4-Substituted 2-amino-3,4-dihydroquinazolines with a 3-hairpin turn side chain as novel inhibitors of BACE-1

Article abstract of DOI:10.1016/j.bioorg.2019.103135

Herein, we report the identification, design, and synthesis of a series of 4-substituted 2-amino-3,4-dihydroquinazolines with hairpin turn side chains as novel inhibitors of BACE-1. The dihydroquinazoline derivatives were rationally designed by modifying the amide group and relocating the α -hydrophobic substituent on the hairpin turn side chain of lead compound 2 to the C4-position on the 3,4-dihydroquinazoline scaffold to facilitate interactions with the S₁, S₂ and S₁′ subsites of BACE-1. Among these derivatives, two compounds exhibited potent BACE-1 inhibitory activity: 4-methyl-substituted (22a, BACE-1 CFA IC₅₀ = 0.38 μM; BACE-1 WCA IC₅₀ = 0.14 μM) and 4-cyclohexylmethyl-substituted (22b, BACE-1 CFA IC₅₀ = 0.49 μM; BACE-1 WCA IC₅₀ = 0.14 μM) 2-amino-3,4-dihydroquinazoline, each bearing a side chain of N-cyclohexyl-N-((1-methyl-1H-pyrazol-4-yl)methyl amide. The results suggest that the structural modifications maintain the hairpin turn topology similar to that of compound 2 and provide an additional interaction with the S₂ subsite.

Full text of DOI:10.1016/j.bioorg.2019.103135

Accepted Manuscript
4
-Substituted 2-amino-3,4-dihydroquinazolines with a 3-hairpin turn side chain
as novel inhibitors of BACE-1
Ajit Dhananjay Jagtap, Nagendra B. Kondekar, Pei-Yun Hung, Chen-En Hsieh,
Chia-Ron Yang, Grace Shiahuy Chen, Ji-Wang Chern
PII:
S0045-2068(19)30709-6
https://doi.org/10.1016/j.bioorg.2019.103135
103135
DOI:
Article Number:
Reference:
YBIOO 103135
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
7 May 2019
13 July 2019
19 July 2019
Please cite this article as: A. Dhananjay Jagtap, N.B. Kondekar, P-Y. Hung, C-E. Hsieh, C-R. Yang, G. Shiahuy
Chen, J-W. Chern, 4-Substituted 2-amino-3,4-dihydroquinazolines with a 3-hairpin turn side chain as novel
inhibitors of BACE-1, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103135
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4
-Substituted 2-amino-3,4-dihydroquinazolines with a 3-hairpin turn
side chain as novel inhibitors of BACE-1.
Ajit Dhananjay Jagtap,a,b,† Nagendra B. Kondekar, † Pei-Yun Hung, Chen-En Hsieh,
a,
b
c
a
c,
a,
Chia-Ron Yang, Grace Shiahuy Chen, * and Ji-Wang Chern *
aSchool of Pharmacy, College of Medicine and Center for Innovative Therapeutics
Discovery, National Taiwan University, No. 33, Lin Sen South Road, Taipei 10050,
Taiwan
bAnnJi Pharmaceutical Co., Ltd., 18, Siyuan Street, Taipei 10087, Taiwan
cDepartment of Applied Chemistry, Providence University, No. 200, Section 7, Taiwan
Boulevard, Taichung 43301, Taiwan
1

Abstract
Herein, we report the identification, design, and synthesis of a series of 4-substituted 2-
amino-3,4-dihydroquinazolines with hairpin turn side chains as novel inhibitors of
BACE-1. The dihydroquinazoline derivatives were rationally designed by modifying the
amide group and relocating the -hydrophobic substituent on the hairpin turn side chain
of lead compound 2 to the C4-position on the 3,4-dihydroquinazoline scaffold to
facilitate interactions with the S , S and S ′ subsites of BACE-1. Among these
1
2
1
derivatives, two compounds exhibited potent BACE-1 inhibitory activity: 4-methyl-
substituted (22a, BACE-1 CFA IC = 0.38 M; BACE-1 WCA IC = 0.14 M) and 4-
5
0
50
cyclohexylmethyl-substituted (22b, BACE-1 CFA IC = 0.49 M; BACE-1 WCA IC =
5
0
50
0
.14 M) 2-amino-3,4-dihydroquinazoline, each bearing a side chain of N-cyclohexyl-N-
((1-methyl-1H-pyrazol-4-yl)methyl amide. The results suggest that the structural
modifications maintain the hairpin turn topology similar to that of compound 2 and
provide an additional interaction with the S subsite.
2
Keywords: Alzheimer’s disease, BACE-1, non-peptidic aspartyl protease inhibitors, 2-
amino-3,4-dihydroquinazolines, structure-activity relationship
Abbreviations: AD, Alzheimer’s disease; BACE-1, β-site amyloid precursor protein
cleaving enzyme 1; Aβ, β-amyloid; APP, amyloid precursor protein; iPr, isopropyl; cPr,
cyclopropyl; Cy, cyclohexyl; Bn, benzyl; CFA, cell free assay; WCA, whole cell assay.
Author Information
2

*
Corresponding authors:
JWC: Phone: +886-2-23939462. Fax: +886-2-23934221. E-mail: jwchern@ntu.edu.tw
GSC: Phone: +886-4-26524370. Fax: +886-4-26327554. E-mail: grace@pu.edu.tw
†
These authors contributed equally
1
. Introduction
Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized
by slow progression of cognitive decline and dementia, ultimately leading to complete
debilitation and death [1]. AD afflicts more than 44 million people worldwide, and the
number of affected individuals is expected to be triple by the year 2050 [2]. Indeed, based
on the projected prevalence and unmet medical need of AD, this disease is becoming a
major socioeconomic burden.
All current clinically approved drugs for AD treatment target symptoms and improve
quality of life rather than alter disease progression [1]. However, these drugs have
relatively short-term and limited benefits, thus highlighting urgent need for novel disease
modifying agents [3, 4]. Extracellular β-amyloid (Aβ) plaques and intracellular hyper-
phosphorylated neurofibrillary tangles of tau protein in the brain are the two major
neuropathological hallmarks of AD [5]. The preeminent amyloid cascade hypothesis
states that the observed increase in Aβ level in AD—either through its excessive
production via dysregulated metabolism of amyloid precursor protein (APP) or defective
clearance—initiates a cascade of events that leads to neurodegeneration and clinical AD
[6, 7]. The two proteases β- and -secretase are responsible for the proteolytic processing
of APP [8]. The enzyme β-site amyloid precursor protein cleaving enzyme 1 (BACE-1,
Asp2 or memapsin) is a membrane-associated aspartyl protease that cleaves the
3

membrane-bound APP at an extramembrane site to yield a soluble N-terminal
ectodomain of APP (sAPPβ) and the C-terminal fragment C99 [9]. Subsequent to β-
secretase cleavage, γ-secretase, a member of a unique class of intramembrane aspartyl
proteases, cleaves the C99 fragment generated from APP by BACE-1 at an
intramembrane site, generating Aβ isoforms (Aβx-40 and Aβx-42) and releasing them
from the membrane [10]. Thus, the inhibition of β- and/or γ-secretase provides attractive
candidates as a disease-modifying, rather than palliative, treatment for AD [11]. γ-
Secretase functions in a variety of physiological processes, and therefore inhibitors of this
enzyme have several mechanism-based toxicity drawbacks owing to a deficiency of
selectivity. On the contrary, the ability to safely and effectively inhibit BACE- which
catalyzes the rate-limiting step in APP processing to Aβ, has become the prevailing
therapeutic approach over the last two decades [12-14]. Moreover, a coding mutation
(A673T) in the gene APP has been shown to have protective effects against AD, thus
providing a proof of principle for the hypothesis that reducing the cleavage of APP may
protect against AD [15].
First-generation BACE-1 inhibitors are transition-state analogs such as
hydroxyethylene, hydroxyethylamines, and aminoethylenes that interact with the catalytic
aspartates Asp32 and Asp228. Although peptidic inhibitors have been proved to be potent
inhibitors of BACE-1, they have poor pharmacodynamics owing to poor oral absorption
and low exposure in the brain. Thus, the development of potent, non-peptidic small-
molecule BACE-1 inhibitors has aroused great interest [16]. Although the development
of drugs to treat AD remains a challenge, various BACE-1 inhibitors such as verubecestat,
4

elenbecestat, lanabecestat, JNJ-54861911, and CNP520 have advanced to Phase III
clinical trials, lending support for BACE-1 as a promising target (Figure 1) [17, 18].
An aspartic protease inhibitory chemotype bearing a 2-amino-3,4-dihydroquinazoline
skeleton, exemplified by 1 (K = 0.9 M, Figure 2), was discovered by high-throughput
i
screening for the inhibition of BACE-1 [19]. An X-ray co-crystal structure of 1 bound to
BACE-1 revealed key pharmacophore elements for target engagement, namely a cyclic
amidine motif interacting with catalytic aspartates and a hairpin turn side chain allowing
the N-cyclohexyl substituent to fit in the S binding pocket of BACE-1. Further
1
modification of 1 yielded (S)-2, having an α-hydrophobic cyclohexyl substituent at the
side chain that could, in concert with the occupancy of subsite S , occupy the S ' subsite
1
1
of BACE-1 and thereby greatly enhance the BACE-1 inhibitory potency (S-2, K = 11 nM,
i
Figure 2) [19]. Macrocyclic 2-amino-3,4-dihydroquinazoline-based BACE-1 inhibitor 3
further demonstrated that introduction of a hydrophobic substituent to fill the S ' pocket
1
improved the inhibitory potency [20]. Moreover, an auxiliary thiazole or pyrazole
substituted at the amidine resulted in an additional binding interaction with Thr232 in the
S subsite [21]. Therefore, the S , S ', and S binding elements represent a non-peptidic
2
1
1
2
aspartyl protease pharmacophore.
We envisioned that a hydrophobic substituent at the C4-position of quinazoline
scaffold might fit in the S ′ pocket as well. Geometry optimization on lead compound 2
1
and the designed 4-substituted quinazoline derivatives I was carried out computationally
at the DFT/B3LYP/6-31G(d) level [22-24]. To our expectations, reallocation of
cyclohexyl group to the C4 position (Ia) did not deviate a lot from the -position at the
side chain of compound 2 (Figure 3). Cyclohexylmethyl group, enlongated by one carbon,
5

at the C4 position (Ib) had an even better superimposition of cyclohexyl group to that of
compound 2. These results verified our design concept. Taking the structure of 1 as a
template, 4-substituted 2-amino-3,4-dihydroquinazolines were evaluated computationally
based on the 2-bound BACE-1 crystal structure [19]. As expected, 2-amino-3,4-
dihydroquinazoline core formed hydrogen bonds to the catalytic aspartyl residues Asp32
and Asp228. As to the S ' pocket, a variety of substituents such as methyl, cyclopropyl,
1
cyclohexyl, and even phenyl on the 4-position are tolerable. Elongated by one carbon
such as 4-cyclohexylmethyl or 4-benzyl groups are more fully filled into S ' pocket.
1
Herein, we report the synthesis and evaluation of a series of 3,4-disubstituted 2-amino-
3
,4-dihydroquinazoline derivatives designed by reallocating the -hydrophobic
substituent at the side chain of lead compound 2 to the C4-position on quinazoline
scaffold to occupy the S ' pocket incorporating N-pyrazolyl amidine to interact the S -
1
2
subsite (Figure 2).
2
. Results and discussion
2
.1. Chemistry
Cyclohexylcyclopropylamine (4) was prepared by the reductive amination reaction of
cyclohexanone and cyclopropylamine in the presence of Adam’s catalyst (Scheme 1) [25].
The formylation of N-methylpyrazole via the Vilsmeier–Haack reaction provided (N-
methylpyrazol-4-yl)methylcarbaldehyde (5), which underwent condensation with
cyclohexylamine and subsequent in situ reduction with NaBH to afford the secondary
4
amine 6 with an (N-methylpyrazol-4-yl)methyl group. Then, N-protected 4-aminobutyric
6

acid, resulting from condensation of 4-aminobutyric acid and phthalic anhydride, was
coupled with cyclohexylmethylamine/4/6 to give the corresponding amides 7a-c,
respectively. A subsequent phthalimide deprotection provided the desired N-alkyl-N-
cyclohexyl-4-aminobutanamides 8a-c (Scheme 1).
To construct the dihydroquinazoline skeleton, commercially available 5-fluoro-2-
nitrobenzaldehdye was subjected to a substitution reaction with phenol to give 2-nitro-5-
phenoxybenzaldehdye (9), which was reacted with Grignard reagents to afford the
corresponding alcohols 10a,b; this latter coupling reaction suffered from poor yields or
impurities, presumably owing to the 2-nitro group of benzaldehyde. An alternative route
was pursued in which the nitro group of 9 was initially reduced and protected as its tert-
butyl carbamate derivative. Thus, 2-nitrobenzaldehyde 9 was reduced to 2-nitrobenzyl
alcohol 11 using NaBH . The nitro group of 11 was then reduced using iron under acidic
4
condition followed by protection with (Boc) O to give tert-butyl carbamate 12 in
2
quantitative yield. Oxidation of benzyl alcohol 12 to benzaldehyde 13 was carried out
using the hypervalent oxidizing agent 2-iodoxybenzoic acid [26] (Scheme 2). This
alternate route achieved higher yields and improved product purity. N-Boc-2-
aminobenzaldehyde 13 and Grignard reagents underwent nucleophilic addition reactions
to efficiently afford the secondary benzyl alcohols 14a and 14c-g.
The trifluoromethyl derivative 14b was obtained by treating 13 with
trimethyl(trifluoromethyl)silane [27]. For 4-(2-hydroxylethyl) derivatives, the hydration
of allyl alcohol 14e by the hydroboration-oxidation reaction provided a mixture of 1,2-
diol 15 and 1,3-diol 16 with the anti-Markovnikov product 16 as the major product. The
7

primary alcohol of 16 was selectively protected using tert-butyldimethylsilyl chloride to
give alcohol 14h (Scheme 2).
Two approaches were used to convert benzyl alcohols 10a,b and 14a-h into suitable
electrophiles for nucleophilic substitution reactions with amines 8a-c. One approach was
the conversion of the alcohol to benzyl bromide using phosphorous tribromide, and the
other was derivatization of the alcohol to its mesyl ester using mesyl chloride. Treatment
of these bromo derivatives and mesyl esters with amines 8a-c under basic condition
afforded the corresponding compounds 17a-h, 18a-d, and 19a-c (Scheme 3). Partial
hydrogenation of 4-ethynyl compounds 17h and 18d using Lindlar’s catalyst [28, 29]
gave 4-vinyl intermediates 17i and 18e, respectively. Complete hydrogenation of 16h
yielded the 4-ethyl intermediate 17j.
Nitro compounds 17a/17f/18a were reduced with iron in AcOH to afford the
corresponding anilines, which was reacted with cyanogen bromide to provide the 2-
aminodihydroquinazoline derivatives 20a, 20f, and 21a. The N-Boc compounds 17b-e,
1
7g-j, 18b-e, and 19a-c were Boc-deprotected under acidic condition followed by
reaction with cyanogen bromide to achieve the corresponding 2-
aminodihydroquinazoline derivatives 20b-e, 20g-j, 21b-e, and 22a-c (Scheme 3).
2
2
.2. Biology
.2.1. Cell Free Assay (Biochemical Assay)
The prepared 2-amino-3,4-dihydroquinazoline derivatives 20–22 were screened for
BACE-1 inhibition in cell free assay (CFA) by the Reaction Biology Corporation [30].
The reported K values of the racemate and S enantiomer of lead compound 2 were 30
i
8

nM and 11 n, respectively [19]. The other well-known BACE-1 inhibitor, β-secretase
inhibitor IV (Calbiochem), has an reported IC50 value of 15 nM [31]. Thus, (S)-2,
prepared in-house, and commercially available β-secretase inhibitor IV were taken as
reference compounds for assays. We measured their IC values as 0.14 M and 0.18 M,
5
0
respectively, which are >10 fold higher than the reported values.
At the outset, for compounds with an N-cyclohexyl-N-methyl amide side chain, i.e.,
present as the hairpin side chain of 1 and 2, 2-amino-3,4-dihydroquinazoline with a 4-
methyl group (20a) gave an IC value of 0.75 μM for BACE-1. This implies equipotent
5
0
inhibition of BACE-1 compared with the originally screened compound 1 (reported K of
i
0
.9 M) [19] and is comparable to the lead compound (S)-2 (IC = 0.14 μM). The 4-
5
0
trifluoromethyl (20b, IC = 1.55 M), 4-isopropyl (20c, IC = 1.77 μM), 4-cyclohexyl
5
0
50
(
20d, IC = 1.36 μM), and 4-ethyl (20j, IC = 1.34 μM) substituents all resulted in a
50 50
decrease of BACE-1 inhibition as compared with the 4-methyl 20a. Use of a -system of
-ethynyl (20h, IC = 1.18 μM) or 4-vinyl (20i, IC =1.24 μM) did not improve the
4
5
0
50
ability to inhibit BACE-1. Nevertheless, introduction of a hydroxyl group such as 4-(2-
hydroxyethyl) (20g, IC50 = 1.03 μM) improved the potency slightly. Surprisingly,
reallocation of the cyclohexyl group from the -carbon at the side chain of 2 to the C4 of
the quinazoline skeleton (20d, IC = 1.36 μM) resulted in 10 fold decrease in inhibition
5
0
of BACE1 than that of (S)-2. Indeed, insertion of a methylene (20e, IC = 0.63 μM)
5
0
between cyclohexyl and the 3,4-dihydroquinazoline ring greatly improved BACE-1
inhibition. It is reported that variety of substituents viz. methyl, ethyl, cyclopropyl etc. are
well tolerated at S1′ subsite with slight difference in BACE1 inhibitory activity[31, 32].
The SAR of compounds 20a-j demonstrated that only methyl (20a, 21a and 22a) and
9

cyclohexylmethyl group (20e, 21b and 22b) were tolerated at the C4 position of 2-amino-
3
3
,4-dihydroquinazoline. A C4-benzyl substituent (20f) was essentially inactive (IC =
50
1.4 M). We reasoned that the C4-benzyl group may not fit in the S ′ pocket. These
1
results are in accord with the aforementioned computational study. In fact, 20d and 20e
are racemic forms of the model compounds Ia and Ib, respectively. The cyclohexyl
group of 20e (Ib) possessed a better superimposition to that of lead compound 2. The
chair conformation of the cyclohexyl of C4-cyclohexylmethyl group may fit better in the
S1' pocket, leading to an increase in potency, whereas the phenyl of C4-benzyl group is
flat and thus probably cannot fully interact with the S ' pocket[32]
1
To optimize interactions with the S pocket, further derivatization was carried out at
2
the amide of the side chain, i.e., N-cyclohexyl-N-cyclopropyl amide (21a-d) and N-
cyclohexyl-N-((1-methyl-1H-pyrazol-4-yl)methyl amide (22a-c). Among these,
consistent with results obtained for the N-cyclohexyl-N-methyl amide, the compounds
with 4-methyl substituents (21a, IC50 = 0.52 μM; 22a, IC50 = 0.38 M) and 4-
cyclohexylmethyl substituents (21b, IC = 0.57 μM; 22b, IC = 0.49 M) exhibited
5
0
50
better inhibition. Evidently, substitution of the N-((1-methyl-1H-pyrazol-4-yl)methyl
group for the N-methyl group on the amide improved the inhibition, suggesting an
additional interaction with the S pocket.
2
2
.2.2. Whole Cell Assay
The selected analogs 20a, 20e, 21a, 21b, 22a, 22b (BACE-1 CFA, IC50 < 1 M) and
lead compound (S)-2 were evaluated for cellular inhibition of -secretase in SH-SY5Y
cells overexpressing human APP . As shown in Table 1, all of the tested analogues
6
95
1
0

show similar potency in the whole cell assay (WCA) relative to their BACE-1 potency in
CFA. Consistent with their BACE-1 CFA potency, the compounds with N-cyclohexyl-N-
(
(1-methyl-1H-pyrazol-4-yl)methyl amide side chain containing 4-methyl (22a, IC =
50
0
.52 μM) and 4-cyclohexylmethyl substituents (22b, IC = 0.38 M) exhibited better
5
0
inhibition of BACE-1 in SH-SY5Y cells than compounds with N-cyclohexyl-N-
cyclopropyl (21a, IC = 0.58 μM; 21b, IC = 0.60 M) and N-cyclohexyl-N-methyl
5
0
50
(
20a, IC = 0.65 μM; 20e, IC = 0.64 M) amide side chain. The analogs 20a, 20e, 21a,
5
0
50
2
1b, 22a, 22b were evaluated for cytotoxicity against wild type SH-SY5Y cells using
MTT assay. All of the tested compounds showed 3-4 fold less cytotoxicity (CC = 8.27
5
0
to 12.53 M) than that of the lead compound (S)-2 (CC = 3.08 M) against SH-SY5Y
5
0
cells (Table 1).
2
.3. Molecular Modelling
To gain an insight into the ligand-binding site interactions, compound 22b was docked
into the active site of BACE1 based on its co-crystal structure with 2 (PDB ID: 2Q15) [19,
3, 34]. As expected, 2-amino-3,4-dihydroquinazoline formed hydrogen bonds to the
3
catalytic aspartyl residues Asp32 and Asp228 (Figure 4A). As to the S ' pocket, 4-
1
cyclohexylmethyl of (R)-22b fully filled in the S ' pocket (Figure 4B). The virtually
1
superimposable binding modes of lead compound 2 and 22b (Figure 4C) demonstrated
that the reallocation of cyclohexylmethyl group at the C4 position of quinazoline
maintain the hydrophobic interaction in S1’ pocket and hairpin turn topology similar to
the lead compound 2.
1
1

3
. Conclusion
Our results demonstrate that a C4-methyl or C4-cyclohexylmethyl substituent on the
,4-dihydroquinazoline with a side chain possessing an N-cyclohexyl-N-((1-methyl-1H-
3
pyrazol-4-yl)methyl amide represents a novel class of BACE-1 inhibitors. In our study,
even racemates of 22a and 22b exhibited BACE-1 inhibition comparable with that of the
enantiomerically pure (S)-2. It has been shown that chirality plays an important role in
BACE-1 inhibition because the potency of (S)-2 is 60% greater than that its racemate. We
posit that compounds 22a and 22b exhibit a similar potency of BACE-1 inhibition
because moieties of these compounds can occupy the S1′ pocket.
4
4
4
. Experimental
.1. Chemistry
.1.1. General Procedures. All reactions were carried out under an argon or nitrogen
atmosphere with dry solvents, unless otherwise noted. An SP-1 stand-alone solvent
purification system (LC Technology Solutions) was used to dry solvents, which then
contained <10 ppm water as determined by Karl-Fischer moisture analysis. Purchased
reagents were used without further purification. Reactions were magnetically stirred and
monitored by subjecting reaction mixture to thin-layer chromatography on 0.25 mm
Merck silica gel plates (60F-254) followed by visual inspection under UV light or
KMnO staining solution. Silica gel (60, academic grade, 0.040–0.063 mm) was used for
4
flash column chromatography. NMR spectra were recorded with Bruker 300, 400, or 600
MHz instruments using residual non-deuterated solvent as an internal reference. High-
resolution mass spectra (HRMS) were recorded with a Esquire 2000 spectrometer.
1
2

Electrospray ionization MS (ESI-MS) spectra were recorded with an Esquire-Agilient-
B1-20 Hystar LC-SPE. The purity of final compounds was confirmed by analytical
HPLC to be >95%.
4
.1.2. Cyclohexylcyclopropylamine (4). To a solution of cyclohexanone (0.5 g, 5.1
mmol) in EtOH (25 mL) was added cyclopropylamine (0.35 mL, 5.1 mmol) and platinum
oxide (0.115 g, 10 mol%). The resulting solution was hydrogenated with H gas at 1 atm.
2
When the uptake of hydrogen was complete (4 h), the solution was filtered, and the
filtrate was evaporated under reduced pressure to afford 4 (0.47 g, 66%), which was used
without purification. The analytically pure sample was obtained by vacuum distillation.
bp 114 –118 °C [lit.[35] bp 114 –118 °C].
4
.1.3. 1-Methyl-1H-pyrazole-4-carbaldehyde (5). A solution of POCl (2.61 mL, 28
3
mmol) in DMF (2.16 mL, 28 mmol) was stirred at 8 °C for 2 h, and the resultant viscous
solution was warmed to 80 °C. To this solution was added N-methylpyrazole (1 g, 12
mmol), and it was stirred at 80 °C for 1 h, at 100 °C for 3 h, and at 115 °C for 1 h. The
solution was then cooled to room temperature and quenched by addition of 60 ml of ice-
cold water. The reaction mixture was stirred for 24 h and then neutralized to pH 7 using 1
N NaOH and extracted with chloroform (3 × 30 mL). The combined organic layer was
washed with brine (30 mL) and dried over MgSO . After filtration, the filtrate was
4
concentrated in vacuo. The residue was purified by column chromatography to afford 5
1
(
0.68 g, 50%), R = 0.2 Hex/EA 7:3. H NMR (400 MHz, CDCl ) δ 9.80 (s, 1H), 7.91 (s,
f
3
+
1
H), 7.89 (s, 1H), 3.93 (s, 3H). MS (ESI) m/z 110.8 [M+H] . HRMS calcd for C H N O
5 6 2
+
[
M+H] 111.0558, found 111.0556.
1
3

4
.1.4. Cyclohexyl-((1-methyl-1H-pyrazol-4-yl)methyl)amine (6). To a solution of 5 (4.91
g, 44.6 mmol) in methanol (45 mL) was added dropwise a solution of cyclohexylamine
5.11 mL, 44.6 mmol) in methanol (45 mL), and the reaction mixture was stirred at room
temperature for 2 h. After the formation of the imine, the reaction mixture was cooled to
°C, and sodium borohydride (6.76 g, 179 mmol) was added portion-wise. The reaction
(
0
mixture was then heated at 80 °C for 1 h, then cooled to room temperature and
evaporated in vacuo. The residue was dissolved in ethyl acetate and washed with 1 N
NaOH and brine (30 mL) and dried over MgSO . After filtration, the filtrate was
4
1
concentrated in vacuo to afford 6 (8.2 g, 95%). H NMR (400 MHz, CDCl ) δ 7.38 (s,
3
1
H), 7.29 (s, 1H), 3.84 (s, 3H), 3.66 (s, 2H), 2.50–2.42 (m, 1H), 1.89–1.59 (m, 6H), 1.25–
1
3
1
.12 (m, 5H). C NMR (100 MHz, CDCl ) δ 138.7, 128.8, 120.7, 56.0, 40.7, 38.8, 33.3,
3
+
+
2
6.0, 24.9. MS (ESI) m/z 194.0 [M+H] . HRMS calcd for C H N [M+H] 194.1657,
11 19 3
found 194.1653.
4
.1.5. General procedure I for the synthesis of 7a-c. Phthalic anhydride (5.92 g, 40
mmol) and 4-aminobutanoic acid (4.12 g, 40 mmol) were heated at 150 °C for 6 h. After
cooling, the resulting solid was dissolved in CH Cl (50 mL) and washed with 0.1 N HCl
2
2
(
3 × 20 mL). The organic layer was dried over MgSO and evaporated in vacuo to yield
4
N-(4-hydroxycarbonylbutyl)phthalimide quantitatively as white solids. Mp 116–118 °C
1
[
lit.[36] mp 117 °C]. H NMR (CDCl , 200 MHz) δ 7.85–7.80 (m, 2H), 7.79–7.64 (m, 2
3
1
3
H), 3.74 (t, J = 7.0 Hz, 2H), 2.43 (t, J =7.0 Hz, 2 H), 2.01 (m, 2 H). C NMR (CDCl ,
3
5
0.0 MHz) δ 178.4, 168.4, 134.0, 123.3, 37.2, 31.4, 23.7. To a solution of N-(4-
1
4

hydroxycarbonylbutyl)phthalimide in CH Cl (0.25 M) was added EDCI (1.4 equiv). The
2
2
resulting mixture was stirred at room temperature for 20 min, and then
alkylcyclohexylamine (1.2 equiv) was added. The reaction mixture was stirred at room
temperature for 17 h, and then each of water (50 mL) and CH Cl (50 mL) were added.
2
2
The organic layer was washed with water (3 × 15 mL) and brine (20 mL) and dried over
MgSO . After filtration, the filtrate was evaporated in vacuo to give the crude amide,
4
which was purified by column chromatography.
4
.1.5.1. N-cyclohexyl-N-methyl-4-(1,3-dioxoisoindolin-2-yl)butanamide (7a). Use of N-
(4-hydroxycarbonylbutyl)phthalimide (0.5 g, 2.14 mmol) and cyclohexylmethylamine
1
(
0.34 mL, 2.57 mmol) afforded 7a (0.61 g, 87%) as a viscous oil. H NMR (400 MHz,
CDCl ) δ 7.87–7.80 (m, 2H), 7.70 (dt, J = 5.4, 3.4 Hz, 2H), 3.76 (t, J = 6.8 Hz, 2H), 2.76
3
(d, J = 11.1 Hz, 3H), 2.41–2.32 (m, 2H), 1.84–1.71 (m, 3H), 2.09–1.98 (m, 2H), 1.67–
1
.46 (m, 4H), 1.40–1.21 (m, 3H), 1.08–1.04 (m, 1H). MS (ESI) m/z 328.2 [M+H]+.
4
.1.5.2. N-cyclohexyl-N-cyclopropyl-4-(1,3-dioxoisoindolin-2-yl)butanamide (7b). Use of
N-(4-hydroxycarbonylbutyl)phthalimide
(0.6
g,
2.57
mmol)
and
cyclohexylcyclopropylamine (4, 0.43 mL, 3.08 mmol) afforded 7b as white solids (0.4 g,
1
4
7
4%), R = 0.51 Hex/EA 6:4. H NMR (400 MHz, CDCl ) δ 7.81–7.79 (m, 2H), 7.68–
f
3
.66 (m, 2H), 3.80–3.71 (m, 3H), 2.53–2.44 (m, 3H), 2.01 (p, J = 6.8 Hz, 2H), 1.71–1.55
1
3
(
m, 7H), 1.30–1.15 (m, 2H), 1.11–1.04 (m, 1H), 0.78–0.72 (m, 4H). C NMR (100 MHz,
CDCl ) δ 174.4, 168.3, 133.7, 132.0, 123.0, 58.0, 37.6, 32.2, 31.2, 27.7, 26.1, 25.6, 23.9,
3
+
+
+
9
.0. MS (ESI) m/z 355.2 [M+H] , 377.2 [M+Na] , 393.1 [M+K] . HRMS calcd for
+
C H N O [M+H] 355.2022, found 355.2008.
2
1
26
2
3
1
5

4
.1.5.3. N-cyclohexyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)-4-(1,3-dioxoisoindolin-2-
yl)butanamide (7c). Use of N-(4-hydroxycarbonylbutyl)phthalimide (0.15 g, 0.66 mmol)
1
and 6 (0.15 g, 0.77 mmol) afforded 7c (0.18 g, 70%), R = 0.33 Hex/EA 6:4. H NMR
f
(
400 MHz, CDCl ) δ 7.81–7.77 (m, 2H), 7.69–7.66 (m, 2H), 7.38–7.24 (m, 2H), 4.37 (s,
3
0
.4 H), 4.23 (d, J = 7.4 Hz, 2H), 3.79 (d, J = 7.4 Hz, 3H), 3.75 (t, J = 6.8 Hz, 1H), 3.68 (t,
J = 6.6 Hz, 1H), 3.50–3.45 (m, 0.6 H), 2.38 (t, J = 7.3 Hz, 1H), 2.31 (t, J = 7.3 Hz, 1H),
.05–1.94 (m, 2H), 1.78–1.42 (m, 6H), 1.31–1.16 (m, 4H), 1.10–1.00 (m, 1H). MS (ESI)
2
+
+
m/z 431.0 [M+Na] . HRMS calcd for C H N O [M+Na] 431.2059, found 431.2046.
2
3
28
4
3
4
.1.6. General procedure II for the preparation of N-alkyl-N-cyclohexy-4-
aminobutanamides 8a-c. To a solution of phthalimides 7a-c in ethanol (0.4 M) was added
hydrazine hexahydrate (10 equiv). The resulting mixture was stirred at room temperature
for 3 h, and the resulting suspension was diluted with diethyl ether (100 mL). The
precipitate was separated by filtration and washed with diethyl ether. The filtrate and
diethyl ether wash solutions were combined and washed with brine (50 mL). The organic
layer was dried over MgSO . After filtration, the filtrate was concentrated in vacuo to
4
give a crude product that was purified by column chromatography (neutral alumina).
1
4
.1.6.1. 4-Amino-N-cyclohexyl-N-methylbutanamide (8a). 88%, a colorless liquid. H
NMR (400 MHz, CDCl ) δ 4.34 (bs, 0.5H), 3.49–3.43 (m, 0.5H), 2.74–2.65 (m, 5H),
3
2
.34–2.20 (m, 2H), 1.79–1.62 (m, 4H), 1.56–1.52 (m, 3H), 1.43–1.38 (m, 3H), 1.30–1.22
1
3
(
m, 3H), 1.06–0.90 (m, 1H). C NMR (100 MHz, CDCl ) δ 171.5, 55.9, 51.5, 41.2, 41.2,
3
3
0.8, 30.3, 30.1, 29.2, 28.8, 28.7, 28.3, 26.4, 25.2, 25.0, 25.0, 24.7.
1
6

4
.1.6.2. 4-Amino-N-cyclohexyl-N-cyclopropylbutanamide (8b). 85%, a colorless liquid.
1
H NMR (200 MHz, CDCl ) δ 3.88 (s, 1H), 2.87–2.47 (m, 7H), 1.83–1.03 (m, 11H),
3
0
.84–0.75 (m, 4H). MS (ESI) m/z 225.1 [M+H]+.
4
7
0
3
.1.6.3. 4-Amino-N-cyclohexyl-N-((1-methyl-1H-pyrazol-4-yl)methyl)butanamide (8c).
1
1%, a colorless viscous liquid. H NMR (400 MHz, CDCl ) δ 7.38 (s, 0.6H), 7.35 (s,
3
.6H), 7.32 (s, 0.4H), 7.19 (s, 0.4H), 4.48–4.41 (m, 0.4H), 4.31(s, 0.8H), 4.25 (s, 1.2H),
.86 (s, 1H), 3.81 (s, 2H), 3.62–3.54 (m, 0.6H), 2.74 (t, J = 6.9 Hz, 1.4H), 2.67 (t, J = 6.9
Hz, 0.6H), 2.40 (t, J = 6.9 Hz, 1.4H), 2.34 (t, J = 6.9 Hz, 0.6H), 1.84–1.08 (m, 12H).
+
HRMS calcd for C H N O [M+H] 279.2185, found 279.2175.
1
5
26
4
4
.1.7. 2-Nitro-5-phenoxybenzaldehyde (9). A mixture of 5-fluoro-2-nitrobenzaldehyde
(
1.00 g, 5.91 mmol), phenol (0.58 g, 6.21 mmol), and K CO (0.90 g, 6.50 mmol) in
2
3
DMF (6 mL, 1.00 M) was stirred under argon at 80 C for 3 h. The reaction mixture was
then cooled to room temperature and evaporated in vacuo. The residue was then poured
into 0.5 N NaOH (5 mL) and extracted with CH Cl (3 × 50 mL). The organic layer was
2
2
washed with brine (30 mL) and dried over MgSO . After filtration, the filtrate was
4
concentrated in vacuo to afford a brown liquid that was purified by column
chromatography (silica gel) to afford 9 as a yellowish solid (1.38 g, 96%), R = 0.65
f
1
Hex/EA 9.5:0.5. H NMR (400 MHz, CDCl ) δ 10.40 (s, 1H), 8.12 (d, J = 9.0 Hz, 1H),
3
7
.42 (t, J = 7.9 Hz, 2H), 7.32 (d, J = 2.8 Hz, 1H), 7.25 (dd, J = 13.0, 5.5 Hz, 1H), 7.17
1
3
(
dd, J = 9.0, 2.8 Hz, 1H), 7.06 (d, J = 8.0 Hz, 2H). C NMR (100 MHz, CDCl ) δ 187.9,
3
1
2
62.8, 154.1, 143.1, 134.3, 130.5, 127.3, 125.9, 120.5, 120.5, 116.6. MS (ESI) m/z
44.21 [M+H]+.
1
7

4
.1.8. (2-Nitro-5-phenoxyphenyl)methanol (11). To a solution of 9 (1 g, 4.11 mmol) in
MeOH (17 mL) at 0 °C was added sodium borohydride (0.077 g, 2.05 mmol), and the
reaction mixture was stirred for 30 min. The reaction mixture was then cooled to 0 °C
and quenched with saturated NH Cl. The mixture was evaporated in vacuo, and the
4
residue was extracted with ethyl acetate (3 × 20 mL). The combined organic layer was
washed with brine and dried over MgSO . After filtration, the filtrate was concentrated in
4
vacuo and purified using column chromatography (silica gel) to afford 11 quantitatively
1
(
1.00 g) H NMR (400 MHz, CDCl ) δ 8.14 (d, J = 9.1 Hz, 1H), 7.45–7.39 (m, 2H),
3
7
.28–7.23 (m, 2H), 7.08 (dd, J = 8.5, 0.9 Hz, 2H), 6.91 (dd, J = 9.1, 2.7 Hz, 1H), 4.94 (d,
1
3
J = 4.7 Hz, 2H), 2.50 (s, 1H). C NMR (100 MHz, CDCl ) δ 162.7, 154.5, 141.1, 140.5,
3
1
30.1, 127.7, 125.2, 120.4, 117.0, 115.6, 62.3. MS (ESI) m/z 246.06 [M+H]+.
4
.1.9. Tert-butyl (2-(hydroxymethyl)-4-phenoxyphenyl)carbamate (12). To a suspension
of 11 (0.527 g, 2.14 mmol) in ethanol/water 5:1 (10.7 mL) was added iron powder (0.24
g, 4.3 mmol) followed by saturated aqueous NH Cl solution (1.1 mL). The mixture was
4
heated at 80 °C for 3 h. After cooling, then reaction mixture was filtered through Celite.
The filtrate was concentrated and partitioned between CH Cl (25 mL) and water (10
2
2
mL). The organic layer was dried over MgSO . The organic layer was then concentrated
4
and purified by column chromatography (neutral alumina) to afford 2-amino-5-
1
phenoxy)phenyl)methanol as a reddish brown solid (0.28 g, 61%). H NMR (300 MHz,
CDCl ) δ 7.34–7.21 (m, 2H), 7.06–6.78 (m, 5H), 6.69 (d, J = 8.3 Hz, 1H), 4.59 (s, 2H),
3
1
3
3
.30 (s, 3H). C NMR (100 MHz, CDCl ) δ 158.5, 148.8, 140.8, 129.5, 126.9, 122.3,
3
1
8

+
1
5
20.7, 120.6, 117.7, 117.4, 63.7. MS (ESI) m/z 216.09 [M+H] . A solution of (2-amino-
-phenoxyphenyl)methanol (0.24 g, 1.14 mmol) in 1,2-dichloroethane (7.1 mL) was
treated with di-tert-butyl dicarbonate (0.287 mL, 1.25 mmol) and heated at reflux for 2 h.
After cooling, the reaction mixture was concentrated in vacuo and purified by column
1
chromatography (silica gel) to afford 12 as a yellow solid (0.35 g, quantitative). H NMR
(
400 MHz, CDCl ) δ 7.78 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.30 (dd, J = 10.7, 5.3 Hz,
3
2
9
1
H), 7.10–7.03 (m, 1H), 7.00–6.92 (m, 3H), 6.89 (d, J = 2.8 Hz, 1H), 4.63 (s, 2H), 1.52 (s,
1
3
H). C NMR (100 MHz, CDCl ) δ 157.5, 153.8, 152.5, 132.9, 131.9, 129.6, 123.2,
3
22.9, 119.5, 119.2, 118.1, 80.4, 63.3, 28.2. MS (ESI) m/z 316.14 [M]+.
4
.1.10. Tert-butyl (2-formyl-4-phenoxyphenyl)carbamate (13). To a solution of 12 (1.98 g,
6
.28 mmol) in EtOAc (44.8 mL, 0.14 M) was added 2-iodoxybenzoic acid (2.1 g, 7.53
mmol), and the resulting mixture was heated at 80 °C for 8 h. The reaction mixture was
then filtered through Celite. The filtrate was concentrated in vacuo and purified by
1
column chromatography (silica gel) to afford 13 in quantitative yield (1.97 g). H NMR
(
400 MHz, CDCl ) δ 10.23 (s, 1H), 9.79 (s, 1H), 8.45 (d, J = 8.8 Hz, 1H), 7.33 (t, J = 7.9
3
Hz, 2H), 7.30–7.24 (m, 2H), 7.10 (t, J = 7.4 Hz, 1H), 6.97 (d, J = 7.9 Hz, 2H), 1.53 (s,
1
3
9
1
H). C NMR (100 MHz, CDCl ) δ 194.1, 157.3, 152.9, 151.0, 137.6, 129.8, 127.3,
3
25.1, 123.4, 122.0, 120.1, 118.2, 80.9, 28.2. MS (ESI) m/z 314.13 [M+H]+.
4
.1.11. General procedure III for the synthesis of benzyl alcohols 10a, 10b, 14a and 14c-
g. Benzaldehyde 9/13 dissolved in anhydrous THF (0.16 M) was cooled to –78 °C. To
this solution was added a Grignard reagent (1.1 equiv) dropwise over 10 min. The
1
9

resulting mixture was stirred for 3 h and then warmed slowly to room temperature.
Saturated NH Cl was then added dropwise. After evaporation, and the resulting solution
4
was extracted with ethyl acetate (2 × 50 mL). The combined organic layer was washed
with brine (30 mL) and dried over MgSO . After filtration, the filtrate was concentrated
4
in vacuo, and the residue was purified by column chromatography (silica gel) to afford
benzyl alcohol.
4
.1.11.1. 1-(2-Nitro-5-phenoxyphenyl)ethanol
(10a).
Use
of
2-nitro-5-
phenoxybenzaldehyde (9, 0.1 g, 0.41 mmol) and 3 M methylmagnesium chloride in THF
1
(
0.20 mL, 0.61 mmol) yielded 10a (0.04 g, 38%). H NMR (400 MHz, CDCl ) δ 8.00 (d,
3
J = 9.0 Hz, 1H), 7.46–7.29 (m, 3H), 7.28–7.13 (m, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.87 (dd,
J = 9.0, 2.7 Hz, 1H), 5.54 (q, J = 6.4 Hz, 1H), 1.95 (bs, 1H), 1.54 (d, J = 6.3 Hz, 3H).
4
.1.11.2. 1-(2-Nitro-5-phenoxyphenyl)-2-phenylethanol (10b). Use of 9 (0.2 g, 0.82 mmol)
and 2 M benzylmagnesium chloride in THF (0.82 mL, 1.20 mmol) yielded 10b (0.10 g,
1
3
7
1
9
1
4
4
6%). H NMR (400 MHz, CDCl ) δ 8.06 (d, J = 9.1 Hz, 1H), 7.48–7.38 (m, 3H), 7.37–
3
.30 (m, 5H), 7.29–7.19 (m, 3H), 7.06 (dd, J = 8.6, 1.0 Hz, 2H), 6.89 (dd, J = 9.1, 2.8 Hz,
H), 5.57 (dd, J = 9.0, 2.8 Hz, 1H), 3.26 (dd, J = 13.7, 2.8 Hz, 1H), 2.73 (dd, J = 13.7,
1
3
.0 Hz, 1H), 2.14 (d, J = 11.7 Hz, 1H). C NMR (100 MHz, CDCl ) δ 162.4, 154.7,
3
43.2, 141.4, 137.7, 130.2, 129.5, 128.6, 127.5, 126.8, 125.2, 120.3, 116.2, 115.9, 70.7,
4.9.
.1.11.3. Tert-butyl (2-(1-hydroxyethyl)-4-phenoxyphenyl)carbamate (14a). Use of 13
(0.1 g, 0.32 mmol) and 3 M methylmagnesium chloride (1.5 mL, 1.28 mmol) in THF
1
yielded 14a (0.052 g, 49%), R = 0.20 Hex/EA 9:1. H NMR (200 MHz, CDCl ) δ 7.85–
f
3
7
.75 (m, 2H), 7.36–7.26 (m, 2H), 7.07 (tt, J = 7.4, 1.2 Hz, 1H), 6.99–6.89 (m, 4H), 4.92
2
0

1
3
(
q, J = 6.6 Hz, 1H), 1.55 (s, 3H), 1.52 (s, 9H); C NMR (50 MHz, CDCl3) δ 157.5,
1
6
4
53.7, 152.4, 135.3, 132.2, 129.6, 129.6, 123.6, 122.8, 118.7, 118.0, 118.0, 117.4, 80.2,
9.0, 28.2, 28.2, 28.2, 22.1; ESI-MS m/z 352.9 [M+Na]+.
.1.11.4. Tert-butyl (2-(1-hydroxy-2-methylpropyl)-4-phenoxyphenyl)carbamate (14c).
Use of 13 (0.6 g, 1.91 mmol) and 2 M isopropylmagnesium chloride in THF (2.1 mL,
1
4
7
.21 mmol) yielded 14c (0.41 g, 60%). H NMR (400 MHz, CDCl ) δ 7.76–7.74 (m, 2H),
3
.24–7.20 (m, 2H), 6.97 (t, J = 7.4 Hz, 1H), 6.87–6.83 (m, 3H), 6.71–6.70 (m, 1H), 4.18
(d, J = 8.4 Hz, 1H), 2.20 (bs, 1H), 2.05–2.00 (m, 1H), 1.43 (s, 9H), 1.01 (d, J = 6.9 Hz,
1
3
3
1
H), 0.67 (d, J = 6.9 Hz, 3H). C NMR (100 MHz, CDCl ) δ 157.8, 153.6, 152.0, 133.3,
3
32.6, 129.6, 123.6, 112.8, 119.6, 119.0, 118.0, 80.9, 80.2, 33.0, 28.4, 19.7, 19.2. HRMS
+
calcd for C H NO [M+Na] 380.1838, found 380.1828.
2
1
27
4
4
.1.11.5. Tert-butyl (2-(1-hydroxyprop-2-yn-1-yl)-4-phenoxyphenyl)carbamate (14d).
Use of 13 (2.0 g, 6.38 mmol) and 0.5 M ethynylmagnesium bromide in THF (31.9 mL,
1
1
1
1
1
7
4
5.94 mmol) yielded 14d (1.8 g, 82%). H NMR (400 MHz, CDCl ) δ 7.70 (d, J = 8.7 Hz,
3
H) 7.33–7.29 (m, 3H), 7.24 (bs, 1H), 7.10–7.06 (m, 1H), 7.00–6.97 (m, 3H), 5.51 (bs,
1
3
H), 2.89 (bs, 1H), 1.69 (d, J = 2.2 Hz, 1H), 1.51 (s, 9H). C NMR (100 MHz, CDCl ) δ
3
57.3, 153.9, 152.9, 131.9, 131.6, 129.6, 124.6, 123.0, 119.7, 118.7, 118.3, 81.5, 80.7,
+
5.9, 62.3, 28.3. HRMS calcd for C H NO [M+Na] 362.1368, found 362.1362.
2
0
21
4
.1.11.6. Tert-butyl (2-(1-hydroxyprop-2-en-1-yl)-4-phenoxyphenyl)carbamate (14e). Use
of 13 (0.75 g, 2.39 mmol) and 0.7 M vinylmagnesium bromide in THF (8.5 mL, 5.98
1
mmol) yielded 14e (0.7 g, 86%). H NMR (400 MHz, CDCl ) δ 7.74 (d, J = 8.4 Hz, 1H),
3
7
6
.55 (s, 1H), 7.32 (t, J = 8.3 Hz, 2H), 7.07 (t, J = 7.3 Hz, 1H), 6.96–6.90 (m, 4H), 6.11–
.02 (m, 1H), 5.35 (d, J = 17.2 Hz, 1H), 5.25 (d, J = 10.3 Hz, 1H), 5.20 (d, J = 5.5 Hz,
2
1

1
3
1
1
H), 2.85 (bs, 1H), 1.50 (s, 9H). C NMR (100 MHz, CDCl ) δ 157.6, 153.7, 152.6,
3
37.5, 133.0, 132.5, 129.7, 124.0, 122.9, 119.2, 118.8, 118.1, 116.0, 80.3, 73.8, 28.3.
+
HRMS calcd for C H NO [M+Na] 364.1525, found 364.1515.
2
0
23
4
4
.1.11.7. Tert-butyl (2-(cyclohexyl(hydroxy)methyl)-4-phenoxyphenyl)carbamate (14f).
Use of 13 (0.50 g, 1.59 mmol) and 0.66 M cyclohexylmagnesium bromide in ether (5.0
1
mL, 3.35 mmol) yielded 14f (0.30 g, 48%). H NMR (200 MHz, CDCl ) δ 7.80 (d, J =
3
8
.5 Hz, 1H), 7.34–7.25 (m, 2 H), 7.09–6.88 (m, 4 H), 6.75 (d, J = 2.8 Hz, 1H), 4.31 (d, J
+
+
=
8.5 Hz, 1H), 2.13–1.15 (m, 20 H). MS (ESI) m/z 420.2 [M+Na] , 436.2 [M+K] .
4
.1.11.8. Tert-butyl (2-(2-cyclohexyl-1-hydroxyethyl)-4-phenoxyphenyl)carbamate (14g).
Use of 13 (2.0 g, 6.38 mmol) and 0.9 M cyclohexylmethylmagnesium bromide in THF
1
(
31.49 mL, 25.53 mmol) yielded 14g (2.31 g, 88%). H NMR (400 MHz, CDCl ) δ 7.78
3
(d, J = 8.7 Hz, 1H), 7.68 (bs, 1H), 7.32–7.28 (m, 2H), 7.08–7.04 (m, 1H), 6.96–6.90 (m,
3
9
1
3
H), 6.85 (d, J = 2.6 Hz, 1H), 4.80 (dd, J = 5.8, 8.4 Hz, 1H), 1.82–1.57 (m, 8H), 1.51 (s,
1
3
H), 1.25–1.15 (m, 4H), 1.05–0.88 (m, 2H). C NMR (100 MHz, CDCl ) δ 157.1, 153.7,
3
52.5, 135.0, 132.4, 129.7, 123.9, 122.8, 119.0, 118.2, 118.0, 80.3, 71.4, 43.5, 34.2, 33.8,
2.9, 28.4, 26.5, 26.2, 26.1. MS (ESI) m/z 434.0 [M+Na]+.
4
.1.12. Tert-butyl (4-phenoxy-2-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)carbamate (14b).
A mixture of 13 (0.2 g, 0.64 mmol) and TMSCF (142.5 L, 0.96 mmol) in 1.28 mL THF
3
(0.5 M) was cooled to 0 °C and then treated with a catalytic amount of TBAF (2.5 mg,
1
.5 mol%). A yellow color developed instantaneously with the initial evolution of
fluorotrimethylsilane, and the reaction mixture was brought to ambient temperature and
stirred. The reaction mixture was then extracted with ether (15 mL). The ether extracts
2
2

were washed with water (5 mL) and brine (5 mL) and dried over MgSO . After filtration,
4
the filtrate was concentrated in vacuo and purified by column chromatography (silica gel)
1
to afford 14b (0.18 g, 74%). H NMR (400 MHz, CDCl ) δ 7.57 (d, J = 8.6 Hz, 1H), 7.32
3
(t, J = 7.7 Hz, 2H), 7.14–7.06 (m, 3H), 7.00–6.96 (m, 3H), 5.04 (q, J = 7.0 Hz, 1H), 1.49
1
3
(
s, 9H). C NMR (100 MHz, CDCl ) δ 157.0, 154.4, 153.9, 131.9, 129.8, 127.6, 127.2,
3
1
2
26.2, 124.3 (q, J = 283.46 Hz), 123.4, 120.2, 119.4, 118.6, 81.2, 71.4 (q, J = 32 Hz),
+
8.2. HRMS calcd for C H F NO [M+H] 384.1423, found 384.1413.
1
9
20
3
4
4
.1.13. Tert-butyl (2-(1,3-dihydroxypropyl)-4-phenoxyphenyl)carbamate (16). To a
solution of 14e (0.79 g, 2.31 mmol) in freshly distilled THF (20.1 mL) at 0 °C was added
M borane in THF (2.42 mL) dropwise. The resulting mixture was stirred at 0 °C for 16
1
h. To this reaction mixture was added 3 N NaOH (2.48 mL) followed by addition of 30%
hydrogen peroxide (2.62 mL) dropwise. The resulting turbid solution was stirred at 0 °C
for 30 min, and the remaining hydrogen peroxide was neutralized by addition of a sodium
bisulfite solution. Ethyl acetate (50 mL) was then added to the reaction mixture, and the
organic layer was washed with saturated aqueous NaHCO and brine and dried over
3
MgSO . After filtration, the filtrate was concentrated in vacuo and purified by column
4
chromatography (silica gel, Hex/EA 7:3) to afford a mixture of 1,2–diol 15 (0.13 g, 16%)
and 1,3–diol 16 (0.36 g, 44%) in 1:3 ratio. NMR analysis revealed that the 1,2–diol 15
1
was a syn/anti mixture in a ratio of 3:1. 1,3–diol 16: H NMR (400 MHz, CDCl ) δ 7.88
3
(bs, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.25 (t, J = 7.4 Hz, 2H), 7.02 (t, J = 7.3 Hz, 1H),
6
1
.92–6.84 (m, 3H), 4.91–4.87 (dd, J = 4.2, 9.2 Hz, 1H), 3.81–3.76 (m, 1H), 3.67–3.62 (m,
1
3
H), 2.14–2.05 (m, 1H), 1.79–1.72 (m, 1H), 1.46 (s, 9H). C NMR (100 MHz, CDCl ) δ
3
2
3

1
6
57.5, 154.0, 152.9, 135.2, 131.8, 129.6, 124.3, 122.9, 118.7, 118.1, 117.7, 80.4, 71.9,
0.9, 37.8, 28.3. MS (ESI) m/z 360.4 [M+H]+.
4
.1.14. Tert-butyl
(2-(3-((tert-butyldimethylsilyl)oxy)-1-hydroxypropyl)-4-
phenoxyphenyl)carbamate (14h). To a –30 °C solution of 16 (0.29 g, 0.80 mmol) and
imidazole (0.12 g, 1.77 mmol) in DMF (1.6 mL) was added a solution of tert-
butyldimethylsilyl chloride (0.13 g, 0.88 mmol) in DMF (1 mL) dropwise. The resulting
mixture was stirred for an additional 24 h and then warmed slowly to room temperature.
Upon completion of the reaction, it was quenched by adding 50 mL water and diluted
with EtOAc (15 mL). The organic layer was washed with brine and dried over MgSO4.
After filtration, the filtrate was concentrated in vacuo and purified by column
1
chromatography (silica gel) to afford 14h as a colorless liquid (0.27 g, 72%). H NMR
(
400 MHz, CDCl ) δ 8.19 (bs, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.31–7.26 (m, 2H), 7.05 (t,
3
J = 7.4 Hz, 1H), 6.95–6.90 (m, 3H), 6.85 (d, J = 2.9 Hz, 1H), 4.98 (dd, J = 3.4, 9.5 Hz,
1
1
1
6
H), 3.96–3.92 (m, 1H), 3.83 (dt, J = 2.8, 10.3 Hz, 1H), 2.27–2.18 (m, 1H), 1.77–1.72 (m,
1
3
H), 1.51 (s, 9H), 0.93 (s, 9H), 0.14 (s, 3H), 0.12 (s, 3H). C NMR (100 MHz, CDCl ) δ
3
57.7, 153.5, 152.2, 133.8, 132.5, 129.5, 123.4, 122.6, 118.7, 118.0, 117.9, 79.8, 73.5,
2.2, 37.8, 28.3, 25.7, 18.0, –5.5, –5.6. MS (ESI) m/z 496.3 [M+Na]+.
4
.1.15. General procedure IV for the synthesis of mesyl esters. To an ice-cold solution of
alcohol 10a/10b/14b/14h in CH Cl (0.25 M) was added dry Et N (3 equiv) dropwise,
2
2
3
and the resulting reaction mixture was stirred at room temperature for 5 min. To this
mixture was added methanesulfonyl chloride (1.1 equiv), with further stirring for 3 h at
2
4

room temperature. Water (5 mL) and CH Cl (5 mL) were then added. The organic layer
2
2
was separated and the aqueous layer extracted with CH Cl (3 × 10 mL). The combined
2
2
organic layer was then dried over MgSO . The MgSO was removed by filtration, and the
4
4
filtrate was concentrated in vacuo to give the mesyl ester that was used directly without
purification.
4
.1.16. General procedure V for the synthesis of bromo intermediate. To a solution of an
alcohol intermediate of interest 14c,d,f,g in diethyl ether (0.04 M) was added PBr (1
3
equiv) dissolved in diethyl ether (21.0 mL) at 0 °C with stirring for 2.5 h. The reaction
mixture was then stirred at 0 °C for 12 h and quenched by addition of aqueous NaHCO3,
after which the aqueous layer was extracted with diethyl ether (3 × 15 mL). The
combined organic layers were washed with water and brine and then dried over MgSO4.
The MgSO was removed by filtration, and the filtrate was concentrated in vacuo to give
4
a crude bromide intermediate, which was used directly for subsequent syntheses without
further purification.
4
.1.17. General procedure VI for the synthesis of N-alkyl-N-cyclohexy-4-((1-(5-
phenoxyphenyl)ethyl)amino)butanamides 17–19. To a solution of mesyl ester or bormo
intermediate in acetonitrile (0.5 M) was added one of the amines 8a-c (1.5-2.0 equiv) and
K CO (1.5 equiv). The resulting reaction mixture was stirred vigorously at 45 °C for 9 h,
2
3
diluted with EtOAc (5 mL) and water (2 mL), and aqueous layer was extracted with
EtOAc (2 × 10 mL). The combined organic layer was dried over MgSO . After filtration,
4
2
5

the filtrate was concentrated and purified by column chromatography to afford the
corresponding compound.
4
.1.17.1. N-cyclohexyl-N-methyl-4-((1-(2-nitro-5-
phenoxyphenyl)ethyl)amino)butanamide (17a). To a solution of 2-(1-mesyloxyethyl)-1-
nitro-4-phenoxybenzene (0.025 g, 0.046 mmol) in acetonitrile (0.5 M) was added 4-
amino-N-cyclohexyl-N-methylbutanamide (8a, 0.019 g, 0.093 mmol) and K CO (0.010
2
3
1
g, 0.070 mmol) to yield 17a (0.025 g, 77%). H NMR (400 MHz, CDCl ) δ 7.79 (d, J =
3
8
.8 Hz, 1H), 7.38–7.32 (m, 3H), 7.15 (t, J = 7.6 Hz, 1H), 7.01 (d, J = 8.0 Hz, 2H), 6.78
(m, 1H), 4.37–4.32 (m, 1H), 4.08–4.02 (m, 0.5H), 3.50–3.45 (m, 0.5H), 2.73 (s, 3H),
2
8
4
.53–2.47 (m, 1H), 2.36–2.17 (m, 3H), 1.97 (s, 1H), 1.74–1.46 (m, 7H), 1.34–0.82 (m,
H). MS (ESI) m/z 440.2 [M+H]+.
.1.17.2. Tert-butyl
(2-(1-((4-(cyclohexyl(methyl)amino)-4-oxobutyl)amino)-2,2,2-
trifluoroethyl)-4-phenoxyphenyl)carbamate (17b). To a solution of tert-butyl (2-(2,2,2-
trifluoro-1-mesyloxyethyl)-4-phenoxyphenyl)carbamate (0.42 g, 0.90 mmol) in
acetonitrile (1.8 mL, 0.5 M) was added 4-amino-N-cyclohexyl-N-methylbutanamide (8a,
1
0
.27 g, 1.35 mmol) and K CO (0.19 g, 1.35 mmol) to yield 17b (0.38 g, 79%). H NMR
2
3
(
400 MHz, CDCl ) δ 9.15 (bs, 1H), 7.92 (bs, 1H), 7.33–7.29 (m, 2H), 7.07 (t, J = 7.2 Hz,
3
1
2
5
H), 7.02–6.92 (m, 4H), 4.44–4.37 (m, 0.6×1H), 4.32 (bs, 1H), 3.50–3.44 (m, 0.4×1H),
.80 (s, 0.6×3H), 2.78 (s, 0.4×3H), 2.72–2.63 (m, 2H), 2.43–2.29 (m, 2H), 1.91–1.75 (m,
H), 1.65–1.55 (m, 4H), 1.49 (s, 9H), 1.39–1.24 (m, 4H),. HRMS calcd for C H F N O
4
3
0
40
3
3
+
[
M+H] 564.3049, found 546.3059.
4
.1.17.3. Tert-butyl
(2-(1-((4-(cyclohexyl(methyl)amino)-4-oxobutyl)amino)-2-
methylpropyl)-4-phenoxyphenyl)carbamate (17c). To a solution of tert-butyl (2-(1-
2
6

bromo-2-methylpropyl)-4-phenoxyphenyl)carbamate (0.25 g, 0.60 mmol) in acetonitrile
(1.2 mL) was added 4-amino-N-cyclohexyl-N-methylbutanamide (8a, 0.18 g, 0.90 mmol)
1
and K CO (0.12 g, 0.90 mmol) to yield 17c (0.12 g, 38%). H NMR (400 MHz, CDCl )
2
3
3
δ 10.37–10.31 (m, 1H), 7.95 (bs, 1H), 7.30–7.26 (m, 2H), 7.03 (t, J = 7.5 Hz, 1H), 6.96–
6
1
9
4
.88 (m, 3H), 6.69 (bs, 1H), 4.44–4.38 (m, 0.6×1H), 3.50–3.43 (m, 0.4×1H), 3.27 (bs,
H), 2.79 (s, 0.6×3H), 2.78 (s, 0.4×3H), 2.55–2.21 (m, 4H), 1.84–1.54 (m, 10H), 1.49 (s,
H), 1.39–1.23 (m, 4H), 1.02 (d, J = 6.2 Hz, 3H), 0.74 (d, J = 6.2 Hz, 3H).
.1.17.4. Tert-butyl
(2-(cyclohexyl((4-(cyclohexyl(methyl)amino)-4-
oxobutyl)amino)methyl)-4-phenoxyphenyl)carbamate (17d). To a solution of tert-butyl
2-(1-bromo-1-cyclohexylmethyl)-4-phenoxyphenyl)carbamate (0.13 g, 0.28 mmol) in
(
acetonitrile (0.55 mL, 0.5 M) was added 4-amino-N-cyclohexyl-N-methylbutanamide (8a,
1
0
.08 g, 0.42 mmol) and K CO (0.06 g, 0.42 mmol) to yield 17d (0.04 g, 26%). H NMR
2
3
(
400 MHz, CDCl ) δ 10.36 (s, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.35–7.21 (m, 2H), 7.09–
3
6
2
1
.99 (m, 1H), 6.97–6.85 (m, 3H), 6.68 (s, 1H), 4.50–4.33 (m, 1H), 3.55–3.27 (m, 1H),
.79 (d, J = 2.2 Hz, 3H), 2.39 (dt, J = 17.7, 9.1 Hz, 4H), 1.91–1.55 (m, 14H), 1.50 (s, 9H),
1
3
.42–1.30 (m, 4H), 1.19–0.98 (m, 4H), 0.96–0.78 (m, 2H). C NMR (100 MHz, CDCl )
3
δ 172.0, 158.0, 153.8, 153.7, 129.5, 122.4, 118.5, 117.8, 117.6, 79.4, 56.6, 52.2, 47.3,
4
2
1.6, 31.9, 31.2, 30.8, 30.4, 29.8, 29.7, 29.4, 28.4, 28.2, 27.1, 26.1, 26.0, 25.7, 25.7, 25.5,
5.5, 25.1 (complexation owing to tautomerization and the cyclohexyl ring). MS (ESI)
+
+
m/z 578.4 [M+H] . HRMS calcd for C H N O [M+H] 578.3958, found 578.3965.
3
5
51
3
4
4
.1.17.5. Tert-butyl
oxobutyl)amino)ethyl)-4-phenoxyphenyl)carbamate (17e). To a solution of tert-butyl (2-
1-bromo-2-cyclohexylethyl)-4-phenoxyphenyl)carbamate (0.31 g, 0.65 mmol) in
(2-(2-cyclohexyl-1-((4-(cyclohexyl(methyl)amino)-4-
(
2
7

acetonitrile (1.30 mL, 0.5 M) was added 4-amino-N-cyclohexyl-N-methylbutanamide (8a,
1
0
.19 g, 0.98 mmol) and K CO (0.13 g, 0.98 mmol) to yield 17e (0.15 g, 46%). H NMR
2
3
(
400 MHz, CDCl ) δ 7.85 (s, 1H), 7.26–7.17 (m, 2H), 6.97 (t, J = 7.3 Hz, 1H), 6.89–6.78
3
(m, 3H), 6.67 (s, 1H), 4.40–4.26 (m, 0.6 H), 3.70 (s, 1H), 3.40 (ddd, J = 11.6, 8.1, 3.8 Hz,
0
1
0
1
3
.4H), 2.72 (d, J = 5.1 Hz, 3H), 2.55 (s, 1H), 2.44–2.27 (m, 2H), 2.27–2.13 (m, 1H),
.81–1.47 (m, 15H), 1.43 (s, 9H), 1.32–1.19 (m, 4H), 1.03 (dd, J = 19.4, 5.8 Hz, 5H),
1
3
.86–0.74 (m, 3H). C NMR (100 MHz, CDCl ) δ 172.0, 171.9, 158.1, 153.6, 134.3,
3
31.2, 131.1, 129.5, 121.5, 120.8, 118.7, 117.6, 79.4, 56.6, 52.3, 47.2, 42.5, 34.4, 33.4,
3.3, 31.9, 31.2, 30.9, 29.8, 29.8, 29.6, 29.4, 28.4, 27.1, 26.4, 26.1, 25.7, 25.6, 25.5, 25.2.
(complexation owing to tautomerization and the cyclohexyl ring). MS (ESI) m/z 592.4
+
+
[
M+H] . HRMS calcd for C H N O [M+H] 592.4114, found 592.4125.
36 53 3 4
4
.1.17.6. N-cyclohexyl-N-methyl-4-((1-(2-nitro-5-phenoxyphenyl)-2-
phenylethyl)amino)butanamide (17f). To a solution of 2-(1-mesyloxy-2-phenylethyl)-1-
nitro-4-phenoxybenzene (0.200 g, 0.483 mmol) in acetonitrile (0.5 M) was added 4-
amino-N-cyclohexyl-N-methylbutanamide (8a, 0.192 g, 0.966 mmol) and K CO (0.100
2
3
1
g, 0.726 mmol) to yield 17f (0.157 g, 63%). H NMR (400 MHz, CDCl ) δ 8.06 (d, J =
3
9
2
2
.1 Hz, 1H), 7.46–7.36 (m, 3H), 7.35–7.28 (m, 5H), 7.27–7.04 (m, 4H), 6.89 (dd, J = 9.1,
.8 Hz, 1H), 4.26 (dd, J = 10.2, 6.2 Hz, 1H), 3.26–2.98 (m, 1H), 2.76–2.65 (m, 1H),
.45–2.36 (m, 1H), 2.25–2.18 (m, 2H), 1.65–1.45 (m, 9H), 1.29–1.15 (m, 7H), 0.99–0.88
(
m, 1H). MS (ESI) m/z 516.2 [M+H]+.
.1.17.7. N-cyclohexyl-N-methyl-4-((3-((tert-butyldimethylsilyl)oxy)-1-(3-
phenoxyphenyl)propyl)amino)butanamide (17g). To a solution of tert-butyl (2-(1-bromo-
-((tert-butyldimethylsilyl)oxy)propyl)-4-phenoxyphenyl)carbamate (0.14 g, 0.25 mmol)
4
3
2
8

in acetonitrile (0.48 mL, 0.5 M) was added 4-amino-N-cyclohexyl-N-methylbutanamide
1
(
8a, 0.07 g, 0.37 mmol) and K CO (0.05 g, 0.37 mmol) to yield 17g (0.06 g, 37%). H
2
3
NMR (400 MHz, CDCl ) δ 10.4 (bs, 1H), 7.93 (bs, 1H), 7.30–7.25 (m, 2H), 7.03 (t, J =
3
7
3
0
1
.4 Hz, 1H), 6.93–6.87 (m, 3H), 6.77 (bs, 1H), 4.43–4.38 (m, 0.6×1H), 3.85 (bs, 1H),
.70–3.67 (m, 1H), 3.54–3.49 (m, 1H), 3.47–3.43 (m, 0.4×1H), 2.78 (s, 0.6×3H), 2.77 (s,
.4×3H), 2.66–2.61 (m, 1H), 2.42–2.29 (m, 3H), 2.05–2.02 (m, 1H), 1.84–1.75 (m, 6H),
.65–1.56 (m, 3H), 1.49 (s, 9H), 1.42–1.25 (m, 5H), 0.88 (s, 9H), 0.5 (s, 3H), 0.03 (s, 3H).
MS (ESI) m/z 654.5 [M+H]+.
.1.17.8. Tert-butyl (2-(1-((4-(cyclohexyl(methyl)amino)-4-oxobutyl)amino)prop-2-yn-1-
yl)-4-phenoxyphenyl)carbamate (17h). To a solution of tert-butyl (2-(1-bromoprop-2-yn-
-yl)-4-phenoxyphenyl)carbamate (0.31 g, 0.76 mmol) in acetonitrile (1.51 mL) was
4
1
added 4-amino-N-cyclohexyl-N-methylbutanamide (8a, 0.23 g, 1.14 mmol) and K CO
2
3
1
(
0.16 g, 1.14 mmol) to yield 17h (0.17 g, 42%). H NMR (400 MHz, CDCl ) δ 9.15–9.08
3
(m, 1H), 7.84 (bs, 1H), 7.34 (t, J = 2.9 Hz, 1H), 7.30 (t, J = 7.8 Hz, 2H), 7.05 (t, J = 7.3
Hz, 1H), 6.97–6.93 (m,4H), 4.73 (dd, J = 2.4, 10.0 Hz, 1H), 3.52–3.48 (m, 0.4×1H), 2.82
s, 0.6×3H), 2.79 (s, 0.4×3H), 2.77–2.72 (m, 3H), 2.54 (d, J = 2.3 Hz, 1H), 2.41–2.35 (m,
H), 1.86–1.76 (m, 5H), 1.50 (s, 9H), 1.44–1.39 (m, 0.6×1H), 1.40–1.26 (m, 5H). MS
(
2
+
+
(
ESI) m/z 520.0 [M+H] . HRMS calcd for C H N O [M+H] 520.3175, found
31 41 3 4
5
4
20.3159.
.1.17.9. N-cyclohexyl-N-cyclopropyl-4-((1-(2-nitro-5-
phenoxyphenyl)ethyl)amino)butanamide (18a). To a solution of 2-(1-mesyloxyethyl)-1-
nitro-4-phenoxybenzene (0.250 g, 0.731 mmol) in acetonitrile (0.5 M) was added 4-
amino-N-cyclohexyl-N-cyclopropylbutanamide (8b, 0.360 g, 1.4 mmol) and K CO
2
3
2
9