Discovery of a Novel Inhibitor of Histone Lysine-Specific Demethylase 1A (KDM1A/LSD1) as Orally Active Antitumor Agent

Article abstract of DOI:10.1021/acs.jmedchem.5b01209

We report the stereoselective synthesis and biological activity of a novel series of tranylcypromine (TCPA) derivatives (14a-k, 15, 16), potent inhibitors of KDM1A. The new compounds strongly inhibit the clonogenic potential of acute leukemia cell lines. In particular three molecules (14d, 14e, and 14g) showing selectivity versus MAO A and remarkably inhibiting colony formation in THP-1 human leukemia cells, were assessed in mouse for their preliminary pharmacokinetic. 14d and 14e were further tested in vivo in a murine acute promyelocytic leukemia model, resulting 14d the most effective. Its two enantiomers were synthesized: the (1S,2R) enantiomer 15 showed higher activity than its (1R,2S) analogue 16, in both biochemical and cellular assays. Compound 15 exhibited in vivo efficacy after oral administration, determining a 62% increased survival in mouse leukemia model with evidence of KDM1A inhibition. The biological profile of compound 15 supports its further investigation as a cancer therapeutic.

Full text of DOI:10.1021/acs.jmedchem.5b01209

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Article
Discovery of a Novel Inhibitor of Histone Lysine-Specific
Demethylase 1A (KDM1A/LSD1) as Orally Active Antitumor Agent
Paola Vianello, Oronza A Botrugno, Anna Cappa, Roberto Dal Zuffo, Paola Dessanti, Antonello
Mai, Biagina Marrocco, Andrea Mattevi, Giuseppe Meroni, Saverio Minucci, Giulia Stazi,
Florian Thaler, Paolo Trifiro', Sergio Valente, Manuela Villa, Mario Varasi, and Ciro Mercurio
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01209 • Publication Date (Web): 24 Dec 2015
Downloaded from http://pubs.acs.org on December 24, 2015
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Discovery of a Novel Inhibitor of Histone Lysine-
Specific Demethylase 1A (KDM1A/LSD1) as
Orally Active Antitumor Agent
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a
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Paola Vianello, * Oronza A. Botrugno, Anna Cappa, Roberto Dal Zuffo, Paola Dessanti,
c,d
c
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a,e
Antonello Mai, Biagina Marrocco, Andrea Mattevi, Giuseppe Meroni, Saverio Minucci,
c
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Giulia Stazi, Florian Thaler, Paolo Trifiró, Sergio Valente, Manuela Villa, Mario Varasi
a,f
and Ciro Mercurio *
a. Department of Experimental Oncology, Academic Drug Discovery, European Institute of
Oncology, Via Adamello 16, Milan 20139, Italy.
b. Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100 Pavia
(Italy).
c. Department of Drug Chemistry and Technologies, Sapienza University of Rome, P.le A. Moro
5
, 00185 Roma (Italy).
d. Pasteur Institute ꢀ Cenci Bolognetti Foundation, Sapienza University of Rome, P.le A. Moro 5,
0185 Roma (Italy).
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e. Department of Biosciences, University of Milan, Via Celoria, 26, 20133 Milan, Italy
f. Genextra Group, DAC s.r.l., Via Adamello 16, 20139 Milano, Italy
*
Email correspondence to paola.vianello@ieo.eu, ciro.mercurio@ieo.eu
KEYWORDS: LSD1, KDM1A, epigenetics; leukemia; lysineꢀspecific demethylaseꢀ1;
tranylcypromine.
ABSTRACT
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We report the stereoselective synthesis and biological activity of a novel series of
tranylcypromine (TCPA) derivatives (14a-k, 15, 16), potent inhibitors of KDM1A. The new
compounds strongly inhibit the clonogenic potential of acute leukemia cell lines. In particular
three molecules (14d, 14e and 14g) showing selectivity versus MAO A and remarkably
inhibiting colony formation in THPꢀ1 human leukemia cells, were assessed in mouse for their
preliminary pharmacokinetic. 14d and 14e were further tested in vivo in a murine acute
promyelocytic leukemia model, resulting 14d the most effective. Its two enantiomers were
synthesized: the (1S, 2R) enantiomer 15 showed higher activity than its (1R, 2S) analogue 16,
both in biochemical and cellular assays. Compound 15 exhibited in vivo efficacy after oral
administration, determining a 62% increased survival in mouse leukemia models with evidence
of KDM1A inhibition. The biological profile of compound 15 supports its further investigation
as a cancer therapeutic.
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INTRODUCTION
Over the last two decades, the relevance of epigenetic modifications for transcriptional control
and normal development, as well as the role of such modifications in cancer development and
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progression, has attracted a great deal of attention. Histone lysine methylation, a dynamic and
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, 5
accurately regulated mechanism, represents one of the several postꢀtranslation modifications
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controlling chromatin conformation and gene transcription. Two enzyme families, lysine
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methyltransferases (KMTs) and demethylases (KDMs) , control histone lysine methylation.
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KDMs encompass the Jumonji C (JmjC) domain–containing proteins and the lysine specific
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demethylase (LSD) family .
Two lysine specific demethylase proteins have been identified to date: KDM1A (LSD1), a flavin
adenine dinucleotide (FAD) dependent amino oxidase which was the first discovered histone
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lysine demethylase , and its homologous KDM1B (LSD2) . KDM1A is often found associated
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with protein complexes functioning as transcriptional repressors, such as CoRest
, and with
. Consistently, KDM1A
demethylates mono and dimethyl histone 3 lysine 4 (H3K4me1, H3K4me2), which are marks
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the nucleosome remodelling and deacetylation complex Nurd
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associated with active gene transcription .
In addition, KDM1A has been found in complexes involved in active gene transcription and
reported to change its substrate specificity from H3K4me1/me2 to mono and dimethyl histone
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H3 lysine 9 (H3K9me1/me2), once associated to estrogen and androgen nuclear receptors
.
KDM1A has also been found in protein complexes involved in transcription elongation such as
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the ELL complex and the MLL super complex .
The H3K4 demethylase activity of KDM1A can be influenced by histone posttranslational
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modifications such as acetylation and phosphorylation . In particular, both histone H3 lysine 9
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acetylation (H3K9Ac) and histone H3 serine 10 phosphorylation (H3S10p) can strongly reduce
or block KDM1A demethylase activity.
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Several non histone substrates of KDM1A have been identified
. For instance, KDM1A
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demethylates and stabilizes DNA methyl transferase 1 (DNMT1) , E2F1 and STAT3 .
Furthermore, KDM1A demethylates the myosin phosphatase MYPT1, a phosphatase involved in
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Rb dephosphorylation , as well as repressing p53 function through the inhibition of its
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interaction with 53BP .
High KDM1A expression correlates with poor prognosis in several tumor types such as
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neuroblastoma, prostate cancer,
non small cell lung cancer, breast cancer,
esophageal
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squamous cancer , and hepatocellular carcinoma . Bladder cancer and colorectal cancer are
also characterized by elevated KDM1A expression. Notably, KDM1A was recently proposed to
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confer stem cell like characteristics upon breast cancer cells as well as maintain stem cellꢀlike
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tumor propagating cells in human glioblastoma . In hematopoietic and lymphoid neoplasias,
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high expression of KDM1A has been observed in acute myeloid leukemia , myeloproliferative
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neoplasms, chronic myelomonocytic leukemia, and myelodysplastic syndromes . In addition,
strong experimental evidence supports a role for KDM1A in the selfꢀrenewal of leukemic stem
cells in acute myeloid leukemia (AML), and, more specifically, in the promotion of the
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oncogenic gene program associated with MLLꢀAF9 leukemia .
The link between KDM1A and tumor pathogenesis, and the discovery that the monoamine
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oxidase (MAO) inhibitor tranylcypromine (TCPA) blocks KDM1A activity , represented the
starting point for intensive drug discovery programs aimed at the development of irreversible
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TCPAꢀbased KDM1A inhibitors
. Two TCPA derivatives (Figure 1), N4ꢀ[(1R,2S)ꢀ2ꢀ
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phenylcyclopropyl]cyclohexaneꢀ1,4ꢀdiamine (22, ORY1001/RGꢀ6016)
and 4ꢀ[[4ꢀ[[[(1R,2S)ꢀ
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2
ꢀphenylcyclopropyl]amino]methyl]ꢀ1ꢀpiperidyl]methyl]benzoic acid (23, GSK2879552)
,
both characterized by nitrogen substitution at the core of TCPA, are currently under clinical
investigation. In addition, several reversible inhibitors of KDM1A have been identified by
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different research groups .
In a previous manuscript, we showed that substitutions on the phenyl moiety of TCPA allowed
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the identification of highly active KDM1A inhibitors . Among these, compound 1 (4ꢀ
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benzamidoꢀcyclopropylamine; 13b in reference ) (Figure 2) demonstrated to be a potent
KDM1A inhibitor, while its ortho and meta regioisomers exhibited a lower activity against
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KDM1A . The enantiomers of compound 1 displayed potent KDM1A inhibition, with IC
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values ranging from 13 to 26 nM, as well as anticlonogenic activity in primary murine acute
promyelocytic leukemia blasts. These results prompted us to expand the scaffold with the aim to
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obtain compounds with a better selectivity profile towards MAO A and good activity in a human
cellular model of MLLꢀAF9 leukemia (THPꢀ1 cells). Compound 14d was found to be the most
promising derivative in this respect, and its biological properties encouraged the synthesis of its
two enantiomers. We report herein the stereoselective synthesis of the two enantiomers (Scheme
1) together with their in vitro and in vivo characterization.
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RESULTS AND DISCUSSION
Chemistry.The synthesis of the final compounds 14a-k, 15 and 16 was carried out following
two routes. According to Scheme 1 (Method A), the oxirane 2 as well its 2ꢀS (3) and 2ꢀR (4)
analogues were added to a solution of triethyl phosphonoacetate and butyl lithium in
dimethoxyethane and the reaction was microwave irradiated to 130°C affording the ethyl esters
of 2ꢀphenylcyclopropanecarboxylic acids. Subsequent hydrolysis of the ethyl esters with LiOH
provided the carboxylic acids 5-7, which were treated with iodine in acetic acid yielding the 4ꢀ
iodo derivatives 8-10. The protected amino derivatives 11-13 were subsequently obtained by
reaction of the iodo derivatives 8-10 with diphenyl phosphorazidate (DPPA) in presence of
triethylamine and in tertꢀbutanol at 90°C. Ullman reaction with primary amides and subsequent
BOC deprotection with hydrochloric acid afforded the cyclopropylamines 14a-g, 15 and 16 as
their hydrochloride salts.
According to Scheme 2 (Method B), the appropriate acids 18hꢀk were activated by benzotriazolꢀ
1ꢀylꢀoxytripyrrolidinophosphonium
hexafluorophosphate
(PyBOP)
or
1ꢀ(3ꢀ
dimethylaminopropyl)ꢀ3ꢀethylcarbodiimide (EDC), in the presence triethylamine and
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condensated with tertꢀbutyl Nꢀ[2ꢀ(4ꢀaminophenyl)cyclopropyl]carbamate 17 . The final
compounds 14h-k were smoothly obtained by removing Boc protection with hydrochloric acid in
dioxane.
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The carboxylic acid or primary amide intermediates were prepared according to Schemes 3 or 4
when not commercially available.
The synthesis of bisubstituted acids 18hꢀk is reported in Scheme 3. tertꢀButyl chloroꢀnitroꢀ
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benzoates were converted into 19hꢀk by reaction with morpholine, 4ꢀmethylpiperazine, or
piperidine in presence of K CO . Reduction of the nitro group to the amine in compounds 20hꢀk
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was achieved by conventional catalytic hydrogenation, e.g. in a Parr hydrogenation or HꢀCube
apparatus using a palladium catalyst. The carbamates 21h-k were then obtained by reacting the
free amines 20hꢀk with benzyl chloroformate in presence triethylamine at room temperature.
Finally, the tertꢀbutyl ester group was hydrolyzed with TFA (trifluoroacetic acid) in
dichloromethane.
The benzamides 19e-f were prepared according to Scheme 4. The commercially available 3ꢀ or
4ꢀ(2ꢀoxoꢀ1,3ꢀoxazolidinꢀ3ꢀyl)benzoic acids 18e and 18f were first activated with thionyl chloride
and then converted into the amides 19e-f with aqueous NH in DMF.
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Biological Evaluation and SAR,.Benzamidoꢀderivatives of TCPA have been previously
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described, reporting the para substitution more potent than the ortho and meta isomers.
Substituents on the phenyl ring of compound 1 were directed towards a major occupancy of the
binding pocket, without impacting on drug like properties of the molecules, thus maintaining
solubility at an acceptable level. For this purpose, both aliphatic (piperazine, morpholine,
piperidine oxazolidinꢀ2ꢀone) and aromatic (furane) heterocycles were prioritized among the set
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of most frequently used ring systems present in small molecule drugs . Alternatively or
concurrently we introduced a carbobenzyloxy residue which could either impact on the
stereoelectronic features of the benzamide end side, or branch the molecule to modulate
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selectivity versus MAOs. As summarized in Table 1 the final compounds 14a-k as well as the
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reference compound 22 were profiled in vitro for their KDM1A, KDM1B, MAO A and B
inhibitory activities. To this end human recombinant KDM1A/CoREST and KDM1B as well as
MAO A and MAO B proteins were employed as enzymatic sources. The compounds were
incubated with the enzymatic complex and the results are expressed as IC values.
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The introduction of different residues did not lead to further increase in KDM1A inhibitory
activity, the most potent being the equipotent furanꢀbearing 14a (IC =0.022 ꢀM to be compared
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to 0.019 ꢀM of compound 1). However all compounds showed IC values equal to or less than
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.1 μM, with the exception of compound 14k (IC =0.308 μM). Similarly, substitution in meta
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versus para did not influence the inhibitory potency of KDM1A (14e vs 14f), whereas the use of
carbobenzyloxy group 14b gave a 2 fold less potent derivative, which slightly improved in its
cyclized analogue meta oxazolidinꢀ2ꢀone 14e. The para oxazolidinꢀ2ꢀone derivative 14f resulted
two times more potent than the morpholine analogue 14c, which in turn was 3 fold more potent
than piperazine 14d. Furthermore, substitution of the benzamide, in particular by a 3ꢀ
benzyloxycarbonylamino (3ꢀCBZ) moiety resulted in an increased KDM1B inhibitory activity.
In specific, compound 1 exhibited an IC value of 58.3 μM, the 3ꢀCBZ substituted derivative
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4b resulted to be roughly five times more active (IC =11.6 μM), while the disubstituted
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inhibitors 14h, 14j, and 14k had IC values of around 5 μM. With the exception of 14a, having
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an IC50 value of 0.09 μM, all other compounds were selective over MAO B: in particular,
compounds 14b, 14g and 14j did not show any MAO B inhibitory activity under the employed
experimental conditions. We hypothesized that the higher activity of 14a versus MAO B can be
related to the aromaticity of the furan, which finds an ideal environment in the highly
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hydrophobic active site of MAO B. MAO A IC s ranged between 0.025 μM (14b) and 1.0 μM
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(14j), among them 14d,14e and 14g-j resulted to be more selective than compound 1 (MAO
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A/KDM1A ratio < 2). 14g and 14j resulted to be the most selective ones, with a selectivity ratio
higher than 10 (MAO A/ KDM1A).
The compounds with good biochemical activity and with an acceptable selectivity versus MAO
A were selected and further tested them towards leukemia cell lines to investigate their ability to
inhibit KDM1A: 14d, 14e and 14g-j (see Table 2). In a first experiment, human promyelocytic
leukemia NB4 cells were incubated with the inhibitors at a concentration equal to their
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corresponding biochemical IC value. NB4 cells were selected on the basis of literature data
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which indicate that the inhibition of KDM1A has a biological relevance both in human
promyelocytic leukemia cell lines (NB4) and murine primary promyelocytic blasts. After 24
hours of incubation, GFI1B mRNA levels were measured by quantitative RTꢀPCR to measure
KDM1A inhibition in cells. GFI1B is a gene associated to hematopoietic differentiation, its
mRNA expression increases following KDM1A down regulation (Figure S2 supplementary data)
and more importantly the gene has been demonstrated to be a direct transcriptional target of
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KDM1A . As shown in Table 2, all the tested compounds determined at least a 4ꢀfold increase
of the mRNA expression of GFI1B, compared to cells treated with the vehicle, proving their
ability to block KDM1A activity in cellular systems. Moreover, considering that KDM1A
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inhibition should induce differentiation of human AML cells we evaluated the effect of the
compounds on the expression level of CD11b, a well known granulocytic differentiation marker
modulated by KDM1A dowregulation (Fig.S2 supplementary data). As reported in Table 2, all
the tested compounds elicited, at their respective biochemical IC50, an increase of CD11b
expression, suggestive of granulocytic differentiation induction of the NB4 cells. Starting from
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experimental data
, which indicate that the KDM1A inhibitors are of potential relevance for
treatment of promyelocytic leukemia, we further investigated the cellular activity of the
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compounds by measuring their ability to inhibit the colony formation of murine acute myeloid
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leukemia blasts (originated by a murine acute promyelocytic leukemia ) in semisolid culture at a
dose of 0.250 μM. All the selected compounds inhibited colony formation, the percentage of
inhibition ranging from 84 to 57% (Table 2). To confirm that the obtained biological data
specifically correlated to KDM1A inhibition, the effect of KDM1A down regulation by shRNA,
both in NB4 cells and murine promyelocytic blast, was analyzed. As reported in the
supplementary section, the effect of KDM1A silencing are in agreement with the reported
biological data obtained with the inhibitors both in terms of transcriptional effects on the
expression of GFI1B and CD11b and on the reduction of colony formation in mouse acute
promyelocytic blast (supplementary data Figure S2 and S3).
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The most active compounds 14d, 14e and 14g, inhibiting the murine promyelocytic blasts at 84
and 79% respectively, as well as the reference compound 1 (inhibition of 73%) and 22
(inhibition of 84%), were additionally tested in a human leukemia cell line. Since the relevance
of KDM1A in sustaining the oncogenic potential of MLLꢀAF9 leukemia stem cells has been
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demonstrated and reported in the literature , we evaluated the anticlonogenic potential of 14d,
14e, 14g, 1 and 22 in a human leukemia cell line, characterized by MLLꢀAF9 translocation
(THPꢀ1). As reported in Table 2, compounds 14d, 14e and 14g as well as 22 continued to show
strong inhibitory efficacy with a percentage of inhibition of 63, 51, 65 and 58 % respectively, at
the fixed dose of 0.50 ꢁM, whereas reference compound 1 unexpectedly only weakly inhibited
the colony forming ability formation of the cells (8%). Of relevance, 14d, 14e and 14g similarly
to 22, induced a clear differentiation of THPꢀ1 cells as demonstrated by altered morphology of
the colonies and by examination of cytospin preparations of cells recovered at the end of semiꢀ
solid culture (Figure 4).
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We therefore advanced through our screening funnel only those compounds with good potency
in the biochemical assay, showing some level of selectivity versus MAO A and demonstrating a
clear cellular efficacy in the anticlonogenic potential assay. In view of their promising overall
activity in both biochemical and cellular assays 14d, 14e and 14g were selected for preliminary
pharmacokinetic assessment in mouse, (Table 3) whilst the parent compound 1 was not included
in the analysis for its very limited anticlonogenic activity observed in THPꢀ1 cells.
0
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The selected inhibitors were administered in single intravenously (iv) or orally (os) to CD1 mice
at 5 mg/kg or 15 mg/kg respectively. The derivatives were dissolved in 5% glucose solution
containing 10% tween 80 for the iv dose or in 5% glucose solution containing 40% PEG for the
oral dose. The plasma PK parameters are summarized in Table 3. Following the iv
administration, the three compounds showed medium to high clearance rates of 94.8±21.3,
4
9.7±12.2 and 80.7±4.8 mL/min/kg for 14d, 14e and 14g respectively. The elimination halfꢀlives
were significantly longer for 14d (with ∼8 h) and for 14g (∼12 h) than for compound 14e
1.92±0.40 h). Moreover, the estimated terminal volume of distribution (V ) was high for all
(
z
three compounds, which indicates an extensive tissue distribution of the molecules. In particular,
14d and 14g had V of 67 and 85 L/kg, respectively, much higher than 14e (V =8.45±3.75 l/kg).
z
z
Compounds 14d and 14g were slowly absorbed after oral administration (tmax were 12 h and 18 h
for 14d and 14g, respectively), whereas ꢀ 14e was taken up more rapidly (tmax of less than 1 h).
Cmax and AUC0–∞ after oral administration were significantly different among the compounds. In
specific, compound 14e exhibits a Cmax of 1.14±0.16 ꢁM, which is around 20 times more than
that obtained for compound 14g. The AUC0–∞ ranged from 0.979±0.131 ꢁMh (14g) to 7.12±2.04
ꢁ
Mh (14e). The higher exposure data after oral administration of 14d and 14e compared to 14g,
both in terms of Cmax and AUC0–∞, and the higher oral bioavailability of compounds 14d (40%)
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and 14e (55.8%) compared to 14g (15.4%) prompted us to select 14d and 14e for a further in
vivo characterization.
To this aim, 14d and 14e were submitted to in vivo efficacy experiment in an established murine
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71, 72
promyelocytic leukemia model.
For the in vivo test, one million leukemic cells were injected
iv into nonꢀirradiated 129SvEv mice. Treatment with 14d and 14e started once blast cells were
detected in the recipients' peripheral blood (10 days after cell injection). To compare the efficacy
between the two selected compounds, 14d and 14e were orally administered at the fixed dose of
27 mg/kg five days per week for two weeks. The survival of mice belonging to different
experimental groups was analyzed and represented by a Kaplan–Meier survival plot. No
significant body weight differences among the groups of mice were observed during the
treatment (data not shown) and, as reported in figure 5, both compounds elicited an increase of
survival of the treated mice compared to the vehicle group. Specifically, 14d and 14e determined
an increase of survival respectively of 35 and 15% with respect to the vehicle group. At this
point, to investigate different schedule of compound administration for the in vivo efficacy, we
considered the irreversible mechanism towards which these derivatives interact with the enzyme
and we postulated that an intermittent treatment could have been desirable. 14d in fact was tested
at the dose of 45 mg/kg for three days per week (Monday, Tuesday and Wednesday) for two
weeks that correspond to the same weekly cumulative dose obtained with the administration of
the compound at the dose of 27 mg/kg for 5 days per week. Interestingly, the in vivo efficacy
evaluated in terms of survival improved significantly (from 35 to 58%) when we followed the
intermittent administration (Figure 5B). Moreover and importantly, 14d shown a significant
better in vivo efficacy in the murine promyelocytic leukemia model if compared to 22 which,
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73
once administered according to schedule/dosage reported by Oryzon was able to increase the
survival of leukemic mice of 14% with respect to the vehicle group (Figure 5C).
Furthermore, in order to correlate the observed in vivo efficacy with KDM1A inhibition, spleens
of the leukemic mice treated with the vehicle and those with 14d were recovered after 3 days of
treatment and cells were collected for RNA analysis. mRNA levels of GFI1B were measured by
quantitative RTꢀPCR using specific primers and normalized against TBP mRNA. Results are
presented as mRNA expression and as illustrated in the graph (Figure 5D) both schedules
determined a clear increase of GFI1B expression, although not statistically significant,
supporting the ability of compound to inhibit KDM1A in vivo after oral administration.
Encouraged by the results on the racemic mixture of compound 14d, we approached the
enantioselective synthesis of 15 (1S, 2R) and 16 (1R, 2S) starting from the enantiomerically pure
styrene epoxides. As evidenced in Table 4, compound 15 resulted to inhibit KDM1A with an
0
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IC of 0.084uM thus three times more potent than the enantiomer 16 (IC =0.23uM), which in
50
50
turn was a weaker inhibitor with respect to the racemate (IC =0.188uM). The activity of the
5
0
racemate and enantiomers versus KDM1B could be considered comparable, being in the high
micromolar range. Selectivity versus MAOs remained instead almost unaltered in term of IC ꢀ
5
0
ratio for the (1S, 2R) enantiomer (ratio MAO A/LSD1=3.5 for 15, to be compared to 3.9 for 14d)
whereas it worsens for compound 16. An even greater difference among the enantiomers was
found in the cellular assays: specifically compound 15 resulted to be more active in reducing the
colony forming ability in the two used cell models, and, importantly, in inducing the
differentiation of THPꢀ1 cells compared to the 1R, 2S analogue 16 (Table 5 and Figure 6).
Compound 15 was then evaluated for its in vivo behavior. For this purpose, the pharmacokinetic
profile in CDꢀ1 mice was analyzed at the same conditions formerly described for inhibitors 14d,
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4e and 14g (Table 6). The compound exhibited a high clearance rate, but somewhat lower than
the racemic mixture 14d (79.7±15.0 mL/min/kg for 15 over 94.8±21.3 mL/min/kg for 14d).
Furthermore 15, when orally administered, exhibited a high V of 58.4±9.78 L/kg and a half life
z
10
11
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14
15
16
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of more than 8 h. Although the oral bioavailability was lower than that shown by 14d (22.5% for
15 over 40% for 14d) both Cmax and AUC0–∞ were comparable (0.147±0.112 and 1.66±0.889 of
15 over 0.138±0.0517 and 2.39±0.770 of 14d).
The single 1S,2R enantiomer 15 was tested in vivo in the murine promyelocytic leukemia model
as previously described for the trans racemate 14d. 15 was dissolved (40% PEG 400 in a 5%
glucose solution) and orally administered 3 days per week (Monday, Tuesday and Wednesday)
for 3 weeks at the doses of 11.25 mg/kg and 22.5 mg/kg. The treatment started once blast cells
were detected in the recipients' peripheral blood (10 days after cell injection). As reported for the
racemic mixture 14d, the survival of mice of the different experimental groups was analyzed and
represented by a Kaplan–Meier survival plot. No significant body weight differences among the
groups of mice were observed during the treatment. A significant dose dependent increase of
survival was obtained and, specifically, 15 determined an increase of 35 and 62% at the dose of
11.25 and 22.50 mg/kg respectively (Figure 7A). Remarkably the observed in vivo efficacy
associated to a good tolerability profile of the compound 15 could be suggestive of its
preferential activity on tumor cells with respect to normal cells. In order to correlate the observed
in vivo efficacy of 15 with KDM1A inhibition, spleens of the leukemic mice treated with the
vehicle or with compound at the highest tested dose of 22.5 mg/kg were recovered after 3 days of
treatment and cells were collected for RNA analysis. A clear increase of GFIB expression,
although not statistically significant, was observed (Figure 7B), which supported the ability of 15
to inhibit KDM1A in vivo after oral administration.
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CONCLUSIONS
A series of novel and potent TCPAꢀderived inhibitors of KDM1A has been identified starting
from the previously disclosed compound 1. Some of the compounds (14d, 14e and 14g)
demonstrated improved selectivity versus MAO A and a higher cellular activity in human THPꢀ1
cells, showing 51ꢀ65% inhibition compared to the 8% inhibitory activity of compound 1.
Pharmacokinetic studies revealed a good oral bioavailability of compounds 14d (40%) and 14e
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(55.8%), in both cases significantly higher than that of derivative 14g (15.4%). Compounds 14d
and 14e were then studied for their in vivo antitumoral activity in a murine acute promyelocytic
leukemia model. Oral treatment with compounds 14d and 14e, at the fixed dose of 27 mg/kg five
days per week for two weeks, led to significant survival increase without signs of overt toxicity
and with evidence of in vivo target modulation. Two enantiomers of 14d were eventually
prepared and profiled, leading to the identification of the (1S, 2R) eutomer 15, which showed
higher potency in both biochemical and cellular assays in comparison to the (1R, 2S) enantiomer
16.
Compound 15, orally admistered in vivo in the murine promyelocytic leukemia model, was well
tolerated and significanly prolonged survival time of the treated mice (35 and 62% at the doses
of 11.25 and 22.50 mg/kg, respectively). Collectively, these results have supported the progress
of compound 15 into preclinical development as a potential oral anticancer agent.
EXPERIMENTAL SECTION
Chemistry
General Procedures.Reagents and solvents used, unless stated otherwise, were of commercially
available reagent grade quality and were used without further purification. Flash chromatography
purifications were performed on Merck silica gel 60 (0.04e0.063 mm). Nuclear magnetic
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resonance spectra ( H NMR and C NMR) were recorded either on a Varian 500 MHz or on a
Varian 400 MHz spectrometer at 300 K and are referenced in ppm (δ) relative to TMS. Coupling
constants (J) are expressed in hertz (Hz). HPLCꢀMS experiments were performed on an Acquity
UPLC apparatus, equipped with a diode array and a Micromass SQD single quadruple (Waters).
Optical rotations were measured at the sodium D line, using a Krüss P3001/3002 RS automatic
digital polarimeter and a 100 mm cell and are reported as follows: concentration (c = g/100 mL),
solvent. Purity was monitored at 220 nm and the purities of the compounds used for biological
tests were found to be at least 95%.
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Materials. tertꢀbutyl Nꢀ[2ꢀ(4ꢀaminophenyl)cyclopropyl]carbamate (48), tert butyl 3ꢀchloroꢀ4ꢀ
6
6
74
75
nitroꢀbenzoate , 4ꢀ(3ꢀfuryl)benzamide
benzyl Nꢀ(3ꢀcarbamoylphenyl)carbamate
were
prepared according to procedures described in the literature. 22 was prepared according to the
5
5
procedure decribed by Oryzon.
(1R,2R)-1-Ethyl-2-phenylcyclopropanecarboxylate (6)
9 mL (173 mmol) of a 2.5 M solution of nꢀbutyllithium in hexane was added at 25 °C dropwise
6
over 5 min to a solution of 39 g (170 mmol) triethyl 2ꢀphosphonoacetate in 75 mL of dry DME.
The reaction mixture was stirred at RT for 5 min, then 16.0 g (133 mmol) of (S)ꢀstyrene oxide
was added in one portion. The reaction was heated at 135°C for 60 min under MW. The orange
reaction mixture was then cooled down to RT, saturated aqueous NH Cl was added and the
4
product was extracted with Et O. The combined organic layers were dried over Na SO , filtered
2
2
4
and the solvent was removed in vacuo. The crude product was filtered on a silica gel pad giving
21g (83%) of ethyl 2ꢀphenylcyclopropane carboxylate as yellow oil.
The ethyl ester was hydrolized according to the following procedure: 10.58 g (441.6 mmol)
LiOH in 200 mL of H O was added to a solution of 21 g (110 mmol) of ethyl 2ꢀ
2
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phenylcyclopropanecarboxylate dissolved in 220 mL of an ethanol/THF mixture (10;1, v:v) and
the solution was heated at 115°C for 50 min under MW irradiation. Then, the solution was
concentrated, diluted with H O and quenched with 2 M HCl at 0°C. The precipitate was filtered
2
0
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3
4
5
6
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off, washed with H O and triturated with Et O providing 14.85 g (83%) the (1R,2R)
2
2
1
cyclopropane carboxylic acid 6 as a white solid. H NMR (CDCl ) δ (ppm): 11.5 (br s, 1 H, ꢀ
3
COOH), 7.34–7.20 (m, 3 H, aromatic protons), 7.16–7.06 (m, 2 H, aromatic protons), 2.66–2.57
(m, 1 H, ꢀCH cyclopropane), 1.98–1.88 (m, 1 H, ꢀCH cyclopropane), 1.73–1.61 (m, 1 H, ꢀCHH
ꢀ
20
cyclopropane), 1.47–1.37 (m, 1 H, ꢀCHHcyclopropane); MS (ESI): m/z: 161 [MꢀH] . [α]D = ꢀ
297° (c 0.753, CHCl3).
The transꢀ1ꢀethylꢀ2ꢀphenylcyclopropanecarboxylate 5 was purchased from SigmaꢀAldrich
(1S,2S)-2-phenylcyclopropanecarboxylic acid 7 was prepared according to the procedure
described for example 6 starting from the appropriate (R)ꢀstyrene oxides and phosphonoacetates
1
Yield: 85%, H NMR (CDCl ) δ (ppm): 11.75 (br s, 1 H, ꢀCOOH ), 7.35–7.08 (m, 5 H, aromatic
3
protons), 2.67–2.56 (m, 1 H, ꢀCH cyclopropane), 1.98–1.86 (m, 1 H, ꢀCH cyclopropane), 1.74–
1.63 (m, 1 H, ꢀCHH cyclopropane), 1.48–1.37 (m, 1 H, ꢀCHH cyclopropane); MS (ESI): m/z:
ꢀ
20
D
1
61 [MꢀH] . [α]
= +294° (c 0.933, CHCl3).
Examination of ethyl esters of 5,6 and 7 by NMR with the aid of the optically active shift reagent
Europium tris[3ꢀ(heptafluoropropylhydroxymethylene)ꢀ(+)ꢀcamphorate], showed 5 to be a
misture 1 to 1 of the two enantiomers, 6 and 7 to be optically pure .
(1R, 2R)ꢀ2-(4-iodophenyl)cyclopropanecarboxylic acid (9)
1
3 g (50 mmol) of iodine, 4.5 g (21 mmol) potassium iodate and 7.5 mL concentrated H SO in
2 4
75 ml of H O was added to a stirred solution of 14.7 g (90.6 mmol) of (1R,2R)ꢀ2ꢀ
2
phenylcyclopropanecarboxylic acid in 300 mL of AcOH. The mixture was heated to reflux for
about 2 h, and then the reaction was stopped by adding 700 mL of 1 M NaHSO . The formed
4
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precipitate was filtered, dried and the resulting solid was triturated with hexane providing 19 g
1
(73%) of the (1R,2R) carboxylic acid 9 as a white solid. H NMR (DMSOꢀd ) δ (ppm): 12.66–
6
10.65 (m, 1 H, ꢀCOOH), 7.72–7.54 (m, 2 H, aromatic protons), 6.96–6.79 (m, 2 H, aromatic
10
11
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15
16
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protons), 2.63–2.46 (m, 1 H, ꢀCH cyclopropane), 1.97–1.80 (m, 1 H, ꢀCH cyclopropane), 1.74–
1.61 (m, 1 H, ꢀCHH cyclopropane), 1.47–1.30 (m, 1 H, ꢀCHHcyclopropane); MS (ESI): m/z: 287
ꢀ
[
MꢀH] .
The following carboxylic acids were prepared according to the procedure for compound 9:
(trans)ꢀ2-(4-iodophenyl)cyclopropanecarboxylic acid (8)
1
Yield: 84% H NMR (DMSOꢀd ) δ (ppm): 12.36 (br s, 1 H, ꢀCOOH), 7.65–7.55 (m, 2 H,
6
aromatic protons), 7.06–6.93 (m, 2 H, aromatic protons), 2.40–2.28 (m, 1 H, ꢀCH cyclopropane),
1.85–1.73 (m, 1 H, ꢀCH cyclopropane), 1.47–1.37 (m, 1 H, ꢀCHH cyclopropane), 1.35–1.24 (m,
ꢀ
1
H, ꢀCHH cyclopropane ) MS (ESI): m/z: 287 [MꢀH]
(1S, 2S)ꢀ2-(4-iodophenyl)cyclopropanecarboxylic acid (10)
1
Yield: 80% H NMR (DMSOꢀd ) δ (ppm): 12.29 (br s, 1 H, ꢀCOOH), 7.60 (d, J=8.3 Hz, 2 H,
6
aromatic protons), 6.99 (d, J=8.3 Hz, 2 H, aromatic protons), 2.41–2.27 (m, 1 H, ꢀCH
cyclopropane ), 1.85–1.71 (m, 1 H, ꢀCH cyclopropane), 1.46–1.36 (m, 1 H, ꢀCHH cyclopropane),
ꢀ
1
.35–1.24 (m, 1 H, ꢀCHH cyclopropane); MS (ESI): m/z: 287 [MꢀH]
tert-butyl N-[trans-2-(4-iodophenyl)cyclopropyl]carbamate (11)
.4 g (27 mmol) Diphenyl phosphoryl azide and 4.4 mL (32 mmol) TEA were added to a
7
solution of 7.05 g (24.5 mmol) of transꢀ2ꢀ(4ꢀiodophenyl) cyclopropanecarboxylic acid in 150
mL dry tꢀBuOH. After stirring at 90 °C for 20 h the solution was concentrated and the residue
was partitioned between 10% aqueous Na CO and Et O. The combined organic layers were
2
3
2
dried over Na SO , filtered, and concentrated. The crude product was purified by flash
2
4
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chromatography (hexane/EtOAc 9:1, v:v) giving 5.6 g (62%) of the tertꢀbutyl carbamate 11 as
1
white solid. H NMR (DMSOꢀd ) δ (ppm): 7.67–7.51 (m, 2 H, aromatic protons), 7.25 (br s, 1 H,
6
NHCOOtBu), 6.98–6.84 (m, 2 H, aromatic protons), 2.57 (br s, 1 H, ꢀCH cyclopropane), 1.89–
0
1
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5
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7
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9
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1
.74 (m, 1 H, ꢀCH cyclopropane), 1.37 (s, 9 H, ꢀCOO(CH ) ), 1.18–0.95 (m, 2 H, ꢀCH
3 3 2
+
cyclopropane); MS (ESI): m/z: 260 [MHꢀ100] .
The following carbamates were prepared according to the procedure for compound 11:
tert-butyl N-[(1R,2S)-2-(4-iodophenyl)cyclopropyl]carbamate (12)
1
Yield: 84%. H NMR (CDCl ) δ (ppm): 7.62–7.53 (m, 2 H, aromatic protons), 6.96–6.82 (m, 2
3
H, aromatic protons), 4.93–4.72 (m, 1 H, ꢀNHCOOtBu ), 2.76–2.60 (m, 1 H, ꢀCH cyclopropane),
2.05–1.91 (m, 1 H, ꢀCH cyclopropane), 1.46 (s, 9 H, ꢀCOO(CH ) ), 1.21–1.07 (m, 2 H, ꢀCH
3
3
2
+
20
cyclopropane); MS (ESI): m/z: 382 [MNa] . [α]D = -74° (c 0.688, MeOH).
tert-butyl N-[(1S,2R)- 2-(4-iodophenyl)cyclopropyl]carbamate (13)
1
Yield: 81%. H NMR (CDCl ) δ (ppm): 7.32–7.22 (m, 2 H, aromatic protons), 7.21–7.09 (m, 3
3
H, aromatic protons), 4.97–4.65 (m, 1 H, ꢀNHCOOtBu), 2.85–2.64 (m, 1 H, ꢀCH cyclopropane ),
2.09–1.99 (m, 1 HꢀCH cyclopropane), 1.47 (s, 9 H, ꢀCOO(CH ) ), 1.23–1.10 (m, 2 H, ꢀCH
3
3
2
+
20
cyclopropane); MS (ESI): m/z: 260 [MHꢀ100] . [α]D = +89.23° (c 1.003, MeOH).
3-(2-Oxooxazolidin-3-yl)benzamide (19e)
A suspension of 0.56 g (2.7 mmol) 3ꢀ(2ꢀoxooxazolidinꢀ3ꢀyl)benzoic acid (18e) (Enamine) in 15
mL dry CH Cl was treated with 0.246 mL (3.38 mmol) thionyl chloride and 2 drops of dry
2
2
DMF. After stirring at reflux for 2 h, the mixture was cooled down to RT and poured into 4 mL
of an aqueous solution of NH (28ꢀ30% in water). After 1 h the resulting mixture was filtered off
3
to afford a white solid that was washed with water. The aqueous phases were extracted with
CH Cl , the combined organic layers were dried over Na SO , filtered and concentrated to give
2
2
2
4
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.549 g (98%) of the benzamide 19e as a white solid. 19e H NMR (DMSOꢀd ) δ (ppm): 8.01 (br
6
s, 1 H, ꢀCONHH), 7.94–7.88 (m, 1 H, aromatic proton), 7.84–7.78 (m, 1 H, aromatic proton),
7
.61 (d, J=7.8 Hz, 1 H, aromatic proton), 7.52–7.34 (m, 2 H, aromatic proton, ꢀCONHH), 4.49–
.39 (m, 2 H, ꢀCH O oxazolidinone), 4.14–3.98 (m, 2 H, ꢀCH N oxazolidinone ); MS (ESI): m/z:
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
2
2
+
207 [M+H] .
4
-(2-Oxooxazolidin-3-yl)benzamide (19f)
4
ꢀ(2ꢀOxooxazolidinꢀ3ꢀyl)benzamide (19f) was prepared according to the procedure for
compound 19e starting from 4ꢀ(2ꢀoxoꢀ1,3ꢀoxazolidinꢀ3ꢀyl)benzoic acid (18f, Enamine). Yield:
1
H NMR (DMSOꢀd ) δ (ppm): 8.01–7.79 (m, 3 H, aromatic protons, ꢀCONHH), 7.67–7.53 (m, 2
6
H, aromatic protons), 7.28 (br s, 1 H, ꢀCONHH), 4.44 (t, J=7.6 Hz, 2 H, ꢀCH O oxazolidinone),
2
+
4
4
0
0
.08 (t, J=7.6 Hz, 2 H, ꢀCH N oxazolidinone); MS (ESI): m/z: 207 [M+H] .
2
-(1-methyl-4-piperidyl)benzamide (19g)
.34 g (5.1 mmol) Sodium cyanoborohydride and 0.75 mL acetic acid was added to a mixture of
.745 g (3.63 mmol) of 4ꢀ(1ꢀmethylꢀ4ꢀpiperidyl)benzoic acid (18g, Fluorochem) and 0.54 mL
37% aqueous formaldehyde in 7.5 mL MeOH. The mixture was stirred for about 30 min, then
quenched with water and concentrated. The crude reaction intermediate was taken up with dry
CH Cl and 0.331 mL (4.54 mmol) thionyl chloride and 2 drops of dry DMF were added. The
2
2
solution was stirred at reflux for about 2 h, then cooled down to RT, and a further portion of 331
ꢁ
2
L (4.538 mmol) thionyl chloride was added. Stirring at reflux was then continued for additional
h. For a complete conversion a third portion of 0.331 mL (4.54 mmol) of thionyl chloride was
added. After further 2 h at reflux, the mixture was cooled down to RT and poured onto an ice
cold solution of 15 mL of aqueous NH (28ꢀ30%); and the resulting mixture was partitioned
3
between CH Cl and an 10% solution of Na CO in water. The organic layer was dried over
2
2
2
3
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2
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2
2
2
2
2
2
2
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3
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5
5
5
5
5
5
6
Na SO and concentrated to give 442 mg, (55% from compound 18g) of benzamide 19g as a
2
4
1
beige solid. H NMR (DMSOꢀd ) δ (ppm): 7.80 ꢀ 7.58 (m, 3 H, ꢀCONHH, aromatic protons),
6
7
.11 ꢀ 6.85 (m, 3 H, ꢀCONHH, aromatic protons), 3.27 ꢀ 3.15 (m, 4 H, ꢀCH N piperazine), 2.46 ꢀ
2
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
+
2
.34 (m, 4 H, ꢀCH N piperazine), 2.21 (s, 3 H, ꢀNCH ) MS (ESI): m/z: 219 [M+H] .
2
3
N-[4-[trans-2-aminocyclopropyl]phenyl]-4-(4-methylpiperazin-1-yl)benzamide
dihydrochloride (14d)
6
mg (0.03 mmol) CuI, 220 mg (0.613 mmol) of the carbamate 11, 0.15 mg (0.67 mmol) 4ꢀ(4ꢀ
methylpiperazinꢀ1ꢀyl)benzamide and 0.17 g (1.23 mmol) K CO were placed in a vial and
2
3
charged with nitrogen. 5 mg (0.06 mmol) N,N'ꢀdimethylethaneꢀ1,2ꢀdiamine and 2 mL dioxane
were added with a syringe, and the vial was heated to 110°C for 20 h. The resulting suspension
was allowed to cool down to RT and was then filtered through a silica gel pad eluting with 25 ml
of 9:1 CH Cl /MeOH. The filtrate was concentrated and the residue was purified by
2
2
chromatography (CH Cl to CH Cl /MeOH 95/5) to give 223 mg (81%) of Nꢀ[transꢀ2ꢀ[4ꢀ[[4ꢀ(4ꢀ
2
2
2
2
1
methylpiperazinꢀ1ꢀyl)benzoyl]amino]phenyl]cyclopropyl]ꢀcarbamate as a white solid. H NMR
DMSOꢀd ) δ (ppm): 9.86 (s, 1 H, ꢀCONH), 7.92–7.77 (m, 2 H, aromatic protons), 7.69–7.57 (m,
(
6
2
3
H, aromatic protons), 7.22 (br s, 1 H, ꢀNHCOOtBu), 7.08–6.90 (m, 4 H, aromatic protons),
.31–3.15 (m, 4 H, ꢀCH N piperazine), 2.62–2.53 (m, 1 H, ꢀCH cyclopropane), 2.47–2.36 (m, 4
2
H, ꢀCH N piperazine), 2.22 (s, 3 H, ꢀNCH ), 1.90–1.79 (m, 1 H, ꢀCH cyclopropane), 1.38 (s, 9
2
3
+
H, ꢀCOO(CH ) ), 1.13–1.02 (m, 2 H, ꢀCH cyclopropane); MS (ESI): m/z: 451 [M+H] .Then, 2
3
3
2
mL of 2 M HCl in Et O was added to a solution of 200 mg (0.44 mmol) of Nꢀ[transꢀ2ꢀ[4ꢀ[[4ꢀ(4ꢀ
2
methylpiperazinꢀ1ꢀyl)benzoyl]amino]phenyl]cyclopropyl]carbamate in 3 mL Et O/MeOH (2:1,
2
v:v) cooled down to 0°C. After stirring at RT for 20 h the formed precipitate was filtered off and
the light yellow solid washed with Et O and dried at 40°C under vacuum giving 167 mg (89%)
2
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of
Nꢀ[4ꢀ[transꢀ2ꢀaminocyclopropyl]phenyl]ꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)benzamide
as
its
1
+
dihydrochloride salt. H NMR (DMSOꢀd ) δ (ppm): 9.97 (s, 1 H, ꢀNH CH methyl piperazine),
6
3
+
9.75 (br s, 1 H, ꢀCONH), 8.22 (br s, 3 H, ꢀNH ), 7.98–7.83 (m, 2 H, aromatic protons), 7.77–
3
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
.63 (m, 2 H, aromatic protons), 7.20–6.98 (m, 4 H, aromatic protons), 4.14–3.93 (m, 2 H, ꢀ
CH N piperazine), 3.60–3.45 (m, 2 H, ꢀCH N piperazine), 3.18–2.98 (m, 4 H, ꢀCH N
2
2
2
piperazine), 2.87 (s, 3 H, ꢀNCH ), 2.83–2.74 (m, 1 H, ꢀCH cyclopropane), 2.27–2.17 (m, 1 H, ꢀ
3
CH cyclopropane), 1.35–1.27 (m, 1 H, ꢀCHH cyclopropane), 1.24–1.14 (m, 1 H, ꢀCHH
+
cyclopropane); MS (ESI): m/z: 351 [M+H] .
The following cyclopropylamines were prepared following the procedure for compound 14d
starting from the carbamate 11 (14a-c, 14e-g), 12 (15) or 13 (16).
N-[4-[trans-2-aminocyclopropyl]phenyl]-4-(3-furyl)benzamide hydrochloride(14a)
1
Yield: 40% from 11. H NMR (DMSOꢀd ) δ (ppm): 10.21 (s, 1 H, ꢀCONH), 8.40–8.24 (m, 4 H, ꢀ
6
+
CH furan, ꢀNH ), 8.01–7.94 (m, 2 H, aromatic protons), 7.81–7.75 (m, 3 H, aromatic protons),
3
7
.74–7.68 (m, 2 H, aromatic protons), 7.17–7.11 (m, 2 H, aromatic protons), 7.09–7.05 (m, 1 H, ꢀ
CH furan), 2.86–2.74 (m, 1 H, ꢀCH cyclopropane), 2.32–2.21 (m, 1 H, ꢀCH cyclopropane), 1.40–
1
.29 (m, 1 H, ꢀCHH cyclopropane), 1.25–1.11 (m, 1 H, ꢀCHH cyclopropane); MS (ESI): m/z:
+
3
19 [M+H] .
Benzyl
N-[3-[[4-[trans-2-aminocyclopropyl]phenyl]carbamoyl]phenyl]carbamate
hydrochloride (14b)
1
Yield: 48% from 11. H NMR (DMSOꢀd ) δ (ppm): 10.24 (s, 1 H, ꢀNHCOO), 9.99 (s, 1 H, ꢀ
6
+
CONH), 8.56 (br s, 3 H, ꢀNH ), 8.06–7.02 (m, 13 H, aromatic protons), 5.17 (s, 2 H, ꢀCH O),
3
2
2
.82–2.68 (m, 1 H, ꢀCH cyclopropane), 2.38–2.26 (m, 1 H, ) ꢀCH cyclopropane, 1.45–1.10 (m, 2
+
H, ꢀCH cyclopropane); MS (ESI): m/z: 402 [M+H] .
2
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2
2
2
2
2
2
2
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3
3
3
3
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5
5
5
5
5
5
5
6
N-[4-[(trans-2-aminocyclopropyl]phenyl]-4-morpholino-benzamide hydrochloride (14c)
1
Yield: 21% from 11. H NMR (DMSOꢀd ) δ (ppm): 9.93 (s, 1 H, ꢀCONH ), 8.31 (br s, 3 H, ꢀ
6
+
NH ), 7.93–7.81 (m, 2 H, aromatic protons), 7.74–7.62 (m, 2 H, aromatic protons), 7.17–7.07
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
(m, 2 H, aromatic protons), 7.05–6.96 (m, 2 H, aromatic protons), 3.79–3.68 (m, 4 H, ꢀCH O
2
morpholine), 3.29–3.19 (m, 4 H, ꢀCH N morpholine), 2.85–2.71 (m, 1 H, ꢀCH cyclopropane),
2
2.31–2.19 (m, 1 H, ꢀCH cyclopropane), 1.40–1.27 (m, 1 H, ꢀCHH cyclopropane), 1.23–1.14 (m,
+
1
H, ꢀCHH cyclopropane); MS (ESI): m/z: 338 [M+H] .
N-[4-[trans-2-aminocyclopropyl]phenyl]-3-(2-oxooxazolidin-3-yl)benzamide hydrochloride
14e)
(
1
Yield: 49% from 11. H NMR (DMSOꢀd ) δ (ppm): 10.29 (s, 1 H, ꢀCONH), 8.31 (br s, 3 H, ꢀ
6
+
NH ), 8.07–8.00 (m, 1 H, aromatic proton), 7.84–7.77 (m, 1 H, aromatic proton), 7.73–7.66 (m,
3
3
H, aromatic protons), 7.59–7.50 (m, 1 H, aromatic proton), 7.19–7.12 (m, 2 H, aromatic
protons), 4.51–4.43 (m, 2 H, ꢀCH O oxazolidinone), 4.20–4.08 (m, 2 H, ꢀCH N oxazolidinone),
2
2
2.87–2.75 (m, 1 H, ꢀCH cyclopropane), 2.32–2.21 (m, 1 H, ꢀCH cyclopropane), 1.39–1.29 (m, 1
+
H, ꢀCHH cyclopropane), 1.27–1.15 (m, 1 H, ꢀCHH cyclopropane); MS (ESI): m/z: 338 [M+H] .
N-[4-[trans-2-aminocyclopropyl]phenyl]-4-(2-oxooxazolidin-3-yl)benzamide
trifluoroacetate (14f)
1
Yield: 10% from 11 and after purification of the final product by preparative HPLC. H NMR
+
(
DMSOꢀd ) δ (ppm): 10.16 (s, 1 H, ꢀCONH), 8.12 (br s, 3 H, ꢀNH ), 8.02–7.96 (m, 2 H,
6
3
aromatic protons), 7.74–7.66 (m, 4 H, aromatic protons), 7.18–7.08 (m, 2 H, aromatic protons),
.55–4.40 (m, 2 H, ꢀCH O oxazolidinone), 4.20–4.02 (m, 2 H, ꢀCH N oxazolidinone), 2.85–2.74
4
2
2
(m, 1 H, ꢀCH cyclopropane), 2.31–2.15 (m, 1 H, ꢀCHH cyclopropane), 1.36–1.13 (m, 2 H, ꢀCHH
+
cyclopropane); MS (ESI): m/z: 338 [M+H] .
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N-[4-[trans-2-aminocyclopropyl]phenyl]-4-(1-methyl-4-piperidyl)benzamide
trifluoroacetate (14g)
1
Yield: 10% from 11 and after purification of the final product by preparative HPLC. H NMR
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
+
(
DMSOꢀd ) δ (ppm): 10.17 (s, 1 H, ꢀCONH), 9.39 (br s, 1 H, ꢀNH CH3 piperidine), 8.23 (br s, 3
6
+
H, ꢀNH ), 7.98–7.87 (m, 2 H, aromatic protons), 7.73–7.61 (m, 2 H, aromatic protons), 7.48–
3
7
.31 (m, 2 H, aromatic protons), 7.19–7.09 (m, 2 H, aromatic protons), 3.61–3.45 (m, 2 H, ꢀ
CH N piperidine), 3.14–2.99 (m, 2 H, ꢀCH N piperidine), 2.94–2.75 (m, 5 H, ꢀNCH , ꢀCH
2
2
3
piperidine, ꢀCH cyclopropane), 2.30–2.19 (m, 1 H, ꢀCH cyclopropane), 2.12–1.98 (m, 2 H, ꢀCH2
piperidine), 1.93–1.78 (m, 2 H, ꢀCH piperidine), 1.36–1.28 (m, 1 H, ꢀCHH cyclopropane), 1.23–
2
+
1.14 (m, 1 H, ꢀCHH cyclopropane); MS (ESI): m/z: 350 [M+H] .
N-[4-[(1S,2R)-2-aminocyclopropyl]phenyl]-4-(4-methylpiperazin-1-yl)benzamide
dihydrochloride (15)
1
+
H NMR (DMSOꢀd ) δ (ppm): 10.85 (br. s., 1 H, ꢀNH CH3 piperazine), 10.00 (s, 1 H, ꢀCONH),
6
+
8
.44 (br. s, 3 H, ꢀNH ), 7.95 ꢀ 7.85 (m, 2 H, aromatic protons), 7.74 ꢀ 7.65 (m, 2 H, aromatic
3
protons), 7.10 (dd, J = 9.0, 12.5 Hz, 4 H, aromatic protons), 4.07 ꢀ 3.96 (m, 2 H, ꢀCH N
2
piperazine), 3.54 ꢀ 3.43 (m, 2 H, ꢀCH N piperazine), 3.25 ꢀ 3.07 (m, 4 H, ꢀCH N piperazine),
2
2
2
.86 ꢀ 2.70 (m, 4 H, ꢀNCH , ꢀCH cyclopropane), 2.33 ꢀ 2.23 (m, 1 H, ꢀCH cyclopropane), 1.41 ꢀ
3
1.30 (m, 1 H, ꢀCHH cyclopropane), 1.22 ꢀ 1.11 (m, 1 H, ꢀCHH cyclopropane); MS (ESI): m/z:
+
20
3
51 [M+H] . [α]D = ꢀ46.48° (c 0.00236, DMSO).
N-[4-[(1R,2S)-2-aminocyclopropyl]phenyl]-4-(4-methylpiperazin-1-yl)benzamide
dihydrochloride (16):
1
+
H NMR (DMSOꢀd ) δ (ppm): 11.00 (br. s., 1 H, ꢀNH CH3 piperazine), 10.01 (s, 1 H, ꢀCONH),
6
+
8.48 (br. s, 3 H, ꢀNH ), 7.97 ꢀ 7.85 (m, 2 H, aromatic protons ), 7.74 ꢀ 7.63 (m, 2 H, aromatic
3
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
protons), 7.17 ꢀ 7.01 (m, 4 H, aromatic protons), 4.08 ꢀ 3.95 (m, 2 H, ꢀCH N piperazine), 3.54 ꢀ
2
3
.42 (m, 2 H, ꢀCH N piperazine), 3.27 ꢀ 3.03 (m, 4 H, ꢀCH N piperazine), 2.87 ꢀ 2.67 (m, 4 H, ꢀ
2
2
NCH , ꢀCH cyclopropane), 2.36 ꢀ 2.23 (m, 1 H, ꢀCH cyclopropane), 1.43 ꢀ 1.30 (m, 1 H, ꢀCHH
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
+
20
cyclopropane), 1.22 ꢀ 1.09 (m, 1 H, ꢀCHH cyclopropane); MS (ESI): m/z: 351 [M+H] . [α]D
=
+41.21° (c 0.00236, DMSO).
tert-Butyl 4-(4-methylpiperazin-1-yl)-3-nitrobenzoate (19j)
A suspension of 2.0 g (7.8 mmol) tert butyl 4ꢀchloroꢀ3ꢀnitroꢀbenzoate, 3.22 g (23.3 mmol) of dry
K CO ) and 2.58 mL (23.3 mmol) of Nꢀmethylpiperazine was stirred in 10 mL of dry DMF at
2
3
90°C for 5 h in a sealed tube. Then, the reaction mixture was quenched with water and extracted
with EtOAc. The combined organic phases were washed with brine, dried over Na SO ,
2
4
concentrated and the residue was purified on silica gel (eluent: EtOAc) and recrystallized from
1
toluene to obtain 2.3 g (92%) of intermediate 19j as a yellow solid. H NMR (CDCl , 400 MHz)
3
δ 8.37 (s, 1H, aromatic proton), 8.03ꢀ8.06 (d, 1H, aromatic proton), 7.07ꢀ7.10 (d, 1H, aromatic
proton), 3.20 (t, 4H, ꢀPhN(CH ) ), 2.58 (t, 4H, ꢀCH N(CH ) ), 2.38 (s, 3H, ꢀNCH ), 1.61 (s, 9H, ꢀ
2 2
3
2 2
3
13
COOC(CH ) ) ppm; C NMR (CDCl , 100 MHz) δ 164.9, 147.8, 137.2, 135.8, 134.0, 119.3,
3
3
3
+
1
15.8, 82.6, 57.7 (2C), 51.9 (2C), 47.0, 28.9 (3C) ppm; MS (EI) m/z: 321.17 [M] ; m.p. 138ꢀ
140°C. The following tertꢀbutyl benzoates were prepared starting from tert butyl 3ꢀchloroꢀ4ꢀ
nitroꢀbenzoate and morpholine (19i) or tert butyl 4ꢀchloroꢀ3ꢀnitroꢀbenzoate and piperidine (19k)
or morpholine (19h) according to the procedure for intermediate 19j.
tert-butyl 4-morpholino-3-nitro-benzoate (19h)
1
Yield: 85%. H NMR (CDCl , 400 MHz) δ 8.38ꢀ8.39 (d, 1H, aromatic proton), 8.06ꢀ8.08 ( dd,
3
1
H, aromatic proton), 7.08ꢀ7.10 ( d, 1H, aromatic proton), 3.87 (t, 4H, ꢀO(CH ) ), 3.16 (t, 4H, ꢀ
2 2
13
N(CH ) ), 1.61 (s, 9H, ꢀCOOC(CH ) ) ppm; C NMR (CDCl , 100 MHz) δ 165.1, 148.7, 134.9,
2
2
3 3
3
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34.8, 131.0, 122.5, 120.1, 81.5, 66.9 (2C), 51.3 (2C), 28.2 (3C) ppm; MS (EI) m/z: 308.14 [M] ;
m.p. 149ꢀ151°C.
tert-butyl 3-morpholino-4-nitro-benzoate (19i)
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1
Yield: 87 %. H NMR (CDCl , 400 MHz) δ 7.79 (d, 1H, aromatic proton), 7.76 (d, 1H, aromatic
3
proton), 7.65ꢀ7.67 (dd, 1H, aromatic proton), 3.86 (t, 4H, ꢀO(CH ) ), 3.10 (t, 4H, ꢀN(CH ) ), 1.63
2
2
2 2
1
3
(
s, 9H, ꢀCOOC(CH ) ) ppm; C NMR (CDCl , 100 MHz) δ 165.1, 142.8, 138.4, 130.9, 127.1,
3 3 3
+
1
21.7, 120.1, 81.5, 66.7 (2C), 51.4 (2C), 28.2 (3C) ppm; MS (EI) m/z: 308.14 [M] ; m.p. 57ꢀ
60°C.
tert-butyl 4-(1-piperidyl)-3-nitro-benzoate (19k)
1
Yield: 88%. H NMR (CDCl , 400 MHz) δ 8.38 (d, 1H, aromatic proton), 7.98ꢀ8.00 (dd, 1H,
3
aromatic proton), 7.07ꢀ7.08 (d, 1H, aromatic proton), 2.89 (t, 4H, ꢀN(CH ) ), 1.62ꢀ1.64 (m, 6H,
2
2
13
piperidine protons), 1.54 (s, 9H, ꢀCOOC(CH ) ) ppm; C NMR (CDCl , 100 MHz) δ 165.1,
3
3
3
1
48.7, 134.9, 134.8, 131.0, 122.5, 120.1, 81.5, 52.6 (2C), 28.2 (3C), 26.1 (2C), 24.0 ppm; MS
+
(
EI) m/z: 306.16 [M] ; m.p. 144ꢀ146°C.
tert-Butyl 3-amino-4-(4-methylpiperazin-1-yl)benzoate (20j)
A suspension of 0.80 g (2.5 mmol) of the tertꢀbutyl nitrobenzoate 19j in 30 mL MeOH and 0.13
g (0.12 mmol) of 10% palladium on carbon were placed in a Parr apparatus and was
hydrogenated at 50 psi and 25°C for 5 h. The palladium was then filtered off and the MeOH was
evaporated to afford an oily residue that was first purified on silica gel (eluent: CHCl /MeOH,
3
1
0:1, v:v) and then recrystallized from cyclohexane to provide 0.29 g (65%) of the tertꢀbutyl 4ꢀ
1
aminoꢀbenzoate 20j as a yellow solid. H NMR (CDCl , 400 MHz) δ 7.41ꢀ7.43 (dd, 1H, aromatic
3
proton), 7.36 (d, 1H, aromatic proton), 6.99ꢀ7.00 (d, 1H, aromatic proton), 3.96 (s, 2H,
Ph(NH )), 2.98 (t, 4H, ꢀPhN(CH ) ), 2.61 (t, 4H, ꢀCH N(CH ) ), 2.38 (s, 3H, ꢀN(CH )),
2
2 2
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1
.58 (s, 9Η,ꢀCOOC(CH ) ) ppm; C NMR (CDCl , 100 MHz) δ 165.1, 141.5, 137.7, 124.5
3
3
3
+
(2C), 119.9, 117.3, 81.5, 53.2 (2C), 50.0 (2C), 44.7, 28.2 (3C) ppm; MS (EI) m/z: 291.19 [M] ;
m.p. 114ꢀ116°C.
0
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9
0
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0
The following tertꢀbutyl benzoates were prepared starting from the tert butyl benzoates 19h, 19i
or 19k according to the procedure for intermediate 20j.
tert-butyl 4-morpholino-3-amino-benzoate (20h)
Yield: 73%. H NMR (CDCl , 400 MHz) δ 7.42ꢀ7.45 (d, 1H, aromatic proton), 7.38 (s, 1H,
1
3
aromatic proton), 6.98ꢀ7.02 (d, 1H, aromatic proton), 4.0 (br s, 2H, ꢀPhNH ), 3.88 (t, 4H, ꢀ
2
13
O(CH ) ), 2.98 (t, 4H, ꢀN(CH ) ), 1.61 (s, 9H, ꢀCOOC(CH ) ) ppm; C NMR (CDCl , 100
2
2
2 2
3 3
3
MHz) δ 164.7, 142.2, 134.4, 120.7, 120.5, 115.4, 115.0, 81.7, 66.3 (2C), 53.4 (2C), 28.8 (3C)
+
ppm; MS (EI) m/z: 278.16 [M] ; m.p. 126ꢀ128°C.
tert-butyl 3-morpholino-4-amino-benzoate (20i)
1
Yield: 84%. H NMR (CDCl3, 400 MHz) δ 7.68 (d, 1H, aromatic proton), 7.63 (dd, 1H, aromatic
proton), 6.69ꢀ6.71 (d, 2H, aromatic proton), 4.39 (br s, 2H, ꢀPhNH ), 3.87 (t, 4H, ꢀO(CH ) ),
2
2 2
1
3
2
.93(t, 4H, ꢀN(CH ) ), 1.59 (s, 9H, ꢀCOOC(CH ) ) ppm; C NMR (CDCl , 100 MHz) δ 165.1,
2 2 3 3 3
140.1, 139.2, 123.8, 123.3, 119.2, 115.4, 81.5, 66.6 (2C), 50.5 (2C), 28.2 (3C) ppm; MS (EI) m/z:
78.16; m.p. 136ꢀ138°C.
2
tert-butyl 4-(1-piperidyl)-3-amino-benzoate (20k)
1
Yield: 67%. H NMR (CDCl , 400 MHz) δ 7.11ꢀ7.13 (dd, 1H, aromatic proton), 6.81 (d, 1H,
3
aromatic proton), 6.58ꢀ6.60 (d, 1H, aromatic proton), 4.35 (s, 2H, ꢀNH ), 2.96 (t, 4H, piperidine
2
proton), 1.77 (m, 4H, piperidine proton), 1.63 (m, 2H, piperidine proton), 1.54 (s, 9H, ꢀ
13
COOC(CH ) ) ppm; C NMR (CDCl , 100 MHz) δ 165.1, 141.5, 137.7, 124.6, 124.5, 120.0,
3
3
3
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17.3, 81.5, 51.0 (2C), 28.2 (3C), 25.6 (2C), 24.0 ppm; MS (EI) m/z: 276.18 [M] ; m.p. 115ꢀ
117°C.
tert-Butyl 3-(benzyloxycarbonylamino)-4-(4-methylpiperazin-1-yl)benzoate (21j)
Benzyl chloroformate (2.1 mmol, 0.3 mL) was slowly added at 0°C to a solution of 0.50 g (1.72
mmol) of tertꢀbutyl 4ꢀaminoꢀ3ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)benzoate (20j) 1in 10 mL of THF and
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0
.29 mL (2.1 mmol) of TEA. After stirring at RT for 1.5 h the solution was then quenched with
water (20 mL) and extracted with CH Cl (3 x 20 mL). The organic phases were washed with
2
2
brine, dried over Na SO and concentrated to afford a residue that was purified on silica gel
2
4
1
(
eluent EtOAc/CHCl , 1:1, v:v) providing 0.53 g (65%) of the tertꢀbutyl benzoate 21j. H NMR
3
(
CDCl , 400 MHz) δ 8.69 (br s, 1H, ꢀPhNHCOO), 7.69ꢀ7.71 (m, 2H, aromatic proton), 7.37ꢀ7.47
3
(m, 5H, ꢀCH Ph), 7.15ꢀ7.17 (d, 1H, aromatic proton), 5.28 (s, 2H, ꢀCOOCH Ph), 2.92 (t, 4H, ꢀ
2
2
PhN(CH ) ), 2.61 (t, 4H, CH N(CH ) ), 2.20 (s, 3H, ꢀN(CH )), 1.60 (s, 9Η, ꢀCOOC(CH ) )
2
2
3
2 2
3
3 3
13
ppm; C NMR (CDCl , 100 MHz) δ 165.1, 155.9, 144.2, 136.3, 130.3, 128.3 (2C), 128.1 (2C),
3
1
27.9, 124.4, 124.0, 120.9, 118.1, 81.5, 67.0, 53.2 (2C), 50.0 (2C), 44.7, 28.2 (3C) ppm; MS (EI)
+
m/z: 425.23 [M] .
The following tertꢀbutyl benzoates were prepared starting from the tert butyl benzoates 20h, 20i
or 20k according to the procedure for intermediate 21j.
tert-butyl 4-morpholino-3-(benzyloxycarbonylamino)-benzoate (21h)
1
Yield: 71%. H NMR (CDCl , 400 MHz) δ 8.71 (br s, 1H, ꢀPhNHCOO), 7.71ꢀ7.73 (m, 2H,
3
aromatic proton), 7.38ꢀ7.47 (m, 5H, ꢀCH Ph), 7.15ꢀ7.17 (d, 1H, aromatic proton), 5.27 (s, 2H, ꢀ
2
COOCH Ph), 3.88 (t, 4H, ꢀO(CH ) ), 2.89 (t, 4H, ꢀN(CH ) ), 1.60 (s, 9Η, ꢀCOOC(CH ) )
2
2 2
2 2
3 3
13
ppm; C NMR (CDCl , 100 MHz) δ 165.1, 155.9, 144.2, 136.3, 130.3, 128.3 (2C), 128.1 (2C),
3
1
28.0, 124.4, 124.0, 120.9, 118.1, 81.5, 67.0, 66.6 (2C), 50.5 (2C), 28.2 (3C) ppm; MS (EI) m/z:
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25.23 [M] .
tert-butyl 3-morpholino-4-(benzyloxycarbonylamino)-benzoate (21i)
1
Yield: 73%. H NMR (CDCl , 400 MHz) δ 8.20 (d, 1H, aromatic proton), 8.15 (br s, 1H, ꢀ
3
0
1
2
3
4
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Ph(NH)COO), 7.81ꢀ7.83 (m, 2H, aromatic protons), 7.38ꢀ7.45 (m, 5H, ꢀCH (Ph)), 5.26 (s, 2H, ꢀ
2
COO(CH )Ph), 3.88 (t, 4H, ꢀO(CH ) ), 2.89 (t, 4H, ꢀN(CH ) ), 1.61 (s, 9Η, ꢀCOOC(CH ) )ppm;
2
2 2
2 2
3 3
13
C NMR (CDCl , 100 MHz) δ 165.1, 155.9, 138.8, 136.3, 128.8, 128.3 (2C), 128.1 (2C), 127.9,
3
1
25.1, 122.4, 120.8, 118.6, 81.5, 67.0, 66.6 (2C), 50.5 (2C), 28.2 (3C) ppm; MS (EI) m/z: 425.23
+
[
M] .
tert-butyl 4-(1-piperidyl)-3-(benzyloxycarbonylamino)-benzoate (21k)
1
Yield: 68%. H NMR (CDCl , 400 MHz) δ 8.08ꢀ8.11 (dd, 1H, aromatic proton), 7.86ꢀ7.87 (d,
3
1
H, aromatic proton), 7.33 (m, 5H, aromatic protons), 7.03 (d, 1H, aromatic proton), 6.80 (s, 1H,
ꢀ
CONHPh), 5.10 (s, 2H, ꢀCOOCH Ph), 2.96 (t, 4H, piperidine protons), 1.77 (m, 4H, piperidine
2
13
protons), 1.63 (m, 2H, piperidine protons), 1.54 (s, 9H, ꢀCOOC(CH ) ) ppm; C NMR (CDCl ,
3
3
3
1
00 MHz) δ 165.1, 155.8, 144.2, 136.3, 130.3, 128.3 (2C), 128.1 (2C), 127.9, 124.4, 124.0,
+
1
20.9, 118.1, 81.5, 67.0, 51.0 (2C), 28.2 (3C), 25.6 (2C), 23.9 ppm; MS (EI) m/z: 410.22 [M] .
3-(benzyloxycarbonylamino)-4-(4-methylpiperazin-1-yl)benzoic acid (18j)
A solution of 0.20 g (0.47 mmol) of tert-butyl benzoate 21j, and 0.72 mL (9.4 mmol) of TFA in
5
mL of dry CH Cl was stirred at RT overnight. The solvent was then removed and the resulting
2 2
solid was first triturated with 10 mL Et O and then crystallized from acetonitrile to give 0.13 g
2
1
(
76%) of the benzoic acid 18j as a colorless solid. H NMR (DMSOꢀd , 400 MHz) δ 12.84 (br s,
6
1H, ꢀCOOH), 9.85 (br s, 1H, ꢀPhNHCOO), 8.36 (s, 1H, aromatic proton), 7.66ꢀ7.68 (d, 1H,
aromatic proton), 7.40ꢀ7.46 (m, 3H, aromatic protons), 7.36ꢀ7.38 (d, 2H, aromatic protons), 7.22ꢀ
7
.25 (d, 1H, aromatic proton), 5.22 (s, 2H, ꢀCOOCH Ph), 3.20 (t, 4H, ꢀPhN(CH ) ), 3.08 (t, 4H,
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CH N(CH ) ), 2.86 (s, 3H, ꢀN(CH )) ppm; C NMR (DMSOꢀ d , 100 MHz) δ 167.6, 155.8,
3
2 2
3
6
145.2, 136.3, 129.3, 128.3 (2C), 128.1 (2C), 127.9, 125.2, 125.1, 119.7, 118.2, 67.0, 53.2 (2C),
+
5
0.0 (2C), 44.7 ppm; MS (EI) m/z: 369.17 [M] ; m.p. 205ꢀ207°C.
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The following tertꢀbutyl benzoates were prepared starting from the tert butyl benzoates 21h, 21i
or 21k according to the procedure for intermediate 18j.
4-morpholino-3-(benzyloxycarbonylamino)-benzoic acid (18h)
1
+
Yield: 83%. H NMR (DMSOꢀd , 400 MHz) δ 12.88 (br s, 1H, ꢀCOOH), 9.72 (br s, 1H, ꢀNH ),
6
8
.76 (br s, 1H, ꢀNHCOOCH Ph), 8.04ꢀ8.06 (d, 1H, aromatic proton), 7.75ꢀ7.77 (d, 1H, aromatic
2
proton), 7.70 (s, 1H, aromatic proton), 7.36ꢀ7.47 (m, 5H, ꢀCH Ph), 5.23 (s, 2H, ꢀ
2
NHCOOCH Ph), 3.46ꢀ3.48 (t, 4H, ꢀPhN(CH ) ), 2.99ꢀ3.12 (m, 4H, ꢀCH N(CH ) ), 2.87 (s, 3H, ꢀ
2
2 2
3
2 2
13
NCH ) ppm; C NMR (DMSOꢀd , 400 MHz) δ 166.8, 154.1, 142.7, 136.3, 128.8 (2C), 127.5,
3
6
127.3 (2C), 126.2, 124.6, 122.7, 120.4, 112.5, 66.9, 57.2 (2C), 52.4 (2C), 46.7 ppm; MS (EI)
+
m/z: 356.14 [M] ; m.p. 234ꢀ236°C.
3-morpholino-4-(benzyloxycarbonylamino)-benzoic acid (18i)
1
Yield: 88%. H NMR (DMSOꢀd , 400 MHz) δ 12.81 (br s, 1H, ꢀCOOH), 8.61 (s, 1H,
6
PhNHCOO), 7.96ꢀ7.98 (d, 1H, aromatic proton), 7.71ꢀ7.73 (m, 2H, aromatic proton), 7.33ꢀ7.46
(
m, 5H, ꢀCH Ph), 5.22 (s, 2H, ꢀCOOCH Ph), 3.77 (t, 4H, ꢀO(CH ) ), 2.81 (t, 4H, ꢀN(CH ) ) ppm;
2
2
2 2
2 2
1
3
C NMR (DMSOꢀd , 100 MHz) δ 167.6, 155.8, 138.7, 136.3, 130.9, 128.3 (2C), 128.1 (2C),
6
+
1
27.9, 126.7, 122.14, 121.0, 116.2, 67.0, 66.6 (2C), 50.5 (2C) ppm; MS (EI) m/z: 369.16 [M] ;
m.p. 218ꢀ220°C.
4-(1-piperidyl)-3-(benzyloxycarbonylamino)-benzoic acid (18k)
1
Yield: 86%. H NMR (DMSOꢀd , 400 MHz) δ 12.99 (s, 1 H, ꢀCOOH), 9.88 (s, 1H, ꢀCONHPh),
6
8.38 (d, 1H, aromatic proton), 8.15ꢀ8.17 (dd,1H, aromatic proton), 7.40ꢀ7.41 (d, 1H, aromatic
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