Potent and Selective Inhibitors of Histone Deacetylase-3 Containing Chiral Oxazoline Capping Groups and a N-(2-Aminophenyl)-benzamide Binding Unit

Article abstract of DOI:10.1021/acs.jmedchem.5b00545

A novel series of potent chiral inhibitors of histone deacetylase (HDAC) is described that contains an oxazoline capping group and a N-(2-aminophenyl)-benzamide unit. Among several new inhibitors of this type exhibiting Class I selectivity and potent inhibition of HDAC3-NCoR2, in vitro assays for the inhibition of HDAC1, HDAC2, and HDAC3-NCoR2 by N-(2-aminophenyl)-benzamide 15k gave respective IC₅₀ values of 80, 110, and 6 nM. Weak inhibition of all other HDAC isoforms (HDAC4, 5, 6, 7, and 9: IC₅₀ > 100000 nM; HDAC8: IC₅₀ = 25000 nM; HDAC10: IC₅₀ > 4000 nM; HDAC11: IC₅₀ > 2000 nM) confirmed the Class I selectivity of 15k. 2-Aminoimidazolinyl, 2-thioimidazolinyl, and 2-aminooxazolinyl units were shown to be effective replacements for the pyrimidine ring present in many other 2-(aminophenyl)-benzamides previously reported, but the 2-aminooxazolinyl unit was the most potent in inhibiting HDAC3-NCoR2. Many of the new HDAC inhibitors showed higher solubilities and lower binding to human serum albumin than that of Mocetinostat. Increases in histone H3K9 acetylation in the human cell lines U937 and PC-3 was observed for all three oxazolinyl inhibitors evaluated; those HDAC inhibitors also lowered cyclin E expression in U937 cells but not in PC-3 cells, indicating underlying differences in the mechanisms of action of the inhibitors on those two cell lines.

Full text of DOI:10.1021/acs.jmedchem.5b00545

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Article
Potent and Selective Inhibitors of Histone Deacetylase-3 containing Chiral
Oxazoline Capping Groups and a N-(2-Aminophenyl)-benzamide Binding Unit
Charles M. Marson, Christopher J Matthews, Stephen J
Atkinson, Nermina Lamadema, and N. Shaun B. Thomas
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Aug 2015
Downloaded from http://pubs.acs.org on August 19, 2015
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Potent and Selective Inhibitors of Histone Deacetylase-3 containing Chiral Oxazoline
Capping Groups and a N-(2-Aminophenyl)-benzamide Binding Unit
,a
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Charles M. Marson,* Christopher J. Matthews, Stephen J. Atkinson, Nermina Lamadema
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and N. Shaun B. Thomas
a
Department of Chemistry, University College London,
Christopher Ingold Laboratories, 20 Gordon Street, London WC1H OAJ, U.K.
b
Epinova DPU, Immuno-Inflammation Therapy Area Unit, GlaxoSmithKline, Gunnels Wood
Road, Stevenage, Herts SG1 2NY, U.K.
d
Department of Haematological Medicine, Leukaemia Sciences Laboratories, Rayne
Institute, King’s College London, 123 Coldharbour Lane, London SE5 9NU, U.K.
Abstract: A novel series of potent chiral inhibitors of histone deacetylase (HDAC) is
described that contains an oxazoline capping group and a Nꢀ(2ꢀaminophenyl)ꢀbenzamide
unit. Among several new inhibitors of this type exhibiting Class I selectivity and potent
inhibition of HDAC3ꢀNCoR2, in vitro assays for the inhibition of HDAC1, HDAC2, and
HDAC3ꢀNCoR2 by the Nꢀ(2ꢀaminophenyl)ꢀbenzamide 15k gave respective IC50 values of
8
0, 110 and 6 nM. Weak inhibition of all other HDAC isoforms (HDAC4, 5, 6, 7 and 9: IC50
>
100,000 nM; HDAC8: IC50 = 25,000 nM; HDAC10: IC50 > 4000 nM; HDAC11: IC50
>
2000 nM) confirmed the Class I selectivity of 15k. 2ꢀAminoimidazolinyl, 2ꢀthioimidazolinyl
and 2ꢀaminooxazolinyl units were shown to be effective replacements for the pyrimidine
ring present in many other 2ꢀ(aminophenyl)ꢀbenzamides previously reported, but the 2ꢀ
aminooxazolinyl unit was the most potent in inhibiting HDAC3ꢀNCoR2. Many of the new
HDAC inhibitors showed higher solubilities and lower binding to human serum albumin
than Mocetinostat. Increases in histone H3K9 acetylation in the human cell lines U937 and
PCꢀ3 was observed for all three oxazolinyl inhibitors evaluated; those HDAC inhibitors also
lowered cyclin E expression in U937 cells but not in PCꢀ3 cells, indicating underlying
differences in mechanisms of action of the inhibitors on those two cell lines.
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Introduction
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Epigenetic mechanisms are relevant to a wide range of diseases including cancer, diabetes,
heart disease and neurological disorders. At physiological pH the alkylammonium units
which form upon deacetylation of the εꢀamino group of lysine residues in histone proteins
bind closely to the negatively charged DNA phosphate groups. Hypoacetylation arising from
increased histone deacetylase (HDAC) activity results in chromatin compaction and
therefore transcriptional repression and aberrant gene regulation associated with
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precancerous or malignant states. Inhibitors of histone deacetylase (HDAC) have been
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much studied as means of epigenetic cancer therapy, and can be effective in the treatment
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,8
of certain leukemias, partly through relief of transcriptional repression. HDAC inhibitors
also reduce cancer cell proliferation by induction of cell cycle arrest, differentiation and/or
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apoptosis.
Class I HDAC inhibitors, especially HDAC1, HDAC2 and HDAC3 are
considered key targets for cancer treatment.
Although Romidepsin, a macrocyclic peptide containing a disulfide bridge, is a
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potent HDAC inhibitor used in the treatment of cutaneous Tꢀcell lymphoma, most Class I
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,6
HDAC inhibitor antiꢀcancer agents are of two structural types: hydroxamic acids,
including Vorinostat (suberoylanilide hydroxamic acid) used in the treatment of cutaneous
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Tꢀcell lymphoma, Belinostat used to treat peripheral Tꢀcell lymphoma, and Panobinostat
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for the treatment of multiple myeloma; and aminoanilides (Fig. 1) such as Chidamide,
approved in China for the treatment of pancreatic cancer; Mocetinosat, in clinical trials for
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the treatment of myelogenous leukemia;
and Entinostat (MSꢀ275) which was in Phase I
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clinical trials for the treatment of metastatic melanoma. Additionally, the HDAC3ꢀselective
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inhibitor RGFP966 has been studied for its effect on cutaneous Tꢀcell lymphoma. Given
the limitations of in vivo efficacy and toxicity observed with many hydroxamic acids, new
aminoanilide inhibitors could be expected to show prolonged inhibitory effects and longer
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intervals between dosing;
the slow, tightꢀbinding of Mocetinostat to HDACs and its
slow decomplexation, may be significant factor, and the observation of a lack of acquired
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resistance of Mocetinostat being a potential advantge. Among aminoanilide Class I HDAC
inhibitors, the factors that govern isoform selectivity have been little investigated,
particularly the effect of chirality on the binding of inhibitors to various HDAC isoforms.
Herein are described the synthesis and preliminary evaluation of a new series of
aminoanilides that contain a chiral oxazoline unit and which possess specificity for HDAC3.
HDAC3 is a requirement for hematopoietic stem/progenitor cells to pass through the S phase
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of the cell cycle, and also for stem cell function and lymphopoiesis. In various cancers
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including acute myelogenous leukemia
and lymphoma
recruitment of SMRT/NꢀCoRꢀ
HDAC3 complexes that induce hypoacetylation leads to significant transcriptional
repression. Additionally, HDAC3 is the target isoform associated with the progressive
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neurodegenerative disease Friedrich’s ataxia.
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Chemistry
A series of analogs of Mocetinostat was sought that possessed the same linker and
zincꢀbinding group but a fiveꢀmembered heterocyclic replacement for the pyrimidine ring.
Chirality would be installed within the fiveꢀmembered heterocycles that also contain a
heteroatom linker at the 2ꢀposition. A 2ꢀaminooxazolinyl unit was selected as a preferred
potential replacement in view of its low clogP (ꢀ0.53 c.w. ꢀ0.22 for 2ꢀaminopyrimidine), high
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solubility, its established use in drug design, and the availability of a variety of enantiopure
ꢀamino alcohols.
For comparison against the new oxazolinyl HDAC inhibitors, selected fiveꢀmembered
rings with either 2ꢀthiomethylꢀ or 2ꢀaminomethylꢀlinkages were prepared. Sꢀalkylation of (S)ꢀ
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4ꢀphenylimidazolidineꢀ2ꢀthione (4) with N-(2ꢀaminophenyl)ꢀ4ꢀ(chloromethyl)benzamide
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(
1) in acetone at reflux afforded the thioimidazoline 5 in 66% yield (Scheme 1), a succinct
route that did not require protection of the arylamino group.
The aminoimidazoline 8 was also prepared for purposes of comparison. Since
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methods for the preparation of 2ꢀ(alkylamino)imidazolines are limited, especially
enantiopure examples, a twoꢀstep approach was developed (Scheme 1). Heating
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isothiocyanate 2 with diamine 3 in THF at reflux afforded the thiourea 6 (57%) which was
quantitatively cyclised in acetone in the presence of iodomethane to give the
aminoimidazoline 7 which was deprotected using TFA in dichloromethane to give the
required aminoimidazoline 8 (47%).
A succinct route to the 2ꢀ(thiomethyl)oxazolines 11 was developed, also without the
need for protection of the arylamino group (Scheme 2). The oxazolidineꢀ2ꢀthiones 10,
prepared by reaction of the 2ꢀaminoalcohols 9 with carbon disulfide in ethanol in the
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presence of K
2
CO
3
at 50 °C, followed by treatment with hydrogen peroxide, underwent Sꢀ
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alkylation with N-(2ꢀaminophenyl)ꢀ4ꢀ(chloromethyl)benzamide (1) in acetone at reflux to
give the corresponding 2ꢀ(thiomethyl)oxazolines 11 (Scheme 2). With suitable ring systems
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now in hand for comparison, attention was turned to the synthesis of 2ꢀalkylaminooxazolines,
the primary replacement motif.
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Reaction of (2S)ꢀ2ꢀaminoꢀ2ꢀphenylethanꢀ1ꢀol (12b)
with tertꢀbutyl Nꢀ{2ꢀ[4ꢀ
(isothiocyanatomethyl)benzamido]phenyl}carbamate (2) in THF afforded the thiourea (S)-
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13b (89%). Yellow mercuric oxide has been used to cyclise Nꢀ(βꢀhydroxyethyl)thioureas to
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the corresponding 2ꢀaminoꢀ4,5ꢀdihydrooxazoles.
However, (S)-13b did not cyclise to (S)-
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4b using the conditions of Uchida. Accordingly, Hirashima’s conditions were
investigated, and modification of those using additional yellow mercuric oxide and a longer
reaction time proved efficient, affording (S)-14b in 76% yield. The benzyl derivative (S)-14a
was similarly prepared (92%). In those reactions, it was found essential to use freshlyꢀ
prepared yellow mercuric oxide in order to obtain satisfactory yields; an aged commercial
sample gave only low yields of dihydrooxazoles (S)-14a and (S)-14b even after extended
reaction periods. Boc deprotection was achieved using TFA in dichloromethane to give the
dihydrooxazoles (S)-15a and (S)-15b in 47% and 74% yields, respectively.
Although the above cyclisations of the Nꢀ(βꢀhydroxyethyl)thioureas (S)-13a and (S)-
13b using yellow mercuric oxide were effective, the need for several equivalents of this toxic
reagent led to a search for a more benign synthetic method. One example of such a
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cyclisation using iodomethane has been described, and this method was found to be
applicable to the preparation of a wide variety of Nꢀ(βꢀhydroxyethyl)thioureas, 14c-l. Boc
deprotection using TFA in dichloromethane afforded the corresponding dihydrooxazoles 15c-
l.
Results and Discussion
Of the few reports of alternative ring systems to the pyrimidine ring in Mocetinostat,
replacement has involved the use of only planar, fused rings that decrease the solubility,
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albeit affording compounds with therapeutic potential. Several analogs with planar rings in
the capping group other than pyrimidine were less potent against HDAC1 and several cell
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lines than was Mocetinostat.
SAR detail regarding the cap region of Mocetinostat
congeners has been limited, especially in regard the potential influence of chirality; to our
knowledge, there is only one publication describing chiral heterocyclic cap analogs of such
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aminoanilides.
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Our previous study showed that fiveꢀmembered semiꢀsaturated ring systems such as
imidazolinꢀ4ꢀone can be effective as replacement for the pyrimidine ring in Mocetinostat,
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and promisingly, even in the absence of the terminal 3ꢀpyridyl ring present in Mocetinostat.
In the absence of a 3ꢀpyridyl group, few analogs of Mocetinostat (in which the pyrimidine
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ring was retained) are potent HDAC inhibitors. Since the carbonyl group of substituted
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imidazolinꢀ4ꢀones rendered an adjacent chiral centre prone to racemisation, 1,2ꢀ
dihydroazole units, as pyrimidine replacements, became the focus of the present work. The
greater saturation of most 1,2ꢀdihydroazoles, and the greater solubility of compounds with
unsaturated oxazoline and imidazoline rings compared to their pyrimidine counterparts were
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additional potential advantages in improving physicochemical properties.
Comparison of the (4S)ꢀ4ꢀphenylꢀ4,5ꢀdihydroꢀ1Hꢀimidazole derivative 8 with
Mocetinostat (Table 1, entries 1 and 2) shows that a smaller and partly saturated imidazoline
ring system is consistent with HDAC inhibition, its IC50 for HDAC3 inhibition showing
threeꢀfold greater potency than Mocetinostat; moreover, whereas inhibition of HDAC1,
HDAC2 and HDAC3ꢀCoR2 by Mocetinostat is similar (and less than fiveꢀfold different for
isoforms 1 and 3), inhibition of HDAC3CoR2 by 8 is 16 times greater than HDAC1 and 18
times greater than HDAC2. Comparison of entry 2 with entries 3ꢀ5 shows that the 2ꢀ
aminoalkyl substituent is important for potent HDAC3 inhibition, the 2ꢀthioalkyl compounds
being appreciably less potent.
For the 2ꢀaminoarylmethyꢀ4ꢀarylꢀsubstituted dihydrooxazole series, little variation
in the potent inhibition of HDAC3ꢀCoR2 (IC = 24ꢀ40 nM) was found. However, the pair of
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enantiomers 15b (entries 7 and 8) show that binding to HDAC1 is significantly influenced
by the absolute configuration, the (4R)ꢀenantiomer being 6ꢀfold more potent, whereas
inhibition of HDAC2 and HDAC3ꢀCoR2 is little affected by the stereochemistry of the
inhibitor. A similar pattern is observed for the enantiomeric pair of 2ꢀaminoarylmethyꢀ5ꢀ
phenyl dihydrooxazoles 15h, the (5R)ꢀenantiomer being 5ꢀfold more potent at inhibiting
HDAC1. The simplest explanation of those results is that rotation about the exocyclic CꢀN
bonds permit the phenyl group of (R)ꢀ15b to adopt the same orientation as that adopted by
the phenyl group in (R)ꢀ15h, bonding of HDAC1 to the respective N and O atoms of the
heterocyclic rings not being the determining factor. The enantiomeric pair 15h exhibit some
preferential inhibition: (S)ꢀ15h of HDAC2, and (R)ꢀ15h of HDAC1.
The relatively bulky and diastereoisomeric 4,5ꢀdiphenylꢀ4,5ꢀdihydrooxazoles 15j
and 15k are potent inhibitors of HDAC3ꢀCoR2, 15k being the more potent, and also with
some 13ꢀ and 18ꢀfold selectivity over HDAC1 and HDAC2, respectively. Compared to 15b,
the additional phenyl ring present in 15k increases by fiveꢀfold the inhibition of HDAC3ꢀ
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CoR2. Those results are consistent with an extended lipophilic region that can accommodate
the cisꢀ1,2ꢀdiphenyl unit.
Other features are also of note. Compared to (R)ꢀ15b, the pꢀ and mꢀfluoro
substituents in 15d and 15e have little effect on the inhibition of HDAC2 and HDAC3ꢀ
CoR2. The terminal 3ꢀpyridyl capping group, present in Mocetinostat, is of little or no
advantage as compared with phenyl, in these fiveꢀmembered heterocyclic systems (compare
entries 4 and 5, and entries 7 and 13). The more flexible benzyl substituent may confer
somewhat greater inhibition of HDAC1 and HDAC2 (entries 4 and 6, and entries 7 and 14)
as compared with phenyl, but showed no advantage over phenyl in regard to inhibition of
HDAC3ꢀCoR2. The same trends were observed for the 4ꢀ(1Hꢀimidazolylmethyl)ꢀ4,5ꢀ
dihydrooxazole pair of enantiomers 15l (entries 20 and 21).
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The appreciable selectivity (up to 18ꢀfold) for HDAC3 over HDAC2 for several of
the compounds in this work is significant, having regard to the close homology of those two
isoforms. All compounds showed feeble inhibition of HDAC6, consistent with the expected
18,27
preference within Class I HDAC isoform selectivity observed for other amino anilides,
18
and very low or negligible inhibition of HDAC8, also as expected, given previous studies.
The class I selectivity of 15k, the most potent HDAC3 inhibitor (IC50 = 0.006 ꢀM) of this
study, was further shown by evaluation against the Class 2 isoforms HDAC 4, HDAC5,
HDAC6, HDAC7 and HDAC9, IC50 values for each enzyme being > 100 ꢀM. Weak
inhibition was observed for HDAC8 (IC50 = 25 ꢀM, Class 1), as well as little activity
against HDAC10 (IC > 2 ꢀM, Class 2) and HDAC 11 (IC > 4 ꢀM, Class 4).
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Physicochemical data (Table 2) for the 5ꢀmembered heterocyclic HDAC inhibitors
are generally favourable, especially with similar and in many cases higher solubilities and
lower binding to human serum albumin as compared to Mocetinostat, with the exception of
the relatively lipophilic inhibitors 11a, 15d and 15iꢀk. The 3ꢀpyridyl compound 15c, the
oxazolinyl analog of Mocetinostat, showed equipotent inhibition of HDAC3ꢀCoR2 but
approximately tenꢀfold selectivity over HDAC1 and HDAC2. HDAC inhibitors with one
aryl substituents at the 4ꢀ or 5ꢀposition of the oxazoline ring showed suitable
physicochemical properties (Table 2) and depending on the stereochemistry, high potency
against HDAC3ꢀCoR2, usually with significant isoform selectivity.
Biology
Changes in histone acetylation status were assayed by analyzing histone H3 acetylated
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on lysine 9 (H3K9Ac) upon addition of Nꢀ(2ꢀaminophenyl)ꢀbenzamides. To determine
whether compounds 15e, (S)-15h, (R)-15h and Mocetinostat increase histone H3 acetylation
levels, the human cell lines U937 and PCꢀ3 were cultured in the presence of each compound
(
10 ꢁM), or an equivalent volume of the diluent (DMSO) added as the control. The cells
were cultured for 24 h and H3K9Ac was analyzed by Western blotting. The data show that at
0 ꢁM each of the above compounds caused an increase in histone H3K9Ac with respect to
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histone H3 expression in U937 and PCꢀ3 cells. (Fig. 1). These data are consistent with an
inhibition of endogenous cellular HDAC activity that causes net acetylation of histone
H3K9.
Previous studies showed that Mocetinostat causes an increase in H3K9Ac in HeLa
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cells and leads to cell death by apoptosis. Consequently, in the present study flow
cytometry analyses of DNA and protein content of cells cultured with the compounds were
carried out to determine whether the compounds 15e, (R)-15h and (S)-15h also cause
apoptosis. The data obtained (Figs. 2 and 3) show that for each of the compounds tested at
1
0 ꢁM in both U937 and PCꢀ3 cells there is an increase in the percentage of cells with sub-
DNA content, consistent with induction of apoptosis. However, PCꢀ3 cells cultured with
each of the compounds 15e, (R)-15h, (S)-15h at 10 ꢁM showed accumulation in G /G
G
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which was not observed for U937 cells treated with those compounds, and indicates a
difference in underlying mechanisms in these two cell types. In contrast, culturing PCꢀ3 cells
with Mocetinostat caused an accumulation of cells in G2/M. When cultured with U937 cells
1
Mocetinostat also resulted in the most significant increase in sub-G DNA content. Cyclin E
expression levels in U937 cells were lowered upon treatment with all compounds, and most
significantly with Mocetinostat and with (R)-15h; however, significant changes in cyclin E
expression levels were not observed in PCꢀ3 cells using any of the above HDAC inhibitors.
Inhibition of HDAC activity by different compounds can cause changes in Cyclin E
48,49
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expression,
among these compounds could be due to greater isoform selectivity of 15e, (R)-15h and (S)-
5h compared to Mocetinostat, although that would need to be investigated in a further
but this does not occur in all cases. Differences in the biological effects
1
study.
Summary
A new capping group, a chiral oxazoline unit bearing one or more aryl substituents, has
been shown to be effective as a substructure of Nꢀ(2ꢀaminophenyl)ꢀbenzamide HDAC
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inhibitors. Inhibition of HDAC1, HDAC2 and HDAC3ꢀNCoR2 (Class I isoforms) was
generally potent, some compounds in this novel class of inhibitors possessing low nanomolar
potency against HDAC3ꢀNCoR2, and greater than that of Mocetinostat, one of the most potent
benzamide HDAC inhibitors previously reported. Highly convergent routes were established to
HDAC inhibitors containing a variety of enantiopure heterocyclic capping groups by use of a
preassembled Nꢀ(2ꢀaminophenyl)ꢀbenzamide unit, new methodology that has been shown to be
applicable to the synthesis of several fiveꢀmembered heterocyclic systems linked by a 2ꢀamino
or 2ꢀthio substituent.
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To our knowledge, this is the first extensive study of the effect of stereochemistry on
HDAC inhibition. These results show that stereochemistry, both absolute and relative, can be
used to distinguish the affinity of inhibitors to various HDAC isoforms, and that in some cases
preferential inhibition of an HDAC isoform can be exhibited by one enantiomer, even when
only one chiral centre is present. Further optimisation of HDAC isoform potency and
selectivity is likely to be achievable, and physicochemical data show promise for the
development of new drugꢀlike benzamide HDAC inhibitors. Such HDAC inhibitors may
benefit from the absence of the commonly required 3ꢀpyridyl terminal capping group and its
metabolism to the Nꢀoxide. This study demonstrates that, at least in terms of HDAC inhibition,
inhibitor solubility and low HSA binding, an oxazoline ring can beneficially replace the
pyrimidine ring present in Mocetinostat, and further that its pyridine ring can also be replaced,
such inhibitors leading to restoration of acetylation status in the two cell lines studied. Thus,
each of compounds 15e, (S)-15h, (R)-15h and Mocetinostat (at 10 ꢁM) increased histone
H3K9 acetylation in U937 and PCꢀ3 cell lines. The results of this study illustrate some
advantages of developing new Class Iꢀselective HDAC inhibitors that incorporate as part of the
capping region a saturated or partly saturated heterocyclic ring with substituents contributing to
a center, or centers, of defined absolute configuration.
Experimental Section
Chemistry. All chemicals were used as supplied, except oꢀphenylenediamine which was
recrystallized from ethanol. Solvents used were reagent grade, and anhydrous solvents were
obtained from Anhydrous Engineering (USA) solvent systems after passing through an
alumina column. Compound homogeneity was monitored by ascending thinꢀlayer
chromatography performed on Merck 0.2 mm aluminumꢀbacked silica gel 60 F254 plates and
visualized using an alkaline potassium permanganate dip or by ultraviolet light. Flash column
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chromatography was performed using Merck 0.040 to 0.063 mm, 230 to 400 mesh silica gel.
Evaporation refers to the removal of solvent under reduced pressure. Melting points were
determined using an Electrothermal digital melting point apparatus. Infrared (IR) spectra were
recorded on a PerkinꢀElmer spectrum 100 FTꢀIR spectrometer as neat powders or as thin films.
1
H NMR spectra were recorded at 300 MHz on a Bruker AMX300 spectrometer, at 400 MHz
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on a Bruker AMX400, at 500 MHz on a Bruker Avance 500 spectrometer, or at 600 MHz on a
Bruker Avance 600 spectrometer in the stated solvent; chemical shifts are reported in δ (ppm)
relative to the internal reference tetramethylsilane. Mass spectra were obtained on a Fisons
VG70ꢀSE mass spectrometer or Thermo Finnigan MAT900xp instrument. Purity of tested
compounds was assessed to be at least 95% by LCMS analysis unless otherwise indicated. For
all samples 0.1% TFA was added to both eluents.
:
The following compounds were prepared according to the literature 2ꢀaminoꢀ2,2ꢀ
5
1
52
diphenylethanol;
(S)ꢀ2ꢀaminoꢀ2ꢀphenylacetamide;
methyl (S)ꢀ2ꢀaminoꢀ2ꢀphenylacetate
5
3
34
hydrochloride;
phenylenediamine;
methylpropaneꢀ2ꢀsulfinamide;
phenylimidazolidineꢀ2ꢀthione (4);
N-(2ꢀaminophenyl)ꢀ4ꢀ(chloromethyl)benzamide
(1);
NꢀBocꢀoꢀ
3
4
(S)ꢀN-((S)ꢀ2ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀ1ꢀ(pyridin-3ꢀyl)ethyl)ꢀ2ꢀ
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(S)ꢀ1ꢀphenylethaneꢀ1,2ꢀdiamine
(3);
(S)ꢀ4ꢀ
(S)ꢀ4ꢀ
3
2
54
(S)ꢀ2ꢀaminoꢀ2ꢀ(pyridinꢀ3ꢀyl)ethanol (9c);
56
56
benzyloxazolidineꢀ2ꢀthione (10a); (S)ꢀ4ꢀphenyloxazolidineꢀ2ꢀthione (10b); (S,E)ꢀN-(2ꢀ
56
((tertꢀbutyldimethylsilyl)oxy)ethylidene)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide;
(2S)ꢀ2ꢀaminoꢀ2ꢀ
3
6
57
phenylethanꢀ1ꢀol (12b); 2ꢀaminoꢀ2ꢀ(3ꢀfluorophenyl)ethanol (12c); tertꢀbutyl (R)ꢀ(2ꢀ(4ꢀ((3ꢀ
34
(2ꢀhydroxyꢀ1ꢀphenylethyl)thioureido)methyl)benzamido)phenyl)carbamate (S)-13a;
tertꢀ
butyl (S)ꢀ(2ꢀ(4ꢀ((3ꢀ(2ꢀhydroxyꢀ1ꢀphenylethyl)thioureido)methyl)benzamido)phenyl)carbamate
3
4
(S)-13b;
Nꢀ(2ꢀaminophenyl)ꢀ4ꢀ({[4ꢀ(pyridinꢀ3ꢀyl)pyrimidinꢀ2ꢀyl]amino}methyl)benzamide
58
(Mocetinostat).
tert-Butyl N-{2-[4-(isothiocyanatomethyl)benzamido]phenyl}carbamate (2). A literature
34
procedure was modified as follows. Carbon disulfide (3.48 mL, 57.5 mmol) was added to a
mixture of 4ꢀ(aminomethyl)benzoic acid (3.0 g, 19.8 mmol) and triethylamine (6.62 mL,
47.5 mmol) in THF (13 mL) and water (13 mL). The mixture was stirred vigorously for 22
h, then cooled to 0 °C and a solution of iodine (5.38 g, 21.2 mmol) in THF (13 mL) was
added dropwise over 10 min. The mixture was stirred at 0 °C for 2 h, then hydrochloric acid
(20 mL, 1M) and sodium sulfite (0.49 g, 3.9 mmol) were added and mixture stirred. The
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mixture was then extracted with ethyl acetate (100 mL, then 50 mL), and the combined
organic layers were washed with hydrochloric acid (2 × 50 mL, 1M) then with brine (50
4
mL), dried (MgSO ) and evaporated to give 4ꢀ(isothiocyanatomethyl)benzoic acid as a cream
solid. To a suspension of the cream solid in dichloromethane (35 mL) was added oxalyl
chloride (2.28 mL, 27.0 mmol) dropwise, followed by a few drops of DMF. The resulting
mixture was stirred at 20 °C for 1 h (until evolution of gas had ceased) and the resulting
solution was stirred at reflux for 1 h. Evaporated gave a residue that was coꢀevaporated with
toluene (20 mL) to ensure complete removal of oxalyl chloride. The residue was dissolved in
dichloromethane (25 mL) and the solution was added dropwise over 10 min to a solution of
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NꢀBocꢀoꢀphenylenediamine (3.75 g, 18 mmol) in dichloromethane (25 mL) and pyridine
25 mL) at 0 °C. The mixture was stirred at 0 °C for 1 h then warmed to 20 °C and stirred for
(
a further 16.5 h. Then aqueous saturated sodium hydrogen carbonate (150 mL) was added
and the mixture was extracted with chloroform (2 × 100 mL). The combined organic layers
were washed with hydrochloric acid (2 × 150 mL, 1M) then with brine (150 mL), and dried
4
MgSO and evaporated to give an orange oil that was purified by column chromatography
(3% ethyl acetate/dichloromethane) to give 2 (5.11 g, 74%) as a cream solid. Spectroscopic
3
4
data were as previously reported.
(S)-N-(2-Aminophenyl)-4-(((4-phenyl-4,5-dihydro-1H-imidazol-2-
yl)thio)methyl)benzamide (5). To a solution of thione 4 (80 mg, 0.45 mmol) and benzyl
chloride 1 (117 mg, 0.45 mmol) in acetone (5 mL) was added potassium carbonate (93 mg,
0.673 mmol). The mixture was then stirred at reflux for 17 h. After allowing to cool the
solvent was evaporated, and the residue was partitioned between water (15 mL) and ethyl
acetate (30 mL). The aqueous layer was extracted with ethyl acetate (2 x 30 mL) and the
combined organic layers were washed with brine (15 mL), dried (Na SO ) and evaporated to
2
4
give a beige solid (200 mg) that was purified by column chromatography on silica gel (5:95
methanol:ethyl acetate) to give a white solid (120 mg, 66%). A small sample was further
purified by recrystallization from chloroform to give 5 as a white solid, mp 150ꢀ151 °C;
ꢁ
ꢂ
ꢀ1
1
[α] ꢀ2.1 (c 1.0, methanol); νmax (cm ) 3239, 1688, 1642; H NMR (600 MHz, chloroformꢀ
ꢀ
d) δ ppm 3.58 (1H, br s), 3.86 (2H, br s), 4.12 (1H, br s), 4.39 (1H, d, J=13.6 Hz), 4.44 (1H,
d, J=13.6 Hz), 4.97 (1H, br s), 6.82 ꢀ 6.89 (2H, m), 7.10 (1H, m), 7.22 ꢀ 7.29 (2H, m), 7.30 ꢀ
1
3
7.36 (3H, m), 7.53 (2H, d, J=7.9 Hz), 7.81 ꢀ 7.92 (3H, m); C NMR (150 MHz, chloroformꢀ
d) δ ppm 35.2, 118.6, 120.0, 124.7, 125.2, 126.4, 127.4, 127.7, 128.8, 129.6, 133.3, 140.7,
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+
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23 4
41.9, 143.4, 162.8, 165.5; m/z (ESI ) 403 ([M+H] , 100%); HRMS C23H N OS requires:
403.1587, found: 403.1577.
(S)-tert-Butyl
(2-(4-((3-(2-amino-2-
phenylethyl)thioureido)methyl)benzamido)phenyl)carbamate (6). To a solution of (S)ꢀ1ꢀ
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phenylethaneꢀ1,2ꢀdiamine (213 mg, 1.56 mmol) in THF (4 mL) was added a solution of 2
(
200 mg, 0.522 mmol) in THF (4 mL). The resulting solution was stirred at 20 °C for 17.5 h,
then evaporated, and the residue was purified by flash column chromatography on silica gel
gradient elution from 1ꢀ8% of methanolic 2M ammonia in dichloromethane) to give 6 (154
(
ꢀ1
1
mg, 57%) as a white solid, mp 105ꢀ106 °C; νmax (cm ) 3276, 1689, 1660; H NMR (500
MHz, chloroformꢀd) δ ppm 1.47 ꢀ 1.54 (9H, m), 2.32 (4H, br s), 3.50 (1H, m), 3.76 (1H, br
s), 4.12 (1H, dd, J=8.4, 4.3 Hz), 4.74 (2H, br s), 7.03 (1H, br s), 7.10 ꢀ 7.20 (3H, m), 7.20 ꢀ
1
3
7
.44 (7H, m), 7.65 (1H, m), 7.78 (2H, d, J=7.7 Hz), 9.35 (1H, br s); C NMR (125 MHz,
chloroformꢀd) δ ppm 28.3, 48.0, 51.8, 55.3, 81.5, 124.7, 125.8, 126.2, 126.3, 127.7, 127.7,
+
+
1
27, 128.8, 130.4, 130.5, 132.9, 142.2, 154.7, 165.9, 183.4; m/z (ESI ) 520 ([M+H] , 100%);
+
HRMS C28
H
34
N
5
O
3
S requires: 520.2377, found: 520.2365.
(
S)-tert-Butyl
yl)amino)methyl)benzamido)phenyl)carbamate (7). To a solution of thiourea 6 (154 mg,
.296 mmol) in acetone (3 mL) was added iodomethane (0.037 mL, 0.593 mmol) and the
(2-(4-(((4-phenyl-4,5-dihydro-1H-imidazol-2-
0
solution stirred at 20 °C for 18 h. Saturated aqueous sodium hydrogen carbonate (1.5 mL)
was added and the mixture was stirred for 10 min. Saturated aqueous sodium hydrogen
carbonate (20 mL) and water (5 mL) were added and the mixture was then extracted with
2 4
ethyl acetate (2 x 20 mL). The combined organic layers were dried (Na SO ) and evaporated
ꢀ1
to give 7 (143 mg, 99%) as a cream solid, mp 130ꢀ132 °C; νmax (cm ) 3171 (NH), 1667
1
(
C=O); H NMR (400 MHz, chloroform-d) δ ppm 1.42 (9H, s, C(CH
3
)
3
), 3.35 (1H, t, J=7.0
Hz), 3.84 (1H, t, J=8.1 Hz), 4.53 (2H, br s), 4.86 (1H, br s), 7.04 (1H, t, J=7.3 Hz), 7.10 ꢀ
7
.22 (3H, m), 7.22 ꢀ 7.37 (6H, m), 7.46 (1H, d, J=7.8 Hz), 7.50 (1H, d, J=7.6 Hz), 7.69 (1H,
1
3
br s), 7.86 (2H, d, J=6.6 Hz), 8.77 (1H, br s), 9.83 (1H, br s); C NMR (126 MHz,
chloroform-d) δ ppm 28.3, 46.3, 51.2, 58.7, 81.1, 124.8, 125.1, 126.1, 126.1, 126.4, 127.4,
+
1
28.3, 128.7, 129.1, 129.8, 131.5, 133.2, 139.0, 140.0, 154.5, 159.3, 166.2; m/z (ESI ) 486
+
+
(
[M+H] , 100%); HRMS C28
32 5 3
H N O requires: 486.2500, found: 486.2496. (90% purity by
LCMS and NMR).
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(
S)-N-(2-Aminophenyl)-4-(((4-phenyl-4,5-dihydro-1H-imidazol-2-
yl)amino)methyl)benzamide (8). To a solution of 7 (140 mg, 0.288 mmol) in
dichloromethane (3 mL) was added trifluoroacetic acid (0.44 mL, 5.77 mmol) dropwise. The
resulting solution was stirred at 20 °C for 2 h, then evaporated and the resulting white solid
purified by MDAP to give 8 (52 mg, 47%) as a white solid, mp 130ꢀ133 °C; [α] ꢁꢀ ꢂ +11.1 (c
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4
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7
8
9
0
ꢀ1
1
1
.0, methanol); νmax (cm ) 3217, 1672, 1613; H NMR (400 MHz, methanolꢀd ) δ ppm 3.41
4
(1H, dd, J=9.9, 7.2 Hz), 4.02 (1H, t, J=9.8 Hz), 4.47 ꢀ 4.59 (2H, m), 5.01 (1H, dd, J=9.5, 7.3
Hz), 6.79 (1H, t, J=7.3 Hz), 6.93 (1H, dd, J=8.1, 0.7 Hz), 7.10 (1H, td, J=7.6, 1.2 Hz), 7.21
1
3
(1H, d, J=7.6 Hz), 7.26 ꢀ 7.41 (5H, m), 7.53 (2H, d, J=8.2 Hz), 8.01 (2H, d, J=8.2 Hz); C
NMR (100 MHz, methanolꢀd
4
) δ ppm 45.7, 54.0, 61.3, 117.4, 118.3, 123.9, 125.9, 126.3,
+
127.0, 127.2, 127.5, 127.8, 128.4, 133.2, 142.4, 142.6, 161.3, 167.2; m/z (ESI ) 386
+
+
([M+H] , 100%); HRMS C H N O requires: 386.1975, found: 386.1974.
23 24 5
(
S)-4-(Pyridin-3-yl)oxazolidine-2-thione (10c). To a flask located behind a blast shield
that contained a stirred mixture of amino alcohol 9c (160 mg, 1.19 mmol), potassium
carbonate (80 mg, 0.58 mmol) and carbon disulfide (0.14 mL, 2.32 mmol) in ethanol (1 mL)
at 50 °C was added dropwise hydrogen peroxide (0.15 mL, 1.72 mmol) (Caution:
Exotherm!). After completion of addition the mixture was cooled to 20 °C and filtered. The
filtrate was diluted with ethyl acetate (30 mL), washed with water (2 x 15 mL), aqueous
2 4
sodium sulfite (10%, 15 mL) and brine, then dried (Na SO ) and evaporated. The yellow
solid was purified by flash column chromatography on silica gel (0ꢀ5% of 2M
ammonia/methanol in dichloromethane) gave 10c (29 mg, 14%) as a white solid, mp 157ꢀ
ꢀ1
1
1
59 °C; νmax (cm ) 3083; H NMR (400 MHz, methanolꢀd
4
) δ ppm 4.52 (1H, dd, J=9.2, 6.2
Hz), 5.04 (1H, t, J=9.2 Hz), 5.28 (1H, dd, J=9.2, 6.2 Hz), 7.53 (1H, dd, J=7.8, 4.9 Hz), 7.87
13
(1H, d, J=7.8 Hz), 8.49 ꢀ 8.63 (2H, m); C NMR (100 MHz, methanolꢀd
4
) δ ppm 57.6, 76.8,
+
+
+
1
8 9 2
24.4, 134.8, 136.0, 147.2, 149.1, 190.2; m/z (ESI ) 181 ([M+H] , 100%); HRMS C H N S
requires:181.0436, found: 181.0430.
(
S)-N-(2-Aminophenyl)-4-(((4-benzyl-4,5-dihydrooxazol-2-yl)thio)methyl)benzamide
11a). To a solution of thione 10a (84 mg, 0.44 mmol) and benzyl chloride 1 (95 mg, 0.36
(
mmol) in acetone (4 mL) was added potassium carbonate (75 mg, 0.543 mmol). The mixture
was then stirred at reflux for 16.5 h, then evaporated. The residue was partitioned between
water (15 mL) and ethyl acetate (20 mL) and the layers then separated. The aqueous layer
was extracted with ethyl acetate (15 mL) and the combined organic layers were washed with
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brine, dried (MgSO
4
) and evaporated to give a solid that was purified by column
chromatography on silica gel (1:1 ethyl acetate:hexane) to give 11a (106 mg, 70%) as a
cream solid, mp 137ꢀ140 °C; [α] ꢁꢀ ꢂ ꢀ32.0 (c 0.5, methanol); νmax (cm ) 3274, 1649; H NMR
ꢀ1
1
(300 MHz, chloroformꢀd) δ ppm 2.66 (1H, dd, J=13.8, 8.3 Hz), 3.08 (1H, dd, J=13.8, 5.5
10
11
12
13
14
15
16
17
18
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60
Hz), 3.86 (2H, br s), 4.03 ꢀ 4.12 (1H, m), 4.19 ꢀ 4.35 (3H, m), 4.43 (1H, m), 6.75 ꢀ 6.89 (2H,
m), 7.07 (1H, m), 7.14 ꢀ 7.35 (6H, m, ), 7.48 (2H, d, J=8.0 Hz, H(11 and 13)), 7.83 (2H, d,
13
J=8.0 Hz), 7.96 (1H, br s); C NMR (125 MHz, chloroformꢀd) δ ppm 36.2, 42.0, 68.2, 74.0,
118.8, 120.2, 125.0, 125.6, 127.0, 127.7, 128.0, 129.0, 129.6, 129.8, 133.7, 138.0, 141.1,
ꢀ
ꢀ
ꢀ
1
41.7, 165.3, 165.8; m/z (ESI ) 416 ([MꢀH] , 100%); HRMS C H N O S requires:
24 22 3 2
416.1438, found: 416.1433.
(S)-N-(2-Aminophenyl)-4-(((4-phenyl-4,5-dihydrooxazol-2-yl)thio)methyl)benzamide
11b). To a solution of thione 10b (185 mg, 1.03 mmol) and benzyl chloride 1 (269 mg, 1.03
(
mmol) in acetone (10 mL) was added potassium carbonate (213 mg, 1.56 mmol). The
mixture was stirred at reflux for 18 h, then evaporated. The residue was partitioned between
water (40 mL) and ethyl acetate (40 mL). The organic layer was washed with brine, dried
4
(MgSO ) and evaporated to give a solid that was purified by column chromatography on
silica gel (ethyl acetate:hexane, 1:1), and then purified again by column chromatography on
silica gel (dichloromethane:ethyl acetate, 4:1 to 3:1) to give 11b as a cream solid (66 mg,
ꢀ1
1
1
6%), mp 64ꢀ65 °C; [α] ꢁꢀ ꢂ ꢀ14.0 (c 0.5, methanol); νmax (cm ) 3292, 1653; H NMR (400
MHz, chloroformꢀd) δ ppm 3.91 (2H, br s), 4.22 (1H, t, J=8.0 Hz), 4.31 ꢀ 4.46 (2H, m), 4.75
1H, t, J=8.9 Hz), 5.25 (6H, dd, J=9.5, 8.0 Hz), 6.83 ꢀ 6.93 (2H, m, H), 7.13 (1H, t, J=7.3
Hz), 7.21 (2H, d, J=7.0 Hz), 7.26 ꢀ 7.41 (6H, m), 7.56 (2H, d, J=8.0 Hz), 7.87 (3H, d, J=7.5
(
1
3
Hz); C NMR (125 MHz, chloroformꢀd) δ ppm 35.9, 69.9, 76.6, 118.5, 119.8, 124.6, 125.4,
126.6, 127.4, 127.7, 127.9, 128.8, 129.5, 133.4, 140.8, 141.4, 141.9, 165.6, 166.0; m/z (CI)
+
+
4
22 3 2
04 ([M+H] , 100%); HRMS C23H N O S requires: 404.1427, found: 404.1426; 66%
purity by LCMS.
(S)-N-(2-Aminophenyl)-4-(((4-(pyridin-3-yl)-4,5-dihydrooxazol-2-
yl)thio)methyl)benzamide (11c). To a solution of amino alcohol 10c (25 mg, 0.139 mmol)
and the arylmethyl chloride 1 (36.2 mg, 0.139 mmol) in acetone (2 mL) was added
potassium carbonate (28.8 mg, 0.208 mmol) and then the mixture was stirred at reflux for 17
h. Since the volume had decreased, acetone (3 mL) was added and then mixture then stirred
at reflux for 6 h. After allowing to cool, the mixture was partitioned between water (15 mL)
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and ethyl acetate (20 mL) and the layers then separated. The aqueous layer was extracted
with ethyl acetate (15 mL) and the combined organic layers were dried (Na SO ) and
2
4
evaporated to give a solid which was purified by MDAP to give 11c (25 mg, 45%) as a white
solid, mp 107ꢀ110 °C; [α]ꢁ +22.0 (c 0.25, methanol); νmax (cm ) 3212, 1645; H NMR (500
ꢂ
ꢀ1
1
ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
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3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, methanolꢀd
J=13.7 Hz), 4.83 (1H, t, J=9.3 Hz), 5.35 (1H, dd, J=9.3, 7.7 Hz), 6.80 (1H, t, J=7.4 Hz), 6.93
1H, d, J=8.0 Hz), 7.11 (1H, t, J=7.4 Hz), 7.22 (1H, d, J=7.7 Hz), 7.43 (1H, dd, J=7.7, 4.9
Hz), 7.60 (3H, d, J=8.0 Hz), 7.96 (2H, d, J=8.0 Hz), 8.40 (1H, s), 8.49 (1H, d, J=4.7 Hz);
4
) δ ppm 4.23 (1H, t, J=8.0 Hz), 4.38 (1H, d, J=13.7 Hz), 4.44 (1H, d,
(
1
3
C NMR (125 MHz, methanolꢀd ) δ ppm 34.8, 67.1, 75.9, 117.3, 118.3, 123.9, 124.1, 126.3,
4
+
1
27.2, 127.7, 128.4, 133.4, 135.1, 138.3, 141.5, 142.4, 147.2, 148.0, 167.1, 167.7; m/z (ESI )
+
+
405 ([M+H] , 100%); HRMS C22
purity by LCMS.
21 4 2
H N O S requires: 405.1380, found: 405.1384; 93%
2-Amino-2-(4-fluorophenyl)ethan-1-ol (12d). A threeꢀnecked flask was fitted with a
magnetic stirrer and a reflux condenser and then flameꢀdried. Under an atmosphere of
nitrogen, sodium borohydride (0.224 g, 5.92 mmol) was added, followed by anhydrous THF
(10 mL). Then a dropping funnel was fitted and the mixture was cooled in an ice bath to 0
°C. A solution of iodine (1.05 g, 2.96 mmol) in THF (10 ml) was added dropwise over 30
min. After the vigorous evolution of gas had ceased, 4ꢀfluoroꢀDLꢀphenylglycine (0.50 g, 2.96
mmol) was added and the mixture was heated at reflux for 17 h. After allowing to cool,
methanol was added slowly until the solution became clear. The mixture was stirred for 30
min and then the solvent was evaporated. The resulting white paste was dissolved in aqueous
potassium hydroxide (20%, 30 mL). After stirring for 6 h the mixture was extracted with
dichloromethane (3 x 30 mL). The combined organic layers were washed with brine (20 mL
then 75 mL), dried (Na
2
SO
4
) and evaporated to give crude 12d as a white solid which was
1
used without further purification (0.22 g, 48%); H NMR (400 MHz, chloroformꢀd) δ ppm
2.51 (3H, br s), 3.54 (1H, dd, J=10.8, 8.3 Hz), 3.72 (1H, dd, J=10.8, 4.3 Hz), 4.07 (1H, dd,
J=8.3, 4.3 Hz), 7.05 (2H, t, J=8.8 Hz), 7.32 (2H, dd, J=8.5, 5.5 Hz).
2-Amino-2-(4-(trifluoromethyl)phenyl)ethanol (12f). To a suspension of 2ꢀaminoꢀ2ꢀ(4ꢀ
(trifluoromethyl)phenyl)acetic acid (1.50 g, 6.84 mmol) in anhydrous THF (15 mL) at 20 °C
was added boraneꢀTHF complex (1M in THF, 17.1 mL) dropwise over 5 min, then the
solution was stirred at 70 °C for 16.5 h. After cooling to 20 ºC methanol (10 mL) was added
cautiously, and the mixture then stirred for 30 min. The solvent was evaporated and the
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resulting yellow paste was stirred with potassium hydroxide (20% aq w/w, 30 mL) for 4 h at
20 °C. The mixture was then extracted with ethyl acetate (3 x 30 mL) and the combined
organic layers were washed with brine, dried (Na SO ) and evaporated to give a yellow
2
4
semiꢀsolid (1.3 g). Purification by flash column chromatography on silica gel (2ꢀ10% of 2M
ammonia/methanol in dichloromethane) gave 12f (0.646 g, 46%) as a white solid, mp 110ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
16 °C; H NMR (500 MHz, chloroformꢀd) δ ppm 2.14 (3H, br s), 3.58 (1H, dd, J=10.7, 8.0
Hz), 3.78 (1H, dd, J=10.7, 4.1 Hz), 4.16 (1H, dd, J=8.0, 4.1 Hz), 7.49 (2H, d, J=8.1 Hz),
13
7
.63 (2H, d, J=8.1 Hz); C NMR (125 MHz, chloroformꢀd) δ ppm 57.0 (s), 67.8 (s), 124.1
+
(
q, J=271.9 Hz), 125.5 (q, J=3.7 Hz), 126.9 (s), 129.8 (q, J=32.5 Hz), 146.6 (s); m/z (ESI )
+
+
2
9 3
06 ([M+H] , 100%); HRMS C H11NOF requires: 206.0787, found: 206.0790.
(
R)-4-(1-Amino-2-hydroxyethyl)phenol (12g). To a suspension of (R)ꢀ2ꢀaminoꢀ2ꢀ(4ꢀ
hydroxyphenyl)acetic acid (1.0 g, 5.98 mmol) in anhydrous THF (10 mL) at 20 °C was
added boraneꢀTHF complex in THF (20 mL, 1M) dropwise over 5 min, then the solution
was stirred at 70 °C for 44 h. After cooling to 20 ºC, methanol (10 mL) was added cautiously
and the mixture was stirred for 30 min. The solvent was then evaporated and the resulting
white paste stirred with potassium hydroxide (20% aqueous w/w, 20 mL) for 4 h at 20 ºC.
The solution was neutralised to pH 7 with hydrochloric acid (2M) then washed with ethyl
acetate (20 mL). The aqueous layer was evaporated and the resulting white solid was stirred
for 1 h with 1:4 ethanol:chloroform (50 mL), then filtered and evaporated to give 12g (300
mg, 33%) as a colorless oil which was used without further purification.
Preparation of yellow mercuric oxide. To a solution of mercury(II) chloride (1.0 g,
CAUTION: TOXIC!) in water (22 mL) was added a sirred solution of sodium hydroxide
(
0.60 g) in water (8 mL). The mixture was stirred at 20 °C for 5 min. The precipitate was
filtered and dried under vacuum overnight over phosphorus pentoxide to give mercuric oxide
as a yellow solid (0.58 g).
tert-Butyl
(S)-(2-(4-(((4-benzyl-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (14a). To a solution of tertꢀbutyl (R)ꢀ(2ꢀ
3
4
(4ꢀ((3ꢀ(2ꢀhydroxyꢀ1ꢀphenylethyl)thioureido)methyl)benzamido)phenyl)carbamate (S)-13a
0.524 g, 0.98 mmol) in ethanol (7 mL) and toluene (14 mL) was added freshly prepared
(
yellow mercuric oxide (0.58 g, 2.6 mmol) the resulting mixture was stirred at reflux for 30
min. After cooling to 20 °C the mixture was filtered through celite and the filtrate evaporated
ꢀ1
1
to give 14a as a white solid (0.45 g, 92%), mp 200ꢀ201 °C; νmax (cm ) 3308, 1664, 1650; H
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NMR (500 MHz, chloroformꢀd) δ ppm 1.51 (9H, s), 2.66 (1H, dd, J=13.5, 8.5 Hz), 3.06 (1H,
dd, J=13.5, 4.7 Hz), 4.03 (1H, t, J=7.1 Hz), 4.22 (1H, t, J=8.2 Hz), 4.32 (1H, m), 4.43 (1H,
d, J=15.3 Hz), 4.47 (1H, d, J=15.3 Hz), 6.93 (1H, br s), 7.13 ꢀ 7.32 (5H, m), 7.38 (2H, d,
1
3
J=7.9 Hz), 7.79 (1H, d, J=7.7 Hz), 7.92 (2H, d, J=7.9 Hz), 9.19 (1H, br s); C NMR (125
MHz, chloroformꢀd) δ ppm 28.4, 42.4, 46.7, 65.1, 72.5, 81.5, 124.6, 125.8, 126.0, 126.1,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
126.5, 127.4, 127.8, 128.6, 129.3, 130.0, 131.0, 133.5, 138.0, 142.9, 154.7, 160.8, 165.4; m/z
ꢀ
ꢀ
ꢀ
(
ESI ) 499 ([MꢀH] , 100%); HRMS C29
H
31
N
O
4 4
requires: 499.2351, found: 499.2358.
(S)-(tert-Butyl
2-(4-(((4-phenyl-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (S)-(14b). To a solution of tertꢀbutyl (S)ꢀ
2ꢀ(2ꢀ(4ꢀ((3ꢀ(2ꢀhydroxyꢀ1ꢀphenylethyl)thioureido)methyl)benzamido)phenyl)carbamate (S)-
3
4
(
13b). (180 mg, 0.345 mmol) in ethanol (3.5 mL) and toluene (7 mL) was added yellow
mercuric oxide (375 mg, 1.73 mmol) in portions. The resulting mixture was stirred at reflux
for 2 h. The cool mixture was filtered through celite and the filtrate evaporated. The residue
was dissolved in ethanol (3.5 mL) and toluene (7 mL), then yellow mercuric oxide (375 mg,
1.73 mmol) was added and the mixture stirred at reflux for 4 h. When cool, the mixture was
filtered through celite and yellow mercuric oxide (375 mg, 1.73 mmol) was added and the
mixture was stirred at reflux for 4 h. Filtration of the mixture through celite and evaporation
of the filtrate afforded (S)-14b as a foamy white solid (0.127 g, 76%), mp 100ꢀ103 °C; νmax
ꢀ1
1
(cm ) 3274, 1654; H NMR (400 MHz, chloroformꢀd) δ ppm 1.51 (9H, s), 4.11 (1H, t, J=7.5
Hz), 4.47 (2H, s), 4.66 (1H, t, J=8.5 Hz), 5.12 (1H, dd, J=9.0, 7.3 Hz), 7.15 (1H, td, J=7.5,
1.6 Hz), 7.19 (1H, td, J=7.5, 1.6 Hz), 7.24 ꢀ 7.30 (5H, m), 7.31 – 7.39 (4H, m), 7.73 (1H, d,
13
J=7.3 Hz), 7.89 (2H, d, J=8.0 Hz), 9.33 (1H, br s); C NMR (125 MHz, chloroformꢀd) δ
ppm 28.7), 47.0, 67.8, 75.8, 81.6, 124.9, 126.1, 126.2, 126.3, 126.8, 127.7, 127.9, 128.1,
ꢀ
ꢀ
1
29.0, 130.5, 131.2, 133.6, 143.3, 143.8, 155.0, 162.0, 165.9; m/z (ESI ) 485 ([MꢀH] , 95%);
ꢀ
HRMS C H N O requires: 485.2194, found: 485.2206.
28
29
4
4
(R)-tert-Butyl
2-(4-(((4-phenyl-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (R)-(14b). To a solution of (R)ꢀ
phenylalaninol (110 mg, 0.80 mmol) in THF (12 mL) was added isothiocyanate 2 (337 mg,
0.88 mmol), and the mixture was stirred at 20 °C for 18 h. Evaporation gave a residue that
was dissolved in acetone (6 mL); iodomethane (0.10 mL, 1.6 mmol) was added and the
solution stirred at 20 °C for 9 h. Saturated aqueous sodium hydrogen carbonate (2 mL) was
then added and the mixture was stirred for 10 min. Saturated aqueous sodium hydrogen
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carbonate (20 mL) was then added and the mixture extracted with ethyl acetate (2 x 20 mL).
The combined organic layers were washed with brine (20 mL), dried (Na SO ) and
2
4
evaporated to give a white solid that was purified by column chromatography on silica gel
(5:95 methanol:ethyl acetate) to give (R)-(14b) (159 mg, 41%) as a white solid, mp 104ꢀ106
ꢀ1
1
°
C; νmax (cm ) 3277, 1653; H NMR (300 MHz, chloroformꢀd) δ ppm 1.48 (9H, s), 4.08
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
1H, t, J=7.5 Hz), 4.44 (2H, s), 4.63 (1H, t, J=8.6 Hz), 5.09 (1H, dd, J=8.9, 7.3 Hz), 7.03 ꢀ
1
3
7
.39 (13H, m), 7.70 (1H, m), 7.85 (2H, d, J=8.1 Hz), 9.30 (1H, br s); C NMR (75 MHz,
chloroformꢀd) δ ppm 28.4, 46.7, 67.6, 75.6, 81.3, 124.7, 125.8, 125.9, 126.1, 126.6, 127.4,
+
1
27.6, 127.9, 128.8, 130.3, 130.9, 133.3, 143.1, 143.6, 154.8, 161.8, 165.7; m/z (ESI ) 487
+
+
([M+H] , 100%); HRMS C28
H
31
N
O
4 4
requires: 487.2340, found: 487.2336.
(S)-tert-Butyl
(2-(4-(((4-(pyridin-3-yl)-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (14c). To a solution of 9c (155 mg, 1.12
mmol) in THF (8 mL) and methanol (3 mL) was added isothiocyanate 2 (330 mg, 0.86
mmol), and the mixture was stirred at 20 °C for 17 h. Evaporation gave a residue that was
dissolved in acetone (4 mL); iodomethane (0.108 mL, 1.72 mmol) was then added and the
solution stirred at 20 °C for 5 h. The solution was then worked up as described for (R)-(14b)
to give a solid that was purified by flash column chromatography on silica gel (gradient
elution from 2ꢀ10% of methanolic 2M ammonia in dichloromethane) gave 14c (86 mg, 21%)
ꢀ1
1
as a white solid, mp 107ꢀ108 °C; νmax (cm ) 3265, 1651; H NMR (400 MHz, chloroformꢀd)
δ ppm 1.50 (9H, s), 4.06 (1H, dd, J=7.8, 7.2 Hz), 4.48 (2H, s), 4.66 (1H, dd, J=7.8, 7.0), 5.12
(1H, dd, J=8.7, 7.2 Hz), 5.43 (1H, br s), 7.09 ꢀ 7.22 (2H, m), 7.26 (1H, m), 7.30 ꢀ 7.42 (4H,
m), 7.56 (1H, d, J=7.8 Hz), 7.73 (1H, d, J=6.8 Hz), 7.88 (2H, d, J=7.8 Hz), 8.42 (1H, s),
13
8
4
1
.49 (1H, d, J=3.7 Hz), 9.39 (1H, br s); C NMR (100 MHz, chloroformꢀd) δ ppm 28.3,
6.6, 65.5, 74.9, 81.1, 123.6, 124.5, 125.6, 125.7, 126.0, 127.4, 127.8, 130.3, 130.7, 133.5,
+
+
34.2, 139.2, 142.6, 148.2, 148.7, 154.6, 162.0, 165.7; m/z (ESI ) 488 ([M+H] , 100%);
+
4
HRMS C27
H
30
N
5
O
requires: 488.2292, found: 488.2285.
tert-Butyl
(2-(4-(((4-(4-fluorophenyl)-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (14d). To a solution of amino alcohol 12d
220 mg, 1.42 mmol) in THF (10 mL) was added isothiocyanate 2 (272 mg, 0.71 mmol), the
(
resulting solution was stirred at 20 °C for 18 h. The solvent was evaporated and the residue
was dissolved in acetone (5 mL); iodomethane (0.09 mL, 1.45 mmol) was then added and
the solution stirred at 20 °C for 16 h. The solution was then worked up as described for (R)-
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(
14b) to give a solid that was purified by column chromatography on silica gel (4:1 ethyl
ꢀ1
acetate:hexane) to give 14d (238 mg, 67%) as a white solid, mp 105ꢀ108 °C; νmax (cm )
1
3
4
7
9
258, 1650; H NMR (400 MHz, chloroformꢀd) δ ppm 1.51 (9H, s), 4.06 (1H, t, J=7.7 Hz),
.50 (2H, s), 4.65 (1H, t, J=8.5 Hz), 5.11 (1H, dd, J=8.9, 7.4 Hz), 7.02 (2H, t, J=8.7 Hz),
.12 ꢀ 7.30 (6H, m), 7.39 (2H, d, J=8.0 Hz), 7.77 (1H, d, J=7.5 Hz), 7.91 (2H, d, J=8.0 Hz),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
.32 (1H, br s); C NMR (125 MHz, chloroformꢀd) δ ppm 28.3, 46.6, 66.8, 75.4, 81.3, 115.4
(d, J=22.1 Hz), 124.5, 125.7, 125.9, 127.3, 127.7, 128.0 (d, J=8.6 Hz), 130.0, 130.8, 133.3,
+
1
39.2, 142.7, 154.6, 162.1 (d, J=244.7 Hz), 161.5, 165.3; m/z (CI) 505 ([M+H] , 93%);
+
HRMS C28
H30FN
4
O
4
requires: 505.2246, found: 505.2254.
tert-Butyl
(2-(4-(((4-(3-fluorophenyl)-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (14e). To a solution of 12e (81 mg, 0.522
mmol) in THF (6 mL) was added isothiocyanate 2 (200 mg, 0.522 mmol), and the resulting
solution was stirred at 20 °C for 16 h. Evaporation gave a residue that was dissolved in
acetone (3 mL); iodomethane (0.10 mL, 1.56 mmol) was then added and the solution stirred
at 20 °C for 4 h. The solution was then worked up as described for (R)-(14b) to give a solid
that was purified by flash column chromatography on silica gel (75ꢀ100% ethyl
ꢀ1
acetate:cyclohexane) gave 14e (200 mg, 76%) as a white solid, mp 104ꢀ107 °C; νmax (cm )
1
3
283, 1652; H NMR (400 MHz, chloroformꢀd) δ ppm 1.51 (1H, s), 3.95 (1H, br s), 4.07
(1H, dd, J=8.1, 7.1 Hz), 4.44 ꢀ 4.54 (2H, m), 4.64 (1H, dd, J=9.0, 8.1 Hz), 5.11 (1H, dd,
J=9.0, 7.1 Hz), 6.91 ꢀ 7.04 (3H, m), 7.11 ꢀ 7.21 (2H, m)), 7.22 ꢀ 7.32 (3H, m), 7.36 (2H, d,
13
J=8.3 Hz), 7.73 (1H, dd, J=7.8, 1.2 Hz), 7.89 (2H, d, J=8.3 Hz), 9.28 (1H, br s); C NMR
(100 MHz, chloroformꢀd) δ ppm 28.3, 46.6, 67.0, 75.2, 81.2, 113.3 (d, J=22.0 Hz), 114.3 (d,
J=21.3 Hz), 122.0 (d, J=2.2 Hz), 124.5, 125.7, 125.8, 125.9, 127.3, 127.8, 130.1 (d, J=8.1
Hz), 130.2, 130.8, 133.3, 142.7, 146.3 (d, J=6.6 Hz), 154.6, 161.8, 163.1 (d, J=245.8 Hz),
+
+
+
1
65.5; m/z (ESI ) 505 ([M+H] , 100%); HRMS C H FN O requires: 505.2246, found:
28 30 4 4
5
05.2240.
tert-Butyl
(2-(4-(((4-(4-(trifluoromethyl)phenyl)-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (14f). To a solution of 12f (107 mg, 0.522
mmol) in THF (6 mL) was added 2 (200 mg, 0.522 mmol). The mixture was stirred at 20 °C
for 17.5 h. Evaporation gave a residue that was dissolved in acetone (3 mL); iodomethane
(0.065 mL, 1.04 mmol) was added and the solution was stirred at 20 °C for 6 h. A further 65
ꢁ
L of iodomethane was then added and the mixture stirred for another 2.5 h at 20 °C. The
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mixture was worked up as described for (R)-(14b) to give a solid that was purified by flash
column chromatography on silica gel (gradient elution from 1ꢀ5% of methanolic 2M
ammonia in dichloromethane) to give 14f (200 mg, 69%) as a white solid, mp 114ꢀ116 °C;
ꢀ1
1
ν
max (cm ) 3282, 1658; H NMR (400 MHz, chloroformꢀd) δ ppm 1.49 (9H, s), 4.04 (1H, t,
J=7.6 Hz), 4.44 (2H, s), 4.65 (1H, t, J=8.7 Hz), 4.94 (1H, br s), 5.14 (1H, t, J=8.1 Hz), 7.05 ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
.18 (2H, m), 7.24 (1H, dd, J=7.6, 1.2 Hz), 7.31 (2H, d, J=8.1 Hz), 7.36 (2H, d, J=8.1 Hz),
.47 (1H, br s), 7.57 (2H, d, J=8.1 Hz), 7.70 (1H, d, J=7.1 Hz), 7.85 (2H, d, J=8.1 Hz), 9.39
7
1
3
(1H, br s); C NMR (100 MHz, chloroformꢀd) δ ppm 28.3, 46.5, 67.0, 75.0, 81.1, 124.1 (q,
J=272.9 Hz), 124.5, 125.5 (q, J=3.7 Hz), 125.6, 125.9, 126.8, 127.2, 127.7, 129.6 (q, J=32.3
+
+
Hz), 130.3, 130.7, 133.2, 142.8, 147.6, 154.7, 162.1, 165.6; m/z (ESI ) 387 ([M+H] , 100%);
+
4 3
HRMS C H N O F requires: 555.2214, found: 555.2214.
29 30
4
(S)-tert-Butyl
(2-(4-(((4-(4-hydroxyphenyl)-4,5-dihydrooxazol-2-
29
yl)amino)methyl)benzamido)phenyl)carbamate (14g). To a solution of the thiourea 13g
270 mg, 0.503 mmol) in acetone (4 mL) was added iodomethane (0.045 mL, 0.75 mmol);
(
the solution was then stirred at 20 °C for 4 h. More iodomethane (0.045 mL, 0.75 mmol) was
then added followed by stirring at 20 °C for an additional 17.5 h. The mixture was worked
up as described for (R)-(14b) to give 14g (250 mg, 99%) as a white solid, mp 133ꢀ139 °C;
ꢀ1
1
ν
max (cm ) 3282, 1650; H NMR (400 MHz, chloroformꢀd) δ ppm 1.49 (9H, s), 4.09 (1H, t,
J=7.5 Hz), 4.33 ꢀ 4.50 (2H, m), 4.48 (1H, br s), (4.58 (1H, t, J=8.6 Hz), 4.98 (1H, dd, J=8.8,
7.1 Hz), 6.58 (2H, d, J=8.5 Hz), 6.91 (2H, d, J=8.5 Hz), 7.11 ꢀ 7.21 (2H, m), 7.27 ꢀ 7.35 (4H,
13
m), 7.68 (1H, m), 7.83 (2H, d, J=8.3 Hz), 9.37 (1H, br s); C NMR (125 MHz, chloroformꢀ
d) δ ppm 28.3, 46.3, 66.2, 75.7, 81.3, 115.9, 124.6, 125.6, 125.7, 126.1, 127.2, 127.5, 127.4,
+
+
1
30.4, 130.6, 132.9, 133.6, 142.7, 154.6, 156.3, 161.5, 165.8; m/z (ESI ) 503 ([M+H] ,
+
5
100%); HRMS C28
H
31
N
4
O
requires: 503.2289, found: 503.2281.
(
R)-tert-Butyl
yl)amino)methyl)benzamido)phenyl)carbamate (R)-(14h). To a solution of (R)ꢀ2ꢀaminoꢀ
ꢀphenylethanol (110 mg, 0.80 mmol) in THF (8 mL) was added isothiocyanate 2 (307 mg,
(2-(4-(((5-phenyl-4,5-dihydrooxazol-2-
1
0.8 mmol); the mixture was stirred at 20 °C for 18 h. Evaporation gave a residue that was
dissolved in acetone (4 mL); iodomethane (0.10 mL, 1.60 mmol) was then added and the
mixture was stirred at 20 °C for 6 h. Saturated aqueous sodium hydrogen carbonate (2 mL)
was added, and the mixture stirred for 10 min, then diluted with saturated aqueous sodium
hydrogen carbonate (20 mL), and extracted with ethyl acetate (2 x 20 mL). The combined
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2 4
organic layers were dried (Na SO ) and evaporated to give a yellow oil that was purified by
flash column chromatography on silica gel (gradient elution from 1ꢀ5% of methanolic 2M
ammonia in dichloromethane) to give (R)-(14h) (291 mg, 75%) as a white solid, mp 97ꢀ99
ꢀ1
1
°
C; νmax (cm ) 3277, 1659; H NMR (400 MHz, chloroformꢀd) δ ppm 1.48 (9H, s), 3.67
(
1H, dd, J=12.2, 7.3 Hz), 4.14 (1H, dd, J=12.2, 9.2 Hz), 4.41 (2H, s), 5.02 (1H, br s), 5.49
1H, t, J=8.3 Hz), 7.04 ꢀ 7.19 (2H, m), 7.22 ꢀ 7.48 (9H, m), 7.65 (1H, m), 7.84 (2H, d, J=8.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
13
Hz), 9.48 (1H, br s); C NMR (100 MHz, chloroformꢀd) δ ppm 28.3, 46.4, 60.1, 80.9, 81.7,
1
1
4
24.5, 125.4, 125.6, 125.7, 125.9, 127.1, 127.8, 128.4, 128.8, 130.5, 130.6, 133.1, 140.3,
+
+
+
31 4 4
42.8, 154.7, 160.8, 165.7; m/z (ESI ) 487 ([M+H] , 100%); HRMS C28H N O requires:
87.2340, found: 487.2342.
(S)-tert-Butyl
(2-(4-(((5-phenyl-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (S)-(14h). To a solution of (S)ꢀ2ꢀaminoꢀ1ꢀ
phenylethanol (110 mg, 0.8 mmol) in THF (8 mL) was added isothiocyanate 2 (307 mg, 0.80
mmol); the mixture was stirred at 20 °C for 18 h. Evaporation gave a residue that was
dissolved in acetone (4 mL); iodomethane (0.10 mL, 1.6 mmol) was then added and the
solution stirred at 20 °C for 4 h. The mixture was worked up as described for (S)-(14h) to
give a solid that was purified by flash column chromatography on silica gel (gradient elution
from 1ꢀ5% of methanolic 2M ammonia in dichloromethane) to give (S)-(14h) (275 mg,
ꢀ1
1
7
1%) as a white solid, mp 100ꢀ102 °C; νmax (cm ) 3274 (NH), 1655 (C=O); H NMR (400
MHz, chloroformꢀd) δ ppm 1.51 (9H, s), 3.73 (1H, dd, J=12.2, 7.3 Hz), 4.20 (1H, dd,
J=12.3, 9.2 Hz), 4.49 (2H, s), 5.52 (1H, dd, J=9.2, 7.3 Hz), 7.12 ꢀ 7.23 (2H, m), 7.27 ꢀ 7.43
1
3
(
9H, m), 7.75 (1H, d, J=7.6 Hz), 7.91 (2H, d, J=8.1 Hz), 9.31 (1H, br s); C NMR (100
MHz, chloroformꢀd) δ ppm 28.3, 46.6, 60.7, 81.2, 81.7, 124.5, 125.6, 125.7, 125.8, 125.9,
+
1
27.3, 127.8, 128.3, 128.8, 130.2, 130.8, 133.3, 140.5, 142.9, 154.6, 160.5, 165.5; m/z (ESI )
+
+
4
4
87 ([M+H] , 100%); HRMS C H N O requires: 487.2340, found: 487.2327.
2
8
31
4
tert-Butyl
(2-(4-(((4,4-diphenyl-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (14i). To a solution of 2ꢀaminoꢀ2,2ꢀ
diphenylethanol (122 mg, 0.574 mmol) in THF (6 mL) was added 2 (200 mg, 0.522 mmol),
the resulting solution was stirred at 20 °C for 24 h, then at reflux for 18 h. Evaporation of the
solvent gave a residue that was dissolved in acetone (3 mL); iodomethane (0.098 mL, 1.565
mmol) was then added and the solution stirred at 20 °C for 4 h. The mixture was worked up
as described for (R)-(14h) to give a solid that was purified by flash column chromatography
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on silica gel (gradient elution from 1ꢀ5% of methanolic 2M ammonia in dichloromethane) to
ꢀ1
1
give 14i (290 mg, 99%) as a white solid, mp 104ꢀ106 °C; νmax (cm ) 3276, 1658; H NMR
(400 MHz, chloroformꢀd) δ ppm 1.50 (9H, s), 4.49 (2H, s), 4.80 (2H, s), 7.08 ꢀ 7.39 (16H,
1
3
m), 7.72 (1H, d, J=6.8 Hz), 7.87 (2H, d, J=8.3 Hz), 9.26 (1H, br s); C NMR (100 MHz,
chloroformꢀd) δ ppm 28.3, 46.5, 80.0, 81.2, 124.6, 125.7, 125.8, 126.0, 126.6, 126.9, 127.4,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
1
27.7, 128.3, 130.2, 130.8, 133.1, 143.3, 146.9, 154.7, 160.1, 165.6; m/z (ESI ) 563
+
+
([M+H] , 100%); HRMS C34
H
35
N
O
4 4
requires: 563.2653, found: 563.2639.
tert-Butyl
(2-(4-((((4S,5S)-4,5-diphenyl-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (14j). To a solution of (1S,2S)ꢀ2ꢀaminoꢀ
,2ꢀdiphenylethanol (122 mg, 0.574 mmol) in THF (6 mL) was added isothiocyanate 2 (200
1
mg, 0.522 mmol); the mixture was then stirred at 20 °C for 16 h. Evaporation gave a residue
that was dissolved in acetone (3 mL); iodomethane (0.01 mL, 1.56 mmol) was then added
and the solution stirred at 20 °C for 5.5 h. The mixture was worked up as described for (R)-
(
(
(
14h) to give a solid that was purified by flash column chromatography on silica gel
gradient elution from 1ꢀ5% of methanolic 2M ammonia in dichloromethane) to give 14j
ꢀ1
1
165 mg, 56%) as a white solid, mp 137ꢀ139 °C; νmax (cm ) 3271, 1656; H NMR (400
MHz, chloroformꢀd) δ ppm 1.51 (9H, s), 4.15 (1H, br s), 4.46 ꢀ 4.60 (2H, m), 4.98 (1H, d,
J=6.8 Hz), 5.23 (1H, d, J=6.8 Hz), 7.08 ꢀ 7.47 (16H, m), 7.75 (1H, dd, J=7.8, 1.0 Hz), 7.92
13
(
2H, d, J=8.1 Hz), 9.32 (1H, br s); C NMR (100 MHz, chloroformꢀd) δ ppm 28.3, 46.5,
7
1
1
6.1, 81.2, 89.6, 124.5, 125.7, 125.8, 125.9, 126.5, 127.3, 127.6, 127.8, 128.6, 128.7, 128.9,
+
+
30.2, 130.8, 133.3, 139.7, 142.7, 142.9, 154.7, 160.8, 165.5; m/z (ESI ) 563 ([M+H] ,
+
4
00%); HRMS C H N O requires: 563.2653, found: 563.2639.
34
35
4
tert-Butyl
(2-(4-((((4R,5S)-4,5-diphenyl-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (14k). To a solution of (1S,2R)ꢀ2ꢀaminoꢀ
1,2ꢀdiphenylethanol (171 mg, 0.8 mmol) in THF (8 mL) was added isothiocyanate 2 (307
mg, 0.80 mmol); the mixture was then stirred at 20 °C for 18 h. Evaporation gave a residue
that was dissolved in acetone (4 mL); iodomethane (0.10 mL, 1.60 mmol) was then added
and the solution stirred at 20 °C for 6 h. The mixture was worked up as described for (R)-
(
14h) to give an oil that was purified by flash column chromatography on silica gel (gradient
elution from 1ꢀ5% of methanolic 2M ammonia in dichloromethane) then with a SCXꢀ2
cartridge (washed with methanol and then eluted with 2M ammonia in methanol) to give 14k
ꢀ1
1
(
421 mg, 85%) as a white solid, mp 121ꢀ123°C; νmax (cm ) 3277, 1653; H NMR (400 MHz,
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chloroformꢀd) δ ppm 1.51 (9H, s), 4.48 ꢀ 4.59 (2H, m), 5.40 (1H, d, J=8.9 Hz), 5.84 (1H, d,
J=8.9 Hz), 6.84 ꢀ 6.92 (4H, m), 6.96 ꢀ 7.09 (6H, m), 7.10 ꢀ 7.22 (2H, m), 7.28 (1H, dd,
J=7.2, 2.1 Hz), 7.35 ꢀ 7.46 (3H, m), 7.74 (1H, d, J=7.6 Hz), 7.90 (2H, d, J=8.1 Hz), 9.36
13
(
1H, br s); C NMR (100 MHz, chloroformꢀd) δ ppm 28.3, 46.5, 71.9, 81.1, 85.8, 124.6,
125.7, 126.0, 126.2, 126.8, 127.4, 127.5, 127.6, 127.6, 127.8, 130.3, 130.7, 133.2, 136.4,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
+
1
35 4 4
39.1, 143.1, 154.7, 161.6, 165.6; m/z (ESI ) 563 ([M+H] , 100%); HRMS C34H N O
requires: 563.2653, found: 563.2641.
(
R)-tert-Butyl
yl)amino)methyl)benzamido)phenyl)carbamate (R)-(14l). To a mixture of (R)ꢀ2ꢀaminoꢀ3ꢀ
1Hꢀimidazolꢀ4ꢀyl)propanꢀ1ꢀol dihydrochloride (123 mg, 0.574 mmol) in THF (10 mL) was
(2-(4-(((4-((1H-imidazol-4-yl)methyl)-4,5-dihydrooxazol-2-
(
added potassium carbonate (0.16 mg, 1.15 mmol) and 2 (200 mg, 0.522 mmol). The mixture
was stirred at 72 °C for 72 h. The mixture was filtered, and evaporation of the filtrate gave a
residue that was dissolved in acetone (6 mL); iodomethane (0.065 mL, 1.04 mmol) was then
added and the solution stirred at 20 °C for 18 h. The mixture was worked up as described for
(
(
(
R)-(14h) to give a solid which was purified by flash column chromatography on silica gel
gradient elution from 2ꢀ20% of methanolic 2M ammonia in dichloromethane) to give (R)-
ꢀ1
14l) (110 mg, 43%) as a white solid, mp 159ꢀ163 °C; νmax (cm ) 3664 (NH), 3382 (NH),
1
1
679, 1641; H NMR (500 MHz, DMSOꢀd
6
) δ ppm 1.46 (9H, s), 2.62 (1H, dd, J=15.4, 5.8
Hz), 3.09 (1H, dd, J=15.4, 7.4 Hz), 3.75 (1H, dd, J=11.0, 5.5 Hz), 4.24 (1H, dd, J=11.0, 7.0
Hz), 4.31 (2H, d, J=6.0 Hz), 4.75 (1H, m), 6.53 (1H, t, J=6.0 Hz), 6.62 (1H, s), 6.66 (1H, d,
J=7.4 Hz), 7.12 ꢀ 7.26 (2H, m), 7.41 (2H, d, J=8.2 Hz), 7.49 ꢀ 7.61 (3H, m), 7.91 (2H, d,
1
3
J=8.0 Hz), 8.69 (1H, br s), 9.81 (1H, s); C NMR (125 MHz, DMSOꢀd ) δ ppm 28.5, 29.6,
6
43.1, 50.7, 55.0, 80.1, 119.8, 124.4, 124.6, 126.0, 126.4, 127.4, 128.0, 130.3, 131.4, 132.1,
+
+
1
33.0, 134.7, 145.5, 153.9, 157.9, 165.6; m/z (ESI ) 491 ([M+H] , 100%); HRMS
+
C H N O requires: 491.2401, found: 491.2390.
26
31
6
4
(S)-tert-Butyl
(2-(4-(((4-((1H-imidazol-4-yl)methyl)-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamido)phenyl)carbamate (S)-(14l). To a mixture of (S)ꢀ2ꢀaminoꢀ3ꢀ
(1Hꢀimidazolꢀ4ꢀyl)propanꢀ1ꢀol dihydrochloride (178 mg, 0.83 mmol) in THF (6 mL) was
added potassium carbonate (230 mg, 1.67 mmol) followed by a solution of 2 (319 mg, 0.833
mmol) in THF (6 mL). The resulting mixture was stirred at 20 °C for 17 h then heated at
reflux for 72 h. The mixture was filtered, and evaporation of the filtrate gave a residue that
was dissolved in acetone (6 mL); iodomethane (0.104 mL, 1.67 mmol) was then added and
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the solution stirred at 20 °C for 5 h. The mixture was worked up as described for (R)-(14h)
to give a solid that was purified by flash column chromatography on silica gel (gradient
elution from 2ꢀ20% of methanolic 2M ammonia in dichloromethane) to give (S)-(14l) (135
ꢀ1
1
mg, 33%) as a white solid, mp 155ꢀ157 ºC; νmax (cm ) 3310, 1635; H NMR (400 MHz,
methanolꢀd ) δ ppm 1.51 (9H, s), 2.74 (1H, ddd, J=15.6, 5.5, 1.0 Hz), 3.23 (1H, ddd, J=15.6,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
7
.3, 1.0 Hz), 3.87 (1H, dd, J=11.4, 5.3 Hz), 4.35 (1H, dd, J=11.4, 6.7 Hz), 4.43 (2H, s), 4.95
(1H, m), 6.69 (1H, s), 7.19 ꢀ 7.29 (2H, m), 7.40 ꢀ 7.49 (3H, m), 7.57 (1H, s), 7.63 (1H, m),
1
3
7
5
1
4
.94 (2H, d, J=8.1 Hz); C NMR (100 MHz, methanolꢀd ) δ ppm 27.2, 28.8, 42.9, 50.6,
4
4.8, 80.3, 118.8, 124.3, 124.9, 125.7, 126.0, 126.9, 127.4, 130.3, 130.9, 131.6, 132.7, 134.6,
+
+
+
31 6 4
44.6, 154.9, 158.8, 166.7; m/z (ESI ) 491 ([M+H] , 100%); HRMS C26H N O requires:
91.2401, found: 491.2382.
(
S)-N-(2-Aminophenyl)-4-(((4-benzyl-4,5-dihydrooxazol-2-yl)amino)methyl)benzamide
15a). To a solution of 14a (0.44 g, 0.88 mmol) in dichloromethane (3.4 mL) was added
(
trifluoroacetic acid (0.6 mL). The solution was stirred at 20 °C for 5 h, then trifluoroacetic
acid (0.20 mL) was added and the solution stirred at 20 °C for a further 30 min. To the
mixture was then added dichloromethane (50 mL) and saturated aqueous sodium hydrogen
carbonate (30 mL), and the mixture was then stirred vigorously for 5 min. The organic layer
was washed with brine (30 mL), dried (Na SO ) and evaporated. The residue was purified by
2
4
column chromatography on silica gel (1:1:98 ammonia (30% aqueous): methanol: ethyl
acetate) to give 15a as a white solid (0.167 g, 47%), mp 146ꢀ147 °C; [α] ꢁꢀ ꢂ +8.8 (c 0.5,
ꢀ1
1
methanol); νmax (cm ) 3202, 1674, 1647; H NMR (500 MHz, chloroformꢀd) δ ppm 2.62
(1H, dd, J=13.5, 8.3 Hz), 3.00 (1H, dd, J=13.5, 5.0 Hz), 3.87 (2H, br s), 3.98 (1H, t, J=7.7
Hz), 4.18 (1H, t, J=8.1 Hz), 4.26 (1H, m), 4.32 ꢀ 4.44 (2H, m), 6.74 ꢀ 6.85 (2H, m), 7.07
(1H, t, J=7.3 Hz), 7.15 (2H, d, J=7.1 Hz), 7.20 (1H, t, J=6.9 Hz), 7.23 ꢀ 7.36 (5H, m), 7.81
+
+
(2H, d, J=7.4 Hz), 8.22 (1H, br s); m/z (CI) 401 ([M+H] , 100%); HRMS C H N O
2
2
4
25
4
requires: 401.1972, found: 401.1963.
(
S)-N-(2-Aminophenyl)-4-(((4-phenyl-4,5-dihydrooxazol-2-yl)amino)methyl)benzamide
(S)-(15b). To a solution of (S)-(14b) (103 mg, 0.21 mmol) in dichloromethane (0.90 mL)
was added trifluoroacetic acid (0.10 mL). The solution was stirred at 20 °C for 1.5 h, then
trifluoroacetic acid (0.05 mL) was added and the solution stirred at 20 °C for a further 2.5 h.
The reaction was diluted with dichloromethane (10 mL) and saturated aqueous sodium
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hydrogen carbonate (10 mL) then stirred vigorously for 5 min. The organic layer washed
with saturated aqueous sodium hydrogen carbonate (10 mL), dried (Na SO ) and evaporated.
2
4
Purification by column chromatography on silica gel (1:1:98 ammonia (30% aqueous):
methanol: ethyl acetate) gave (S)-15b as a white solid (60 mg, 74%), mp 85ꢀ86 °C; [α] ꢀꢁ ꢂ
ꢀ1
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+12.2 (c 0.5, methanol); νmax (cm ) 3254, 1649; H NMR (500 MHz, chloroformꢀd) δ ppm
.91 (2H, br s), 4.09 (1H, t, J=7.6 Hz), 4.42 (2H, br s), 4.62 (1H, t, J=8.5 Hz), 5.09 (1H, t,
J=7.9 Hz), 6.75 ꢀ 6.88 (2H, m), 7.09 (1H, t, J=7.6 Hz), 7.20 ꢀ 7.38 (8H, m), 7.80 (2H, d,
3
13
J=6.3 Hz), 8.32 (1H, br s); C NMR (125 MHz, chloroformꢀd) δ ppm 46.9, 67.9, 75.7,
118.7, 120.0, 125.0, 125.8, 126.8, 127.6, 127.7, 127.8, 128.1, 129.0, 133.5, 141.3, 143.6,
ꢀ
ꢀ
ꢀ
1
44.0, 162.1, 166.2; m/z (ESI ) 385 ([MꢀH] , 100%); HRMS C23
H
21
N
4
O
2
requires:
385.1670, found: 385.1673.
(
R)-N-(2-Aminophenyl)-4-(((4-phenyl-4,5-dihydrooxazol-2-yl)amino)methyl)benzamide
R)-(15b). To a solution of (R)-(14b) (150 mg, 0.308 mmol) in dichloromethane (5 mL) was
(
added trifluoroacetic acid (0.50 mL, 6.2 mmol) dropwise, then the mixture was stirred at 20
C for 3 h. Saturated aqueous sodium hydrogen carbonate (20 mL) was then added and the
mixture was extracted with ethyl acetate (2 x 30 mL). The combined organic layers were
washed with brine (30 mL), dried (Na SO ) and evaporated to give a white solid that was
°
2
4
purified by column chromatography on silica gel (1:1:98 ammonia (30% aqueous):
methanol: ethyl acetate) to give (R)-(15b) as a white solid (38 mg, 32%), mp 115ꢀ118 °C;
ꢁ
ꢂ
ꢀ1
1
[α] ꢀ14.0 (c 1.0, methanol); νmax (cm ) 3287, 1649; H NMR (300 MHz, chloroformꢀd) δ
ꢀ
ppm 4.04 (1H, t, J=7.5 Hz), 4.11 (1H, br s), 4.33 (2H, s), 4.56 (1H, t, J=8.6 Hz), 5.02 (1H,
dd, J=8.8, 7.3 Hz), 6.68 – 6.61 (2H, m), 7.04 (1H, td, J=7.6, 1.0 Hz), 7.12 ꢀ 7.36 (8H, m),
13
7.73 (2H, d, J=7.9 Hz), 8.52 (1H, br s); C NMR (75 MHz, chloroformꢀd) δ ppm 46.5, 67.4,
75.5, 118.3, 119.6, 124.6, 125.8, 126.6, 127.4, 127.6, 127.9, 128.8, 133.1, 141.2, 143.2,
+
+
+
1
43.6, 161.9, 166.1; m/z (ESI ) 387 ([M+H] , 100%); HRMS C23
H
23
N
4
O
2
requires:
387.1816, found: 387.1808.
(S)-N-(2-Aminophenyl)-4-(((4-(pyridin-3-yl)-4,5-dihydrooxazol-2-
yl)/amino)methyl)benzamide (15c). To a solution of 14c (80 mg, 0.164 mmol) in
dichloromethane (1.0 mL) was added trifluoroacetic acid (0.25 mL, 3.25 mmol). The
resulting solution was stirred at 20 °C for 1.5 h. The mixture was worked up as for (R)-(15b)
to give a solid that was purified by flash column chromatography on silica gel (gradient
elution from 2ꢀ10% of methanolic 2M ammonia in dichloromethane) to give to give 15c (38
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mg, 60%) as a white solid, mp 89ꢀ90 °C (dec.); [α] ꢁꢀ ꢂ ꢀ20.0 (c 1.0, methanol); νmax (cmꢀ1)
1
3214, 1652; H NMR (400 MHz, methanolꢀd
4
) δ ppm 4.12 (1H, dd, J=8.3, 6.6 Hz), 4.42 ꢀ
4
.58 (2H, m), 4.73 (1H, dd, J=9.1, 8.3 Hz), 5.16 (1H, dd, J=9.1, 6.6 Hz), 6.79 (1H, td, J=7.6,
.0 Hz), 6.93 (1H, dd, J=8.1, 1.0 Hz), 7.10 (1H, td, J=7.6, 1.3 Hz), 7.22 (1H, d, J=7.6 Hz),
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7.41 (1H, dd, J=7.8, 5.1 Hz), 7.53 (2H, d, J=8.1 Hz), 7.70 (1H, dt, J=7.8, 1.8 Hz), 8.00 (2H,
1
3
d, J=8.1 Hz), 8.41 ꢀ 8.49 (2H, m); C NMR (100 MHz, methanolꢀd ) δ ppm 45.5, 64.5, 74.6,
4
1
17.3, 118.3, 123.9, 126.3, 126.9, 127.1, 127.7, 127.7, 133.1, 135.0, 140.1, 142.4, 143.3,
+
+
+
147.2, 147.7, 163.3, 167.3; m/z (ESI ) 388 ([M+H] , 100%); HRMS C22
22 5 2
H N O requires:
388.1768, found: 388.1774.
N-(2-Aminophenyl)-4-(((4-(4-fluorophenyl)-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamide (15d). To a solution of 14d (220 mg, 0.436 mmol) in
dichloromethane (6 mL) was added trifluoroacetic acid (0.67 mL, 8.7 mmol) dropwise, then
the mixture was stirred at 20 °C for 3 h. The mixture was then worked up as for (R)-(15b) to
give a solid that was purified by column chromatography on silica gel (1:1:98 ammonia
(
30% aqueous): methanol: ethyl acetate) and again (1:6:93 ammonia (30% aqueous):
methanol: ethyl acetate) to give 15d as a white solid (108 mg, 61%), mp 104ꢀ108 °C; νmax
ꢀ1
1
(cm ) 3245, 1649; H NMR (500 MHz, chloroformꢀd) δ ppm 4.02 (1H, m), 4.09 (2H, br s),
4
.39 (2H, s), 4.60 (1H, m), 5.08 (1H, m), 6.71 ꢀ 6.86 (2H, m), 6.93 ꢀ 7.03 (2H, m), 7.07 (1H,
1
3
m), 7.12 ꢀ 7.22 (2H, m), 7.22 ꢀ 7.36 (3H, m), 7.70 ꢀ 7.87 (2H, m), 8.44 (1H, br s); C NMR
(125 MHz, chloroformꢀd) δ ppm 46.4, 66.7, 75.3, 115.3 (d, J=22.1 Hz), 118.1, 119.5, 124.4,
1
25.5, 127.2, 127.6, 127.9 (d, J=8.6 Hz), 133.0, 139.2, 140.9, 142.9, 162.0 (d, J=245.7 Hz),
+
+
+
1
4 2
61.7), 165.7; m/z (ESI ) 405 ([M+H] , 100%); HRMS C23H22FN O requires: 405.1721,
found: 405.1710.
N-(2-Aminophenyl)-4-(((4-(3-fluorophenyl)-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamide (15e). To a solution of 14e (190 mg, 0.377 mmol) in
dichloromethane (3 mL) was added trifluoroacetic acid (0.60 mL, 7.5 mmol) dropwise. The
resulting solution was stirred at 20 °C for 2 h. The mixture was then worked up as for (R)-
(
15b) to give a solid that was purified by flash column chromatography on silica gel
(gradient elution from 1ꢀ6% of methanolic 2M ammonia in dichloromethane) to give 15e
ꢀ1
1
(
131 mg, 86%) as a white solid, mp 84ꢀ86 °C; νmax (cm ) 3276, 1652; H NMR (400 MHz,
methanolꢀd ) δ ppm 4.05 (1H, dd, J=8.2, 6.4 Hz), 4.41 ꢀ 4.56 (2H, m), 4.68 (1H, dd, J=9.1,
8.3 Hz), 5.09 (1H, dd, J=9.0, 6.4 Hz), 6.79 (1H, td, J=7.6, 1.0 Hz), 6.88 ꢀ 7.02 (3H, m), 7.03
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ꢀ
7.14 (2H, m), 7.21 (1H, d, J=7.8 Hz), 7.33 (1H, td, J=7.9, 5.9 Hz), 7.53 (2H, d, J=8.1 Hz),
13
8
.00 (2H, d, J=8.1 Hz); C NMR (100 MHz, methanolꢀd ) δ ppm 45.6, 66.2, 74.9, 112.7 (d,
4
J=22.4 Hz), 113.7 (d, J=20.8 Hz), 117.3, 118.3, 121.9 (d, J=3.2 Hz), 124.0, 126.3, 127.0,
1
27.2, 127.7, 130.0 (d, J=8.0 Hz), 133.0, 142.4, 143.4, 146.8 (d, J=7.2 Hz), 163.0 (d,
+
+
+
J=245.3 Hz), 163.0, 167.2; m/z (ESI ) 405 ([M+H] , 100%); HRMS C H FN O requires:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
23 22
4
2
4
05.1721, found: 405.1723; 94% purity by LCMS.
N-(2-Aminophenyl)-4-(((4-(4-(trifluoromethyl)phenyl)-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamide (15f). To a solution of 14f (190 mg, 0.343 mmol) in
dichloromethane (4 mL) was added trifluoroacetic acid (0.53 mL, 6.85 mmol). The resulting
solution was stirred at 20 °C for 2.5 h. The mixture was then worked up as for (R)-(15b) to
give a solid that was purified by flash column chromatography on silica gel (gradient elution
from 1ꢀ6% of methanolic 2M ammonia in dichloromethane) to give 15f (110 mg, 71%) as a
ꢀ1
1
white solid, mp 198ꢀ199 °C; νmax (cm ) 3265, 1653; H NMR (500 MHz, methanolꢀd
ppm 4.08 (1H, dd, J=8.2, 6.6 Hz), 4.44 ꢀ 4.58 (2H, m), 4.73 (1H, dd, J=9.2, 8.2 Hz), 5.18
1H, dd, J=9.2, 6.6 Hz), 6.80 (1H, t, J=7.4 Hz), 6.93 (1H, d, J=8.0 Hz), 7.11 (1H, t, J=7.1
4
) δ
(
Hz), 7.22 (1H, d, J=7.7 Hz), 7.43 (2H, d, J=8.0 Hz), 7.54 (2H, d, J=8.0 Hz), 7.64 (2H, d,
13
J=8.0 Hz), 8.01 (2H, d, J=8.0 Hz); C NMR (125 MHz, methanolꢀd
4
) δ ppm 45.6, 66.3,
7
4.8, 117.4, 118.3, 124.3 (q, J=271.0 Hz), 124.0, 125.1 (q, J=3.9 Hz), 126.3, 126.7, 127.0,
+
1
27.2, 127.7, 129.2 (q, J=32.4 Hz), 133.0, 142.4, 143.4, 148.3, 163.2, 167.2; m/z (ESI ) 455
+
+
([M+H] , 100%); HRMS C24
22 3 4 2
H F N O requires: 455.1689, found: 455.1686; 92% purity
by LCMS.
(S)-N-(2-Aminophenyl)-4-(((4-(4-hydroxyphenyl)-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamide (15g). To a solution of 14g (250 mg, 0.497 mmol) in
dichloromethane (6 mL) was added trifluoroacetic acid (0.77 mL, 10 mmol). The resulting
solution was stirred at 20 °C for 2.5 h. The mixture was then worked up as for (R)-(15b) to
give a solid that was purified by flash column chromatography on silica gel (gradient elution
from 1ꢀ10% of methanolic 2M ammonia in dichloromethane) to give 15g (141 mg, 70%) as
a white solid, mp 158ꢀ160 °C; [α] ꢁꢀ ꢂ +17.6 (c 0.5, methanol); νmax (cm ) 3262, 1646; H
NMR (400 MHz, methanolꢀd ) δ ppm 4.03 (1H, dd, J=8.0, 6.8 Hz), 4.39 ꢀ 4.54 (2H, m), 4.62
1H, dd, J=9.0, 8.0 Hz), 4.99 (1H, dd, J=9.0, 6.8 Hz), 6.74 (2H, d, J=8.6 Hz), 6.79 (1H, td,
J=7.6, 1.0 Hz), 6.92 (1H, dd, J=7.8, 1.2 Hz), 7.02 ꢀ 7.14 (3H, m), 7.22 (1H, d, J=7.8 Hz),
ꢀ1
1
4
(
1
3
7
4
.52 (2H, d, J=8.1 Hz), 7.98 (2H, d, J=8.3 Hz); C NMR (100 MHz, methanolꢀd ) δ ppm
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5.6, 66.3, 75.3, 114.9, 117.4, 118.3, 124.0, 126.3, 127.0, 127.1, 127.2, 127.6, 132.9, 134.4,
+
+
+
142.3, 143.6, 156.5, 162.5, 167.3; m/z (ESI ) 403 ([M+H] , 100%); HRMS C H N O
23 23 4 3
requires: 403.1765, found: 403.1765.
(
R)-N-(2-Aminophenyl)-4-(((5-phenyl-4,5-dihydrooxazol-2-yl)amino)methyl)benzamide
R)-(15h). To a solution of (R)ꢀ14h (280 mg, 0.575 mmol) in dichloromethane (4 mL) was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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52
53
54
55
56
57
58
59
60
(
added trifluoroacetic acid (0.60 mL, 7.8 mmol) dropwise. The resulting solution was stirred
at 20 °C for 3.5 h. The mixture was then worked up as for (R)-(15b) to give a solid that was
purified by flash column chromatography on silica gel (gradient elution from 1.6ꢀ8% of
methanolic 2M ammonia in dichloromethane) to give a white solid (140 mg). Further
purification by MDAP gave (R)-(15h) (75 mg, 34%) as a white solid, mp 196ꢀ197 °C; [α] ꢀꢁ ꢂ
ꢀ1
1
ꢀ23.0 (c 1.0, methanol); νmax (cm ) 3398, 3238, 3340, 1668, 1641; H NMR (400 MHz,
DMSOꢀd ) δ ppm 3.44 (1H, dd, J=12.2, 6.6 Hz), 4.04 (1H, dd, J=12.2, 9.2 Hz), 4.38 (2H, s),
6
4.90 (2H, br s, NH
2
), 5.51 (1H, dd, J=9.2, 6.6 Hz), 6.62 (1H, t, J=7.3 Hz,), 6.80 (1H, d,
J=7.8 Hz), 6.98 (1H, t, J=7.3 Hz), 7.10 ꢀ 7.24 (2H, m), 7.26 ꢀ 7.36 (3H, m), 7.40 (2H, t,
13
J=7.1 Hz), 7.46 (2H, d, J=8.0 Hz), 7.96 (2H, d, J=8.0 Hz), 9.64 (1H, s); C NMR (100
MHz, DMSOꢀd ) δ ppm 46.1, 61.2, 80.3, 116.6, 116.8, 123.9, 125.9, 126.9, 127.1 , 127.3,
6
+
+
128.2, 128.3, 129.1, 133.5, 142.2, 143.6, 144.2, 160.5, 165.7; m/z (ESI ) 387 ([M+H] ,
+
2
100%); HRMS C23
H
23
N
4
O
requires: 387.1816, found: 387.1813.
(S)-N-(2-Aminophenyl)-4-(((5-phenyl-4,5-dihydrooxazol-2-yl)amino)methyl)benzamide
(S)-(15h). To a solution of (S)ꢀ14h (0.275 g, 0.565 mmol) in dichloromethane (6 mL) was
added trifluoroacetic acid (0.90 mL, 11.5 mmol). The resulting solution was stirred at 20 °C
for 3 h. The mixture was then worked up as for (R)-(15b) to give a solid that was purified by
MDAP to give (S)-(15h) (75 mg, 34%) as a cream solid, mp 214ꢀ215 °C; [α] ꢁꢀ ꢂ +22.0 (c 0.5,
ꢀ1
1
methanol); νmax (cm ) 3338, 3237, 1668, 1641; H NMR (500 MHz, DMSOꢀd
1H, dd, J=11.8, 7.0 Hz), 4.08 (1H, dd, J=11.8, 9.1 Hz), 4.41 (2H, s), 4.91 (2H, br s), 5.61
(1H, t, J=8.0 Hz), 6.62 (1H, t, J=7.3 Hz), 6.80 (1H, d, J=7.7 Hz), 6.99 (1H, t, J=7.0 Hz),
6
) δ ppm 3.49
(
7.19 (1H, d, J=7.4 Hz), 7.31 ꢀ 7.38 (3H, m), 7.39 ꢀ 7.44 (2H, m), 7.46 (2H, d, J=8.0 Hz),
1
3
7
6
.97 (2H, d, J=7.7 Hz), 9.67 (1H, s); C NMR (125 MHz, DMSOꢀd ) δ ppm 46.0, 59.5,
80.9, 116.6, 116.7, 123.8, 126.1, 126.9, 127.2, 127.3, 128.3, 128.6, 129.1, 133.7, 141.3,
+
+
+
1
43.6, 143.6, 160.7, 165.6; m/z (ESI ) 387 ([M+H] , 100%); HRMS C H N O requires:
23 23 4 2
387.1816 , found: 387.1822.
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N-(2-Aminophenyl)-4-(((4,4-diphenyl-4,5-dihydrooxazol-2-yl)amino)methyl)benzamide
(15i). To a solution of 14i (280 mg, 0.498 mmol) in dichloromethane (6 mL) was added
trifluoroacetic acid (0.80 mL, 10 mmol). The resulting solution was stirred at 20 °C for 2.5
h. The mixture was then worked up as for (R)-(15b) to give a solid that was purified by flash
column chromatography on silica gel (gradient elution from 1ꢀ6% of methanolic 2M
ammonia in dichloromethane) to give 15i (135 mg, 59%) as a white solid, mp 176ꢀ177 °C;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
1
ν
4
max (cm ) 3194, 1670, 1649; H NMR (400 MHz, methanolꢀd ) δ ppm 4.54 (2H, s), 4.79
(2H, s), 6.79 (1H, td, J=7.6, 0.8 Hz), 6.92 (1H, dd, J=8.1, 1.2 Hz), 7.10 (1H, td, J=7.7, 1.3
13
Hz), 7.17 ꢀ 7.37 (11H, m), 7.49 (2H, d, J=8.2 Hz), 7.96 (2H, d, J=8.2 Hz); C NMR (100
4
MHz, methanolꢀd ) δ ppm 45.4, 76.4, 80.0, 117.4, 118.3, 124.0, 126.3, 126.6, 126.6, 127.0,
+
+
1
27.1, 127.6, 127.8, 132.9, 142.4, 143.6, 146.4, 161.4, 167.2; m/z (ESI ) 463 ([M+H] ,
+
1
27 4 2
00%); HRMS C29H N O requires: 463.2129, found: 463.2118.
N-(2-Aminophenyl)-4-((((4S,5S)-4,5-diphenyl-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamide (15j). To a solution of 14j (155 mg, 0.275 mmol) in
dichloromethane (2 mL) was added trifluoroacetic acid (0.42 mL, 5.5 mmol) dropwise. The
resulting solution was stirred at 20 °C for 2 h. The mixture was then worked up as for (R)-
(15b) to give a solid that was purified by flash column chromatography on silica gel
(gradient elution from 1ꢀ6% of methanolic 2M ammonia in dichloromethane) to give 15j
(
125 mg, 98%) as a white solid, mp 110ꢀ113 °C; [α] ꢁꢀ ꢂ ꢀ33.2 (c 1.0, methanol); νmax (cmꢀ1)
+
+
1
3264, 1657; m/z (ESI ) 463 ([M+H] , 100%); H NMR (400 MHz, methanolꢀd
4.63 (2H, m), 4.89 (1H, m), 5.23 (1H, d, J=6.6 Hz), 6.79 (1H, t, J=7.5 Hz), 6.92 (1H, dd,
J=8.1, 1.3 Hz), 7.10 (1H, td, J=7.7, 1.5 Hz), 7.19 ꢀ 7.44 (11H, m), 7.57 (2H, d, J=8.3 Hz),
4
) δ ppm 4.46
ꢀ
1
3
8.02 (2H, d, J=8.1 Hz); C NMR (100 MHz, methanolꢀd ) δ ppm 45.6, 75.9, 89.3, 117.4,
4
118.3, 124.0, 125.3, 126.2, 126.3, 127.1, 127.2, 127.4, 127.7, 128.2, 128.4, 128.5, 133.0,
+
139.9, 142.4, 142.8, 143.4, 162.0, 167.3; HRMS C29
H
27
N
4
O
2
requires: 463.2129, found:
463.2128; 94% purity by LCMS.
N-(2-Aminophenyl)-4-((((4R,5S)-4,5-diphenyl-4,5-dihydrooxazol-2-
yl)amino)methyl)benzamide (15k). To a solution of 14k (414 mg, 0.736 mmol) in
dichloromethane (6 mL) was added trifluoroacetic acid (1.15 mL, 15 mmol) dropwise. The
resulting solution was stirred at 20 °C for 5 h. To the mixture was added saturated aqueous
sodium hydrogen carbonate (20 mL) and ethyl acetate (50 mL). After extraction, the aqueous
layer was separated and extracted with another portion of ethyl acetate (20 mL). The
2
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Page 29 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
combined organic layers were washed with brine, dried (Na
2 4
SO ) and evaporated to give a
white solid. Purification by flash column chromatography on silica gel (gradient elution from
3ꢀ6% of methanolic 2M ammonia in dichloromethane) followed by MDAP gave 15k (107
mg, 31%) as a white solid, mp 112ꢀ113 °C; [α] ꢁꢀ ꢂ +51.6 (c 0.5, methanol); νmax (cm ) 3270,
ꢀ1
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1652; H NMR (400 MHz, chloroformꢀd) δ ppm 3.90 (2H, br s), 4.39 ꢀ 4.63 (2H, m), 5.38
(
1H, d, J=8.8 Hz), 5.81 (1H, d, J=8.8 Hz), 6.73 ꢀ 6.94 (6H, m), 6.94 ꢀ 7.17 (7H, m), 7.30
1
3
(1H, m), 7.40 (2H, d, J=7.8 Hz), 7.84 (2H, d, J=7.8 Hz), 8.30 (1H, br s); C NMR (100
MHz, chloroformꢀd) δ ppm 46.5, 72.0, 85.7, 118.3, 119.7, 124.6, 125.4, 126.2, 126.8, 127.2,
27.4, 127.5, 127.5, 127.6, 127.6, 127.7, 133.2, 136.4, 139.2, 140.9, 143.3, 161.5, 165.8; m/z
1
+
+
+
27 4 2
(ESI ) 463 ([M+H] , 100%); HRMS C29H N O requires: 463.2129, found: 463.2128.
(R)-4-(((4-((1H-Imidazol-4-yl)methyl)-4,5-dihydrooxazol-2-yl)amino)methyl)-N-(2-
aminophenyl)benzamide (R)-(15l). To a suspension of (R)-(14l) (100 mg, 0.204 mmol) in
dichloromethane (2 mL) was added trifluoroacetic acid (0.31 mL, 4.1 mmol) dropwise. The
resulting solution was stirred at 20 °C for 2 h, then evaporated, and the residue was purified
by MDAP to (R)-(15l) (43 mg, 54%) as a white solid, mp 191ꢀ193 °C; [α] ꢁꢀ ꢂ +6.3 (c 1.0,
ꢀ1
1
methanol); νmax (cm ) 3227, 1673, 1627; H NMR (500 MHz, methanolꢀd ) δ ppm 2.74 (1H,
4
dd, J=15.5, 5.4 Hz), 3.24 (1H, dd, J=15.5, 7.3 Hz), 3.88 (1H, dd, J=11.3, 5.2 Hz), 4.36 (1H,
dd, J=11.1, 6.7 Hz), 4.43 (2H, s), 4.95 (1H, m), 6.70 (1H, s), 6.79 (1H, t, J=7.3 Hz), 6.93
(1H, d, J=7.7 Hz), 7.10 (1H, m), 7.21 (1H, d, J=7.4 Hz), 7.45 (2H, d, J=7.7 Hz), 7.59 (1H,
1
3
s), 7.97 (2H, d, J=8.0 Hz); C NMR (125 MHz, methanolꢀd
4
) δ ppm 28.8, 42.9, 50.6, 54.7,
1
1
17.3, 118.3, 118.8, 123.9, 126.2, 126.8, 127.1, 127.6, 130.9, 132.8, 134.6, 142.4, 144.2,
+
+
+
58.8, 167.3; m/z (ESI ) 391 ([M+H] , 100%); HRMS C21
H
23
N
6
O
2
requires: 391.1877,
found: 391.1881.
(S)-4-(((4-((1H-Imidazol-4-yl)methyl)-4,5-dihydrooxazol-2-yl)amino)methyl)-N-(2-
aminophenyl)benzamide (S)-(15l). To a suspension of 14k (130 mg, 0.265 mmol) in
dichloromethane (2 mL) was added trifluoroacetic acid (0.20 mL, 2.6 mmol) dropwise. The
resulting solution was stirred at 20 °C for 3 h, then additional trifluoroacetic acid (0.20 mL,
2
.6 mmol) was added, and the mixture stirred at 20 °C for 2 h. Addition of saturated aqueous
sodium hydrogen carbonate (15 mL) and then ethyl acetate (20 mL) gave an insoluble white
precipitate. The organic layer was separated and the aqueous layer was adjusted to pH 10
with sodium hydroxide (2M), then extracted with 1:4 ethanol:chloroform (4 x 25 mL). The
2 4
combined organic layers were washed with brine, dried (Na SO ) and evaporated to give a
2
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