478.3.
4.4.2 Synthesis of 2-oxocyclopentyl 2-(tert-butoxycarbonylamino)-2-(1-((tert-
butyldiphenylsilyloxy) methyl)cyclopropyl) acetate 17
28