Discovery of N 1-(4-((7-Cyclopentyl-6-(dimethylcarbamoyl)-7 H -pyrrolo[2,3- d] pyrimidin-2-yl)amino)phenyl)- N 8-hydroxyoctanediamide as a Novel Inhibitor Targeting Cyclin-dependent Kinase 4/9 (CDK4/9) and Histone Deacetlyase1 (HDAC1) against Malignant Cancer

Article abstract of DOI:10.1021/acs.jmedchem.8b00209

A series of novel, highly potent, selective inhibitors targeting both CDK4/9 and HDAC1 have been designed and synthesized. N1-(4-((7-Cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d] pyrimidin-2-yl)amino)phenyl)-N8-hydroxyoctanediamide (6e) was discovered. The lead compound 6e with excellent CDK4/9 and HDAC1 inhibitory activity of IC₅₀ = 8.8, 12, and 2.2 nM, respectively, can effectively induce apoptosis of cancer cell lines. The kinase profiling of compound 6e showed excellent selectivity and specificity. Compound 6e induces G2/M arrest in high concentration and G0/G1 arrest in low concentration to prevent the proliferation and differentiation of cancer cells. Mice bared-breast cancer treated with 6e showed significant antitumor efficacy. The insight into mechanisms of 6e indicated that it could induce cancer cell death via cell apoptosis based on CDK4/9 and HDAC1 repression and phosphorylation of p53. Our data demonstrated the novel compound 6e could be a promising drug candidate for cancer therapy.

Full text of DOI:10.1021/acs.jmedchem.8b00209

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Discovery of N1-(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-
d] pyrimidin-2-yl) amino) phenyl)-N8-hydroxyoctanediamide as a
Novel Inhibitor Targeting Cyclin Dependent Kinase 4/9 (CDK4/9)
and Histone Deacetlyase1 (HDAC1) against Malignant Cancer
Yongtao Li, Xiaohe Luo, Qingxiang Guo, Yongwei Nie, Tianqi Wang, Chao Zhang, Zhi Huang, Xin
Wang, Yanhua Liu, Yanan Chen, Jian-Yu Zheng, Shengyong Yang, Yan Fan, and Rong Xiang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00209 • Publication Date (Web): 08 Mar 2018
Downloaded from http://pubs.acs.org on March 9, 2018
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Discovery
of
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N1-(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]
pyrimidin-2-yl) amino) phenyl)-N8-hydroxyoctanediamide as a
Novel Inhibitor Targeting Cyclin Dependent Kinase 4/9 (CDK4/9)
and Histone Deacetlyase1 (HDAC1) against Malignant Cancer
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Yongtao Li, Xiaohe Luo, Qingxiang Guo, Yongwei Nie, Tianqi Wang, Chao Zhang, Zhi Huang,
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Xin Wang, Yanhua Liu, Yanan Chen, Jianyu Zheng, Shengyong Yang, Yan Fan*
and Rong
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Xiang*
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Department of Medicinal Chemistry, School of Medicine, Nankai University, 94 Weijin Road, Tianjin
300071, China
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State Key Laboratory of Medicinal Chemical Biology, 94 Weijin Road, Tianjin 300071, China
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011 Project Collaborative Innovation Center for Biotherapy of Ministry of Education, 94 Weijin Road,
Tianjin 300071, China
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State Key Laboratory and Institute of ElementoꢀOrganic Chemistry, Collaborative Innovation Center
of Chemical Science and Engineering, Nankai University, Tianjin 300071, China.
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Medical Oncology, Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan
University, Chengdu 610041, China
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Abstract
A series of novel, highly potent, selective inhibitors targeting both CDK4/9 and HDAC1 have been
designed and synthesized. N1ꢀ(4ꢀ((7ꢀcyclopentylꢀ6ꢀ(dimethylcarbamoyl)ꢀ7Hꢀpyrrolo[2,3ꢀd]
pyrimidinꢀ2ꢀyl) amino) phenyl)ꢀN8ꢀhydroxyoctanediamide (6e) was discovered. The lead compound 6e
with excellent CDK4/9 and HDAC1 inhibitory activity of IC50 = 8.8 nM, 12 nM and 2.2 nM
respectively, can effectively induce apoptosis of cancer cell lines. The kinase profiling of compound 6e
showed excellent selectivity and specificity. 6e induces G2/M arrest in high concentration and G0/G1
arrest in low concentration to prevent the proliferation and differentiation of cancer cells. Mice
baredꢀbreast cancer treated with 6e showed significant antitumor efficacy. The insight into mechanisms
of 6e indicated that it could induce cancer cell death via cell apoptosis based on CDK4/9 and HDAC1
repression and phosphorylation of p53. Our data demonstrated the novel compound 6e could be a
promising drug candidate for cancer therapy.
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Keywords: CDK4, HDAC1, CDK9, Inhibitor, Cancer
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. INTRODUCTION
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Cancer is believed to result from dysregulation of normal cell cycle that leads to abnormal proliferation
and inability of cells to undergo differentiation and/or apoptosis. The cell cycle consists of four phases,
G1, S, G2 and M phase, which play a critical and central role in control of cell growth and
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proliferation. Cyclins and their catalytic binding partners, cyclinꢀdependent kinases (CDKs) maintain
the regulation of the cell cycle. CDK4 formed a complex with cyclinD1 is important component of cell
cycle activation and control the key progression step, G1 phase, where cells grow and synthesize
proteins in preparation for DNA synthesis. Deregulation of the CDK4ꢀcyclinDꢀRb pathway and CDK4
overexpression has been observed in cancer. Inhibition of CDK4 associated with cell cycle regulation by
inhibitors can lead to a G1 arrest and cell cycle progression halt, which provides an effective approach
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ꢀ11
to the control of tumor growth.
Many researchers have focused their efforts on the development of small molecule inhibitors of
CDK4. The first generation of CDK4 inhibitors was nonselective and caused severe toxic side effects in
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clinical trials. Toxicity due to poor specificity has limited their therapeutic efficacy.
In an attempt to
overcome the toxicity, potent and selective CDK4 inhibitors have received significant attentions.
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palbociclib and ribociclib that entered clinical trials are highly specific for CDK4.
They all need to
combine with letrozole for the treatment of ER+/HER2− metastatic breast cancer. These studies suggest
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9, 20
that monotherapy targeting on CDK4 to induce tumor cell death may be insufficient.
Additionally,
many studies have shown synergistic outcomes in reduction of tumor burden by simultaneous inhibition
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of more than one activated protein.
Therefore, we reasoned that development of single drugs with
multiple biological targets based on CDK4 might improve efficacy in cancer treatment.
Histone deacetylases (HDACs) catalyze the deacetylation from lysine residues in histones tails,
leading to chromatin condensation, thus controlling the transcriptional regulation, cell cycle progression
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4ꢀ27
and apoptosis.
Inhibition of HDACs significantly suppress cancer cell proliferation, angiogenesis,
and metastasis and induce apoptosis. Interestingly, HDAC1 inhibitors that possess synergistic or
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additive antitumor effects in rational combinations with anticancer drugs have been investigated.
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Following the success of synergy between HDAC inhibitor and CDK inhibitor in treating
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neuroblastomas and melanoma, we conceived that multitarget inhibition using of HDAC1 inhibitor in
combination with CDK4 target agent to increase the efficiencies the CDK inhibitor. There is few report
in the literature concerning bifunctional targeting of CDK and HDAC1 by single chemical inhibitors.
With this approach, a novel single molecule targeting CDK4 and HDAC1, was designed to avoid side
effect of more separate agents, such as drugꢀdrug interaction or different physicochemical properties.
In our study, a series of novel dualꢀaction compounds targeting CDK4 and HDAC1were designed and
synthesized. In vitro antiproliferative activity and kinase profiling revealed that the most active
compound, 6e was a multikinase inhibitor with excellent CDK4 inhibitory activity (IC = 8.8 nM),
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HDAC1 inhibitory activity (IC = 2.2 nM) and CDK9 inhibitory activity (IC = 9 nM). The lead
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compound can induce apoptosis of a wide variety of tumor cell lines and significantly induce G2/M
arrest in high concentration and induces G /G arrest in low concentration. In vivo it is effective in a
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T1 xenograft model in BALB/c mice. On the basis of these findings, it appears that compound 6e
offers potential for the treatment of a variety of tumor diseases. Herein, we report the synthesis,
structureꢀactivity relationship (SAR), kinase inhibitory profile, in vitro cytotoxicity, and tumor
regression studies of this lead compound. The selection of key drugꢀlike parameters comprising Log P
and Log D were measured. This work provides a novel approach in the development of new
chemotherapy for further exploration of dual CDKꢀHDAC1 pathway inhibition achieved with single
molecule.
2
. RESULTS AND DISCUSSION
2.1. Dual CDK4-HDAC1 Inhibitors Design Strategy. Rather than linking two inhibitors together
with a long chain, a merged pharmacophore is more desirable order to avoid high molecular weight.
Since there is no 3D structure of CDK4ꢀligand complex reported at present, the structure of CDK6 in
33
complex with an inhibitor (PDB entry: 4EZ5) was used as a template for homology modeling.
Analyzing the Xꢀray cocrystal structure and the third generation of CDKꢀdirected drugs, we found all of
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them interact with the hinge region using the 2ꢀaminopyrimidine group. Analysis of the structural
information in Figure S1 and Figure 1 suggested that His100 in CDK6 (His92 in CDK4), an amino acid
that is rarely conserved in other kinases. Previously reported structural studies with CDK inhibitors
bound to CDK6 propose 2ꢀamino and Nꢀ3 position of the pyrrolo[2,3ꢀd] pyrimidine ring forming two
hydrogen bonds with His100 (His92 in CDK4) as an important determinant for CDK enzyme inhibition.
The 2ꢀaminopyrimidine group, which interacts with the hinge region as its core CDK6ꢀbinding scaffold
provides a suitable moiety to explore our hypothesis. Thus, it was postulated that inserting the HDAC1
binding group here would retain CDK activity. An intrinsic property that affects kinase selectivity for
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many drugs is lipophilicity , quantified by cLogP. As the cLogP of ribociclib (2.3) is significantly
lower than abemaciclib (5.5) or palbociclib (2.7), we selected pyrrolo[2,3ꢀd] pyrimidinesꢀ2ꢀamine as the
backbone because this class of compound showed to possess higher CDK kinase inhibitory activity and
kinase selectivity. Most HDACis, such as vorinostat, are characterized by a widely accepted
pharmacophore model including three moieties, the capping group, linker region and zinc binding
group. The capping group that interacts with residues at the entry point of the active site tunnel. The
linker chain plus a connecting unit interacting mainly with hydrophobic residues of the tunnel. The zinc
binding functional group, such as hydroxamic acid, binds strongly to the zinc cation or a monodentate
group at the active site. According to the structural information, the zinc binding group plays a critical
35
role in chelation of zinc ion for HDAC1 inhibition. The hydroxamic acids moiety consistently inserts
into the catalytic outer tunnel of HDAC and chelates a zinc ion, which is important for the catalytic
process of HDAC. We further designed a merged molecule (Figure 1), in which the two essential
pharmacophoric elements, pyrrolo[2,3ꢀd] pyrimidinesꢀ2ꢀamine and hydroxamic acid moiety. Thus, we
design single compound that selectively inhibits both CDK and HDAC1 enzymes to improve efficacy in
cancer treatment compared to a single inhibitor alone.
2
.2. Chemistry. All target compounds were prepared by routes outlined in Schemes 1−3. First, we
synthesized the key intermediates and the general synthetic routes are illustrated in Scheme 1. Briefly,
commercially available 1a-1d reacted with phenylenediamine (2a-2c) to provide aniline intermediate
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a-3f (40−87% yield
)
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Commercial compound 4 with intermediate anilines 3a-3f through palladium
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catalyzed crossꢀcoupling reaction provided compounds 5a-5f. Similarly, commercially available 7a-7d
reacted with 4ꢀaminobenzoic acid (8) which were purchased from market to provide aniline
intermediate 9a-9d. After that, 10a-10d were obtained by 9a-9d with one step. Those methyl carbonate
analogues (5a-5f, 10a-10d) were directly converted into the hydroxamic acids analogues (6a-6f,
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1a-11d) by NH OH in CH OH. Aniline intermediate 13a-13f were acquired by 12a-12d with
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pꢀphenylenediamine (2c). Then, intermediate 13a-13d with compound 4 to provid compounds
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1
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A primary amine 7b was reacted with triphosgene and trimethylamine to provide
isocyanate derivative 15, which was then reacted with pꢀphenylenediamine to produce aniline
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intermediate 16. 16 underwent S Ar with compound 4 to produce 17, which was then directly
N
converted into 18 by NH OH in CH OH.
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In Scheme 2, Compound 4 was coupled to the diphenylmethanimine to obtain 8, which was not
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necessary to further purify and treated with concentrated HCl to obtain 20. Compound 4 was heated
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with N H ·H O in EtOH to give 21. Compound 1c was condensed with 20, 21, ribociclib,
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ꢀ(Aminomethyl)anilinedihydrochloride and tertꢀbutyl (5ꢀaminopyridinꢀ2ꢀyl)carbamate to provide 22,
3, 24, 26 and 29, respectively. Similarly, 26 underwent S Ar with 4 to produce 27, which was then
2
N
directly converted into 28 by NH OH in CH OH. Removal the protecting group of 29 provided aniline
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0, which underwent S Ar with 4 to produce 31. 31 was heated with NH OH in MeOH to give 32. 33
N 2
was obtained by the procedure from 1c reacting with 5ꢀnitropyridinꢀ2ꢀamine, and the nitro was reduced
to produce 34. The syntheses procedure of 35 is similar with 31.
In Scheme 3, to viewing the contribution of the N,Nꢀdimethylamide substituent, the unsubstituted
intermediates
5ꢀchloroꢀ3ꢀcyclopentylꢀ3Hꢀ[1,2,3]triazolo[4,5ꢀd]pyrimidine (43) were required. Commercially available
,4ꢀdichloroꢀ5ꢀnitropyrimidine (36) as the starting material reacted with cyclopentylamine (37) to give
2ꢀchloroꢀ9ꢀcyclopentylꢀ9Hꢀpurine
(40)
and
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46
compound 38, which was treated with SnCl ·2H O for reduction of nitro to obtain compound 39.
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The compound 40 was prepared from 39 by treatment with trimethyl orthoformate in 47% yield.
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Compound 39 was treated with NaNO under concentrated HCl conditions to produce compound 43. A
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palladium catalyzed crossꢀcoupling reaction of compounds 40 and 43 with intermediate anilines 3e
provided compounds 41 and 44 followed by converted into the hydroxamic acids compounds 42 and 45
by NH OH in CH OH. The compound 46 was prepared by reduction of compound 5e with lithium
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aluminum hydride in THF. Hydrolysis of 5e gave the acetic acid derivative 47, followed by
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condensation with dimethylamine in the presence of EDCI and HOBt and DIEA to afford the amide
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derivative 48.
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.3. Enzyme Inhibitory Activity and SAR. It is evident from Table 1 that the nature of substituents
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Yꢀ6 position in the general structure specifies the enzyme activity of the molecules on CDK4/cyclinD.
Placing dimethylcarbamoyl in Yꢀ6 position and keeping other positions constant, the molecule showed
better activity targeting CDK4. As the dimethylcarbamoyl was displaced, inhibitory properties of
compounds as shown 41, 42, 44 and 45, were decreased by several fold compared to other molecules.
These data clearly show that a dimethylcarbamoyl group at Yꢀ6 position is the most optimal substituent,
suggesting that the dimethylcarbamoyl group at the Yꢀ6 position of the pyrrolo[2,3ꢀd] pyrimidine ring is
critical for its activity.
Once we identified suitable substituents for the Yꢀ6 position, we then investigate whether the
aromatic ring moiety, between the pyrrolo[2,3ꢀd] pyrimidine and zinc binding group, was important for
potent inhibition of CDK4/cyclinD. The results of enzyme inhibitory activity studies showed that all of
the molecules with phenyl substituent between the pyrrolo[2,3ꢀd] pyrimidine and zinc binding group
were similar active with CDK inhibitor ribociclib. These compounds fared better with halfꢀmaximal
inhibitory concentrations (IC ) values ranging from 4.8 nM to 30 nM, such as compounds 5b-f, 6b-6f,
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0a-d, 11a-d, 14a-b, 18, 27, 28, and 46 to 48. Other derivatives focusing on the position connecting to
the Cꢀ2 position of the pyrrolo [2,3ꢀd] pyrimidineꢀ6ꢀone was varied in an attempt to further improve the
potency for CDK4/cyclinD. When the position changed to pyridine moiety, the molecules (31 and 32)
were severalꢀfold less active than aboved compounds. Compound 5a and 6a, which possess an
orthoꢀaniline group instead of a paraꢀaniline, showed lower binding affinity (IC > 5000 nM), most
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likely because of unfavorable steric interactions with the pocket of CDK4/cyclinD. Analogues 22 and
3 with progressive decrease of IC values were also observed. 5ꢀ(Piperazinꢀ1ꢀyl) pyridinꢀ2ꢀyl group in
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24 and 25 conferred high CDK4/cyclinD potency.
Several substituents, such as hydroxamic acids, methyl ester, dimethylcarbamoyl group, alkyl and
hydroxy, were designed as a zinc binding group. We have selected some representative compounds to
detect the HDAC1 inhibition activities. Among these compounds, the ester group, dimethylcarbamoyl
group, alkyl and hydroxy were completely inactive against HDAC1 at the highest concentration tested
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(5 ꢁM) in Table 1, due to the weak chelating effect to the zinc cation in the HDAC1 active site.
Therefore, hydroxamic acid is essential for good zinc binding and activity, thus the hydroxamic
derivatives showed significant inhibition of HDAC1 activity in Table 1. These compounds were
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0ꢀ100ꢀfold more potent than positive control, HDAC1 Inhibitor (vorinostat). Compounds afforded
strong HDAC1 inhibition, such as 6b, 6e, 6f, 11c, 11d and 44, especially compound 6b (IC = 0.84 nM)
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and 11b (IC = 0.53 nM). The SAR appears to mirror that observed for a series of homologues in which
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the linking section between the hydroxamic acid and amide must be at least five carbon atoms in length,
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with length of five being optimal. Compound 6c (IC50 = 27 nM) did not demonstrated outstanding
HDAC1 inhibition activity, probably due to the short distance between the acid and the pyrrolo[2,3ꢀd]
pyrimidine. Compound 6a was not particularly active, probably because the dimethylcarbamoyl is ortho
to 2ꢀaminopyrimidine group resulting in steric hindrance. With HDAC1 inhibitory of IC = 53 nM, the
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compound 28 with a methylene spacer was found to be less active than the parent molecule, probably
because of the benzyl amine group increasing the flexibility of the zinc binding moiety. In comparison
with vorinostat, compound 11a-d was made as reversed amide. The observed low HDAC1 inhibitoion
of 25, 32 and 45 is probably due to the differences in the cap binding region of the CDK.
2.4. Cellular Assays. We focused our efforts on the evaluation of our bifunctional inhibitors.
Whether or not the kinase inhibitory activity of the dualꢀaction molecules transfer to tumor cell lines
was next explored with cancer cell proliferation assays. We screened dualꢀacting compounds exhibiting
better kinase inhibitory than ribiciclib and vorinostat to do the preliminary cell inhibition assays (Table
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S1). Then, IC values were further detected on compounds that exhibit better cell inhibition in a panel
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of cancer cell lines growth using standard CCK8 assay. Despite being highly potent against both CDK4
and HDAC1, analogues 6d, 6f and 11b, with five or seven carbon atoms in length of the linking section
between the hydroxamic acid and amide, were only weakly antitumor activities in these cells (Table S2).
The best tumor cell potency of these analogues was obtained for six carbon linked molecule 6e, which
was also outstanding inhibitor of CDK4 and HDAC1 with IC50 values of 8.8 and 2.2 nM. It was
probably due to compound 6e with a comfortable and optimal merged pharmacophore for cell uptake.
As attributes of a truly merged bispecific pharmacophore and the results of antitumor activity in cell
lines showed that 6e is the best in this class, we performed all subsequent in vitro and in vivo biological
studies using this compound. The compound was next evaluated in a dose response study against breast
cancer cell lines (4T1, T47D and MDAꢀMBꢀ231), lung cancer cell lines (A549 and H460), and liver
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cancer cell lines (HepG2 and Hep3B) to test cytotoxic activity (Table 2 and Table S3). Compounds
ribociclib and vorinostat were used as references. Compound 6e showed potent profile in vitro, with
around 1 ꢁM IC50 values in the 4T1, MDAꢀMBꢀ231, A549, H1299 and Hep3B cell lines and about
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ꢀ3ꢁM in other cell lines. The CDK4 selective inhibitor ribociclib was not potent in these cells, which
indicated CDK4 monotherapy lack sensitivity. This result demonstrated that combination CDK and
HDAC1 inhibition in a single molecule, led to synergy at the cellular level more efficiently.
2
.5. Kinase Selectivity Profiling. To investigate the kinase selectivity, kinase inhibition profiling
assays with a fixed concentration of 1 ꢁM of compound 6e were first carried out against a series of 375
kinases through the Eurofins kinase profiling. The results are provided in Figure 2 and Table S4. Further
IC values of the kinases that showed a high inhibitory rate at 1 ꢁM, were examined and the results are
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shown in Table 3. Interestingly, ribociclib with highly selective for CDK4/6 relative to other human
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protein, was detected with the IC of CDK4/cyclinD, 13 nM and IC of CDK9/cyclinT, 197 nM. Our
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compound 6e showed higher inhibitory activity against CDK4/cyclinD (IC50 = 8.8 nM) and
CDK9/cyclinT (IC = 9 nM) over other CDKs. Resulting from high homology, identical substrate
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specificities and enzymatic activities CDK4 and CDK6 have overlapping functions in development. In
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light of this fact, many of the third generation CDK4 inhibitors such as abemaciclib, dinaciclib,
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ribociclib, palbociclib and AGꢀ024322 also inhibited CDK6. Therefore, the development of
ATPꢀcompetitive CDK4 inhibitors that are selective over CDK6 may offer an improved therapeutic
potential as well as dual CDK4/CDK6 inhibitors. CDK4 along with cyclins D is involved in the
regulation of cell cycle, while CDK9 along with cyclins T is key player in transcription regulation/RNA
processing. Compound 6e that target cell cycleꢀrelated CDK4 and transcriptionꢀrelated CDK9, might
contribute to the antitumor activity. Compound 6e has additional kinase activities (6 human protein
kinases have IC <50 nmol/L), all of which are intimately associated with the growth, survival, and
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metastasis in tumor cells.
2.6. Molecular Modeling of Compound 6e. Molecular docking studies were carried out to
investigate the binding modes of compound 6e in CDK4, CDK9, and HDAC1, respectively. Since there
is no 3D structure of CDK4ꢀligand complex reported at present, the structure of CDK6 in complex with
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an inhibitor (PDB entry: 4EZ5) was used as a template for homology modeling. MODELLER in the
Discovery Studio (DS, Accelrys Inc., USA) was employed for the homology modeling. Then the
compound 6e was docked to CDK4, CDK9 (PDB code: 4BCF) and HDAC1 (PDB code: 4BKX) by
GOLD (version 5.0). Hydrogen atoms were added to the proteins by using DS 3.1. GoldScore was
selected as the scoring function, and other parameters were set as default. The image was created using
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PyMOL.
Figures 3 illustrate the predicted binding modes and the detailed proteinꢀinhibitor interactions of
compound 6e with CDK4, HDAC1 and CDK9, respectively. For comparison, the binding mode of 6e
was superimposed on that of ribociclib or vorinostat. Compound 6e is a potent inhibitor of CDK4 with
an IC of 8.8 nM and ribociclib shows a similar level of CDK4 inhibition, with an IC of 13 nM. As
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shown in Figure 3A, 6e could tightly bind to the ATPꢀbinding site of CDK4 in a binding mode similar to
that of ribociclib. Hydrogen bonds appear to be formed between the aminopyrimidine and the backbone
residue of VAL96 and HIS95. Given that these hydrogen bonds are critical for binding to CDK4, it is
satisfying to find that NH of aminopyrimidine may also be serving a similar role in its interactions with
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HDAC1. From this point of view, 6e displays attributes of a truly merged bispecific pharmacophore. In
an attempt to find the reasons for the HDAC1 inhibitory of the compound 6e, we docked compound 6e
into the HDAC1 protein. As shown in Figure 3B, the hydroxamic acid could insert into the catalytic
outer tunnel of HDAC1 and the group also chelates a zinc ion, which is important for the catalytic
process of HDAC1. In addition, the 2ꢀaminopyrimidine group contribute to the hydrogenꢀbonding
interaction with the residue in the catalytic tunnel. In contrast to vorinostat that contain simple
heterocycle to exert cap action, our results of molecular modeling reveal that placing 2ꢀaminopyrimidine
substituents at proper locations forms a hydrogen bonding interaction with GLUꢀ98. This deeper and
closer binding of compound 6e to HDAC1 may explain the better binding affinity of compound 6e for
HDAC1, about 10 fold than vorinostat. The CDK9 inhibitory IC50 of compound 6e is 9 nM, while
ribociclib is 197 nM. From Figure 3C, we can see that, compared with ribociclib, compound 6e gains
another new hydrogen bonding interaction between hydroxamic acid group and THRꢀ29, which could
be used to interpret the phenomena that 6e showed a stronger activity against the CDK9 kinase than
ribociclib.
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In order to illustrate the SARs more rational, molecular modeling studies are employed to select 5c
and 11b as the best candidate for CDK4 and HDAC1 enzyme inhibition, respectively. As shown in
Figure S2. Compared with 6e, compound 5c gains another new hydrogen bonding interaction with
ARG101, which could explain 5c showed a stronger CDK4 inhibition than 6e and ribociclib. As shown
in Figure S3, the length of the linking section with four carbon atoms between the hydroxamic acid and
amide made the structure of compound 11b expanded, which lead to it is more suitable in the active
pocket of HDAC1.That may be the reason for the inhibition difference between compound 6e and 11b.
The SARs findings according to the docking poses are coordinating with our SAR results discussed
above.
2
.7. Effect of Compound 6e on Cell Cycle Progression. We therefore wondered whether this cell
apoptotic and kinase inhibition effect could partly result from growth arrest induction and cell cycle
inhibition. We next investigated the effect of compound 6e treatment on breast cancer cell lines (T47D
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and MDAꢀMBꢀ231), lung cancer cell lines (A549 and H460), and liver cancer cell lines (HepG2 and
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Hep3B) cell cycle kinetics (Figure 4). Cells were treated with compound 6e with varying
concentrationso according to IC values, ribociclib and vehicle (DMSO) for 24 h, which were stained
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with propidium iodide, and subjected to flow cytometric analysis to determine the distribution of cells in
various phases of the cell cycle. Similar effects were observed in three type cells. Compared to the
vehicleꢀtreated cells, cancer cells treated with compound 6e demonstrate a loss of Sꢀphase cells and an
increase in the percentage of cells in G2 and G0/G1. Ribociclib treatment led to a massive accumulation
of cells in G0/G1. It was evident that compound 6e blocked the cells in G2 at high dose of 2.5 and 10
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M, while at low dose of 625 nM, induced an increase in cells in G0/G1 phase, which resulted in
decreased Sꢀphase populations. It is well known that CDK4 plays a critical role in the G1/S transition of
the cell cycle. Inhibition of cellular CDK4 activity is expected inhibition of cell cycle arrest in the G1
phase. Analyses of our result demonstrated the ability of compound 6e to affect the activities of CDK4
in cells and led to support the hypothesis of a blockage during the G1 phase of compound 6e with lower
concentration. The effects on G0/G1ꢀphase inhibition are consistent with the inhibition of CDK4.
Compound 6e blocked both G0/G1 and G2ꢀM transitions. The G2 phase inhibition is indicative of cells
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undergoing apoptosis. Additionally, there may be a role for inhibition of CDK9 and HDAC1, which
are inhibited by compound 6e with similar potency as CDK4. This result indicated that compound 6e
has cellular HDAC1 or CDK9 inhibitory activity at a higher concentration and a cellular CDK4
inhibitory activity with a lower concentration. The results confirmed that compound 6e had
antiproliferative properties, suggesting the existence of multiꢀintracellular targets.
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.8. Effect of Compound 6e on Cell Apoptosis. The Hoechst 33342 staining was used to verify if
the compound 6e was able to affect cell growth through apoptosis. As shown by representative pictures
of T47D (Figure 5A), the apoptotic bodies of cells have been investigated that revealed a
doseꢀdependent increase of apoptosis in cell cultures after 48h of incubation with compound 6e,
consistent with the effect on cell proliferation. Compound 6e generated an increase of the presence of
apoptotic cells compared to untreated cells.
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It was subjected to an apoptotic assay for further evaluation of the antiproliferative activities of our
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compound against breast, lung and liver cancer cell lines. Cells were treated with compound 6e,
vorinostat as reference drugs and vehicle (DMSO) for 48h, which were analyzed by annexin V/PI
staining (Figure 5B). Compound 6e induced cell apoptosis in a doseꢀdependent manner. The percentage
of apoptotic cells was defined as the sum of early and late apoptosis (annexin Vꢀpositive and PIꢀpositive
cells). Compared with the vehicle condition, compound 6e was significantly more active in apoptosis
induction as the concentration increased. 6eꢀtreated T47D, H460 and HepG2 cells exhibited
significantly better inhibition. Taken together, compound 6e could induce cell death via apoptosis.
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.9. Molecular Mechanisms of Activity for Compound 6e. As compound 6e exhibited clear cell
cycle suppression and significant induction of apoptosis, we decided to characterize the cellular effects
of compound 6e in more detail. To further confirm the CDK inhibitory, we first performed western blot
analysis to evaluate the protein levels of CDK4 inhibition (Figure 6A). Similar results were obtained for
six types cell. Compound 6e exhibited stronger inhibition of CDK4 enzymes. Treated cells exhibited
doseꢀdependent reductions in the level of the protein CDK4 and Cyclin D1, which is consistent with the
known mechanisms of action for CDK4 inhibitors. Compound suppressed CDK4 at 1 ꢁM in cells,
which is in accord with the IC value in cell viability assays. To evaluate if the antiproliferative activity
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of compound 6e is caused by inhibition of cellular CDK4 and CDK9, we selected inhibition of Rb
phosphorylation at the CDKꢀspecific sites Ser807/811. The results of this study (Figure 6A) show that
compound 6e effectively suppressed phosphorylation of Rb Ser807/811 at 1 ꢁM, confirming that CDK
is one target of this compound. The intracellular histone acetylation status is a direct marker of class I
HDAC1 inhibition. To further investigate whether the dual enzyme inhibition profile of compound 6e
translates into intracellular inhibition against HDAC1 pathway, we performed western blot analysis to
study the effect of compound 6e in the six type cell lines. As shown in Figure 6B, compound 6e affected
acetylꢀhistone H3 expression level, resulting in upregulation of acetylꢀhistone H3, especially in breast
cancer cell lines and lung cancer cell lines. In liver cancer cell line, the assay showed that compound 6e
showed a similar level of acetylꢀhistone H3 with vorinostat.
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As illustrated in Figure 6B, to confirm further the apoptosis induction of compound 6e, we examined
the expression of apoptotic proteins Bclꢀ2 and the cleavage states of caspase 3. Cells were treated with
or without compound 6e and control for 48h and then lysed and analyzed by Western blot. It was
revealed that treatment with compound 6e dramatically increased the relative levels of antiapoptotic
Bclꢀ2 expression in a doseꢀdependent manner. Furthermore, compound 6e resulted in more significant
cleavaged caspase 3 to different extents than the control group. Additionally, the inhibition of both
HDAC1 and transcriptional CDK9 might also induce cell death through the downꢀregulation of these
shortꢀlived antiꢀapoptotic proteins Bclꢀ2. This implies that compound 6e may induce cell death by both
transcriptional and HDAC1 repression. It is well known that phosphorylation of p53 results of DNA
damage induced by treatment with HDAC1 inhibitors. Next, we wanted to detected whether compound
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e induced apoptosis depends on the expression of p53. Protein level of Pꢀp53 detected by western blot,
was enhanced in compound 6eꢀtreated cells in respect to control groups. This finding led to the
conclusion that compound 6eꢀinduced apoptosis is associated with p53 phosphorylation.
The above results reꢀemphasized that compound 6e inhibits CDK4/9 and HDAC1 activity and is
efficient in cultured breast cancer cells, lung cancer cells and liver cancer cells. The correlation between
the suppression of CDK4, Rbꢀphosphorylation and onset of apoptosis suggests that the mechanism of
action of compound 6e, in these six cancer cell lines proceed through inhibition of CDK and HDAC1
critical.
Immunofluorescence (Figure 7) were carried out to demonstrate the inhibitory activity of compound
6e on CDK4 and deacetylation of histone H3. A549 cell line has been selected as a representative model
because it resulted as being nearly the most sensitive to the compound 6e antiproliferative effect. It was
demonstrated above that compound 6e induced an increase in cells in G0/G1 phase at low concentration,
which is consistent with the inhibition of CDK4. The 100 nM concentration and 1 ꢁM concentrations of
compound 6e were chosen and used in the immunofluorescence assay to detect the effect on CDK4 and
acetylꢀhistone H3, respectively. After 24 h of treatment with compound 6e and vehicle condition, a
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significant inhibition of CDK4 and increase of acetylation, observable staining of CDK4 and
acetylꢀhistone H3, were detected by immunofluorescence analysis (Figure 7).
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2.10. Lipophilicity. Log P and Log D describe a compound’s lipophilicity, and values often correlate
with a number of key biopharmaceutical parameters in drug discovery. We detected the experimental
Log P, Log D7.4 values and calculated Log P (cLogP) value for compound 6e. The values were measured
using the traditional shake flask method. The LogP value for 6e is 2.62 and LogD7.4 value is 2.02. Using
ChemDraw Ultra 12 software, the cLogP for the virtual compound 6e, is 2.08. Our results indicated that
compound 6e has a reasonable lipophilicity. We also detected the solubility of 6e. The solubility of 6e in
PBS at pH 7.4 is 0.35 ꢁg/mL and in the methanesulfonic acid formulation is 526 ꢁg/ mL.
Methanesulfonic acid formulation is compound 6e combined with one equiv methanesulfonic acid. As
the solubility of compound 6e in other acid formulation was not good, data were not shown. Despite its
limited solubility, 6e exhibited good lipophilicity.
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2.11. Pharmacokinetic Properties of 6e in Rats. Considering the excellent antiꢀtumor activities of
compounds 6e both in vitro and in vivo, pharmacokinetic (PK) properties of these compound was
further evaluated. Key PK parameters are summarized in Table 4. After per iv administration of 20
mg/kg compound 6e, the area under the concentration−time curve (AUC0−∞) was about 6158 ꢁg/L*h
and the halfꢀlife (t ) was about 10.76 h. After per ip administration of 20 mg/kg compound 6e, the area
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under the concentration−time curve (AUC0−∞) was about 2133.7 ꢁg/L*h and the halfꢀlife (t ) was about
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.65 h. In addition, after per ip compound 6e achieved a maximum plasma concentration (Cmax) of
359.6 ꢁg/L and showed a clearance rate (CL) of 12.61 L/h/kg and apparent distribution volume (Vss) of
19.94 L/kg. When administered 20 mg/kg compound 6e orally, the area under the concentration−time
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curve (AUC0−∞) was about 1130.6 ꢁg/L*h and the halfꢀlife (t ) was about 14.91 h. After per op
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compound 6e achieved a maximum plasma concentration (Cmax) of 136.8 ꢁg/L and showed a clearance
rate (CL) of 20.62 L/h/kg and apparent distribution volume (Vss) of 431.52 L/kg. All of these indicated
that compound 6e has moderate PK properties.
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2
.12. In Vivo Anticancer Activity of Compound 6e. We next carried out studies to determine the
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maximum tolerated dose of compound 6e in mice. BALB/c female mice (n = 4) received different doses
(45 mg/kg, 90 mg/kg and 130 mg/kg) of compound 6e intraperitoneally for 16 consecutive days and
were monitored over signs of toxicity (Figure 8A). No signs of toxicity or weight loss were observed.
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transduction. To determine the efficacy of compound 6e in vivo using tumor xenograft models,
Flucꢀ4T1 cells were orthotopically implanted into the mammary fat pads of 6−8 weeks old female
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administered daily via IP injection based on the toxicity results and literature
. The
bioluminescence imagings were performed on the representative animals at 8 days and 25 days for Fluc
reporter gene expression to reflect the number of viable tumor cells (Figure 8B). The tumor treated with
compound 6e therapy demonstrated a great decrease in the strength of Fluc signal which suggested a
considerable extent of tumor inhibition. Quantitative analysis of bioluminescence imaging signals at day
2
5 demonstrated that compound 6e showed significantly better inhibition (Figure 8C). The results of this
study (Figure 8D) showed that compound 6e administered on this schedule led to a doseꢀdependent
inhibition of tumor growth. Tumor growth inhibitions of 78.4% and 84.8% were observed at doses of 90
and 130 mg/kg, respectively. In contrast, vorinostat and ribociclib as the positive controls were less
potent (76.8%) and (68.5%) than 6e at the same dose, respectively. As positive controls, 130 mg/kg
vorinostat and ribociclib showed weaker antitumor activities compared with that of the compound 6e. A
decrease in tumor weight was also observed at the endꢀpoint of the study (Figure 8E). No overt signs of
toxicity were observed in the 6e treated groups (body weights was not shown), indicating that the
compound 6e is wellꢀtolerated in vivo. Administration of compound 6e by oral gavage was also
effective. IHC staining showed that the expression level of pꢀRb and Bclꢀ2 was suppressed in 6e
treatment groups (Figure 9). Tumor growth inhibitions of 70.3%, 79.3% were observed in the 4T1
model at doses of 90 and 130 mg/kg, respectively. In contrast, vorinostat and ribociclib as the positive
controls were less potent (75.6%) and (38.9%) than 6e at the same dose, respectively. 6e exhibited
1
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potent activity in vivo after both intraperitoneal and oral administration. Intraperitoneal administration
showed a high antitumor efficacy compared with oral administration at the same doses, which are
coordinating with our bioavailability results as shown in Table 4.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
. CONCLUSIONS
Through designing and structural optimization targeting on CDK4 and HDAC1, we finally
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
discovered a new compound 6e. Compound 6e is a multikinase inhibitor, which potently inhibited
CDK4, CDK9 and HDAC1 with IC50 values of 8.8 nM, 9 nM and 2.2 nM, respectively. 6e also
inhibited several kinases, such as the AuroraꢀA/B/C, Flt4, LIMK1 and TrkA, with IC < 50 nmol/L.
5
0
Even so, it is not a promiscuous agent and showed good selectivity in a kinase profiling assay against
375 kinases. In vitro cellular assays, 6e showed potent activities against breast cancer cell lines, T47D
and MDAꢀMBꢀ231 (triple negative breast cancer cell line), lung cancer cell lines, A549 and H460, liver
cancer cell lines, HepG2 and Hep3B. In cell cycle assays, compound 6e could block the six tumor cells
mentioned above in G2 at high dose of 2.5 and 10 ꢁM, while at low dose of 625 nM, induce an increase
in cells in G0/G1 phase, which resulted in decreased Sꢀphase populations. The effect on the cell cycle
progression confirmed that compound 6e has cellular HDAC1 or CDK9 inhibitory activity at a higher
concentration and a cellular CDK4 inhibitory activity with a lower concentration. In vivo antitumor
activity assays showed that intraperitoneal and oral administration of compound 6e led to potent tumor
regression in the 4T1 xenograft models. Studies of mechanisms of action indicated that 6e
downꢀregulated the activity of CDK4 and cyclinD1 kinase and the phosphorylation of protein Rb. In
addition, 6e significantly upregulation of acetylꢀhistone H3 and the relative levels of antiapoptotic Bclꢀ2
expression and cleavedꢀcaspaseꢀ3. 6e also enhanced the protein level of Pꢀp53, which led to the
conclusion that compound 6eꢀinduced apoptosis is associated with DNA damage. All of the studies
presented here support 6e as a novel drug candidate targeting on CDK4/9 and HDAC1 and deserve
further research and development.
4
. EXPERIMENTAL SECTION
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4
.1 Chemistry Methods. All reagents and solvents were obtained from commercial suppliers and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
used without further purification unless otherwise indicated. Melting points (mp) were taken in open
capillaries on a Mettler MP 50 meltingꢀpoint system. All reactions were monitored by thinꢀlayer
chromatography (TLC), and silica gel plates with fluorescence Fꢀ254 were used and visualized with UV
1
13
light. H NMR (400 MHz) and C NMR (101 MHz) spectra were taken on a Bruker AVꢀ400 MHz
spectrometer and chemical shifts were reported in ppm downfield from internal Me4Si. Highꢀresolution
mass spectra (HRMS) were recorded on a VG ZABꢀHS mass spectrometer under electron spray
ionization (ESI). All of the solvents were purified and distilled according to the standard procedure. The
commercially obtained materials were used directly without further purification unless otherwise noted.
All of the final compounds were purified to > 95% purity, as determined by highꢀperformance liquid
chromatography (HPLC). The purity by HPLC analysis on a Shimadzu Prominenceꢀi LCꢀ2030C 3D
system (column, InertSustain C18, 4.6 mm × 250 mm, 5 ꢁM; mobile phase, gradient elution of
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
methanol/H O; low rate, 1.0 mL/min; UV wavelength, 190−800 nm; temperature, 40 °C; injection
2
1
13
volume, 10 ꢁL). H and C spectra of test compounds are provided in Supporting Information
Appendix.
General Procedure for the Synthesis of 3a-f. A mixture of acid 1a-1d (30 mmol, 1 equiv), amine
2
b-2c (60 mmol, 2 equiv), EDCI (8.27 g, 45 mmol, 1.5 equiv), HOBt (4.87 g, 36 mmol, 1.2 equiv), and
DIEA (7.76 g, 60 mmol, 2 equiv) was stirred in DMF (60 mL) at room temperature for 12 hours. After
completion of the reaction, the mixture was quenched by water and extracted by ethyl acetate. The
combined organic layer was washed with brine solution and dried by MgSO . The solvent was
4
evaporated, and the residue was purified by silica gel column chromatography (dichloromethane –
methanol, gradient 100:0 → 98:2) to yield the desired aniline reagents 3a-3f.
4
.1.1. methyl 8-((2-aminophenyl)amino)-8-oxooctanoate (3a). Lightꢀbrown solid; 69% yield; mp
1
8
1.7 °C; H NMR (400 MHz, Chloroformꢀd) δ 7.40 (d, J = 12.0 Hz, 1H), 7.19 – 7.12 (m, 1H), 7.04 (td,
J = 7.7, 1.4 Hz, 1H), 6.81 – 6.73 (m, 2H), 3.66 (s, 3H), 2.39 – 2.27 (m, 4H), 1.77 – 1.57 (m, 4H), 1.43 –
1
3
1
.29 (m, 4H). C NMR (101 MHz, CDCl ) δ 174.26, 171.85, 140.74, 127.14, 125.27, 124.40, 119.54,
3
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+
118.23, 51.53, 36.78, 33.95, 28.81, 28.74, 25.56, 24.70. ESIꢀHRMS m/z calcd for C H N O
15 23 2 3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
2
79.1703, found 279.1703 [M + H] . HPLC purity 97%.
1
4.1.2. methyl 8-((3-aminophenyl)amino)-8-oxooctanoate (3b). Yellow oil; 78% yield; H NMR
(
400 MHz, Chloroformꢀd) δ 7.52 – 7.41 (m, 1H), 7.17 (t, J = 2.2 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H), 6.67
(
dd, J = 7.9, 2.1 Hz, 1H), 6.40 (dd, J = 8.2, 2.1 Hz, 1H), 3.65 (s, 3H), 2.32 – 2.26 (m, 4H), 1.65 (dt, J =
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
3
3
1
0.0, 7.1 Hz, 4H), 1.40 – 1.28 (m, 4H). C NMR (101 MHz, CDCl ) δ 174.31, 171.42, 147.22, 139.08,
3
29.63, 110.90, 109.63, 106.56, 51.53, 37.63, 33.96, 28.78, 28.76, 25.38, 24.71. ESIꢀHRMS m/z calcd
+
+
for C H N O 279.1703, found 279.1705 [M + H] . HPLC purity 96%.
1
5
23
2
3
4
.1.3. methyl 6-((4-aminophenyl)amino)-6-oxohexanoate (3c). Grey solid; 39% yield; mp 70.7 °C;
1
H NMR (400 MHz, Chloroformꢀd) δ 7.26 – 7.20 (m, 2H), 6.59 (dt, J = 8.8, 2.0 Hz, 2H), 3.65 (s, 3H),
13
2
.35 – 2.23 (m, 4H), 1.74 – 1.61 (m, 4H). C NMR (101 MHz, CDCl ) δ 174.14, 170.90, 143.28,
3
+
1
29.35, 122.14, 115.34, 51.63, 36.86, 33.71, 25.08, 24.39. ESIꢀHRMS m/z calcd for C H N O
3
1
3
19
2
+
251.1390, found 251.1390 [M + H] . HPLC purity 97%.
.1.4. methyl 7-((4-aminophenyl)amino)-7-oxoheptanoate (3d). Grey solid; 77% yield; mp 80.3 °C;
H NMR (400 MHz, Chloroformꢀd) δ 7.26 – 7.19 (m, 2H), 6.62 – 6.54 (m, 2H), 3.63 (s, 3H), 3.57 (br,
4
1
13
2
H), 2.33 – 2.21 (m, 4H), 1.74 – 1.55 (m, 4H), 1.39 – 1.29 (m, 2H). C NMR (101 MHz, CDCl ) δ
3
1
74.28, 171.23, 143.18, 129.42, 122.08, 115.37, 51.56, 37.05, 33.84, 28.65, 25.32, 24.55. ESIꢀHRMS
+
+
m/z calcd for C H N O 265.1547, found 265.1546 [M + H] . HPLC purity 95%.
1
4
21
2
3
4
.1.5. methyl 8-((4-aminophenyl)amino)-8-oxooctanoate(3e). Offꢀwhite solid; 63% yield; mp
1
77.6 °C; H NMR (400 MHz, DMSOꢀd ) δ 9.42 (s, 1H), 7.19 (d, J = 8.5 Hz, 2H), 6.47 (d, J = 8.5 Hz,
6
2
1
H), 4.82 (s, 2H), 3.57 (s, 3H), 2.29 (t, J = 7.4 Hz, 2H), 2.19 (t, J = 7.4 Hz, 2H), 1.53 (h, J = 7.4 Hz, 4H),
1
3
.35 – 1.19 (m, 4H). C NMR (101 MHz, DMSOꢀd ) δ 173.82, 170.64, 144.98, 129.04, 121.31, 114.22,
6
+
51.62, 36.60, 33.69, 28.83, 28.72, 25.61, 24.80. ESIꢀHRMS m/z calcd for C H N O 279.1703, found
15
23
2
3
+
2
79.1704 [M + H] . HPLC purity 99%.
4
.1.6. methyl 9-((4-aminophenyl)amino)-9-oxononanoate(3f). Reddish solid; 80% yield; mp
1
9
3.7 °C; H NMR (400 MHz, Chloroformꢀd) δ 7.23 (d, J = 8.2 Hz, 2H), 6.57 (d, J = 7.4 Hz, 2H), 3.63 (s,
1
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3
H), 2.31 – 2.19 (m, 4H), 1.68 – 1.50 (m, 4H), 1.34 – 1.24 (m, 6H). C NMR (101 MHz, CDCl ) δ
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
74.41, 171.45, 143.20, 129.46, 122.02, 115.31, 51.52, 37.37, 34.03, 29.03, 28.97, 28.92, 25.67, 24.85.
+
+
ESIꢀHRMS m/z calcd for C H N O 293.1860, found 293.1861 [M + H] . HPLC purity 98%.
16
25
2
3
General
Procedure
for
the
Synthesis
of
5a-f.
To
a
suspension
of
2
ꢀchloroꢀ7ꢀcyclopentylꢀN,Nꢀdimethylꢀ7Hꢀpyrrolo[2,3ꢀd]pyrimidineꢀ6ꢀcarboxamide (4) (586 mg, 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
mmol, 1 equiv) in 20 mL 1,4ꢀdioxane was added compound 3a-f (2 mmol, 1 equiv), Pd(OAc) (11 mg,
2
0
.05 mmol, 0.25 equiv), BINAP (62 mg, 0.1 mmol, 0.05 equiv) and Cs CO (1.37 g, 4.2 mmol, 2.1
2 3
equiv) and the flask was purged with N . Then the flask was sealed and the mixture was heated for 12 h
2
at 100 °C. The reaction was cooled to room temperature, and the solvent was removed under reduced
pressure. The residue was purified by silica gel column chromatography (dichloromethane – methanol,
gradient 100:0 → 96:4) to obtain the desired product 5a-f.
4.1.7.
methyl
8-((2-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]
1
pyrimidin-2-yl)amino) phenyl)amino)-8-oxooctanoate (5a). White solid; 83% yield; mp 52.1 °C; H
NMR (400 MHz, Chloroformꢀd) δ 8.59 (s, 1H), 8.10 (s, 1H), 7.85 – 7.79 (m, 1H), 7.65 – 7.57 (m, 1H),
7
6
4
1
.38 (s, 1H), 7.15 (dq, J = 6.8, 4.1, 2.6 Hz, 2H), 6.38 (s, 1H), 4.78 – 4.61 (m, 1H), 3.63 (s, 3H), 3.12 (s,
H), 2.36 – 2.19 (m, 6H), 2.02 – 1.91 (m, 2H), 1.88 – 1.76 (m, 2H), 1.70 – 1.46 (m, 6H), 1.36 – 1.20 (m,
13
H). C NMR (101 MHz, CDCl ) δ 174.19, 171.76, 164.71, 163.98, 156.81, 152.36, 151.77, 132.08,
3
31.84, 131.74, 125.19, 124.63, 124.08, 112.52, 100.86, 57.62, 51.45, 37.35, 33.95, 30.61, 28.79, 28.74,
+
+
25.39, 24.86, 24.69. ESIꢀHRMS m/z calcd for C H N O 535.3027, found 535.3030 [M + H] . HPLC
2
9
39
6
4
purity 99%.
.1.8.
4
methyl
8-((3-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)
1
phenyl)amino)-8-oxooctanoate (5b). White solid; 84% yield; mp 128.5 °C; H NMR (400 MHz,
Chloroformꢀd) δ 8.63 (s, 1H), 7.83 (s, 1H), 7.56 – 7.45 (m, 3H), 7.23 (t, J = 8.1 Hz, 1H), 7.16 (d, J = 8.1
Hz, 1H), 6.40 (s, 1H), 4.91 – 4.70 (m,1H), 3.64 (s, 3H), 3.13 (s, 6H), 2.60 – 2.45 (m, 2H), 2.36 – 2.24
1
3
(m, 4H), 2.11 – 1.92 (m, 4H), 1.76 – 1.55 (m, 6H), 1.42 – 1.28 (m, 4H). C NMR (101 MHz, CDCl ) δ
3
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1
74.24, 171.26, 164.16, 155.40, 152.09, 151.57, 140.75, 138.57, 131.88, 129.18, 114.52, 113.27, 112.39,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
110.05, 101.00, 57.73, 51.50, 37.58, 33.97, 30.26, 28.82, 28.79, 25.34, 24.71, 24.69. ESIꢀHRMS m/z
+
+
calcd for C H N O 535.3027, found 535.3030 [M + H] . HPLC purity 97%.
2
9
39
6
4
4
.1.9.
Methyl
6
-((4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)
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
phenyl)amino)-6-oxohexanoate (5c). White solid; 89% yield; mp 164.9 °C; H NMR (400 MHz,
Chloroformꢀd) δ 8.51 (s, 1H), 8.20 (s, 1H), 7.96 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.0 Hz,
2
6
H), 6.29 (s, 1H), 4.64 (q, J = 8.8 Hz, 1H), 3.63 (s, 3H), 3.11 (s, 6H), 2.57 – 2.46 (m, 2H), 2.33 (t, J =
1
3
.7 Hz, 4H), 2.04 – 1.91 (m, 4H), 1.78 – 1.57 (m, 6H). C NMR (101 MHz, CDCl ) δ 174.04, 170.89,
3
164.46, 155.43, 151.97, 151.48, 136.69, 131.95, 131.42, 120.99, 118.87, 112.02, 100.88, 57.99, 51.58,
+
3
6.87, 33.74, 30.07, 25.10, 24.60, 24.44. ESIꢀHRMS m/z calcd for C H N O 507.2714, found
27
35
6
4
+
5
07.2714 [M + H] . HPLC purity 99%.
4.1.10.
methyl
7
-((4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)
1
phenyl)amino)-7-oxoheptanoate (5d). White solid; 73% yield; mp 151.7 °C; H NMR (400 MHz,
Chloroformꢀd) δ 8.54 (s, 1H), 8.07 (s, 1H), 7.82 (s, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz,
2
H), 6.32 (s, 1H), 4.66 (p, J = 8.9 Hz, 1H), 3.64 (s, 3H), 3.13 (s, 6H), 2.59 – 2.44 (m, 2H), 2.35 – 2.27
13
(m, 4H), 2.03 – 1.96 (m, 4H), 1.76 – 1.63 (m, 6H), 1.47 – 1.32 (m, 2H). C NMR (101 MHz, CDCl ) δ
3
1
74.19, 171.19, 164.40, 155.47, 151.99, 151.56, 136.65, 132.03, 131.46, 120.87, 118.90, 112.06, 100.91,
+
57.97, 51.52, 37.10, 33.84, 30.08, 28.70, 25.29, 24.61, 24.57. ESIꢀHRMS m/z calcd for C H N O
4
2
8
37
6
+
5
21.2871, found 521.2872 [M + H] . HPLC purity 99%.
4
.1.11.
methyl
8-((4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)
1
phenyl)amino)-8-oxooctanoate (5e). White solid; 82% yield; mp 171.0 °C; H NMR (400 MHz,
Chloroformꢀd) δ 8.57 (s, 1H), 7.77 (s, 1H), 7.65 – 7.53 (m, 3H), 7.37 (d, J = 8.4 Hz, 2H), 6.36 (s, 1H),
4
.70 (p, J = 8.9 Hz, 1H), 3.65 (s, 3H), 3.14 (s, 6H), 2.55 (t, J = 10.7 Hz, 2H), 2.31 (dt, J = 11.1, 7.5 Hz,
2
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5
H), 2.08 – 1.93 (m, 5H), 1.77 – 1.57 (m, 4H), 1.36 (dq, J = 8.6, 4.5 Hz, 4H). C NMR (101 MHz,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
DMSOꢀd ) δ 173.76, 171.13, 163.38, 156.04, 152.52, 151.74, 136.78, 133.41, 131.79, 119.80, 118.91,
6
111.73, 101.18, 57.41, 51.58, 36.71, 35.03, 33.68, 30.03, 28.84, 28.72, 25.49, 24.79, 24.65. ESIꢀHRMS
+
+
m/z calcd for C H N O 535.3027, found 535.3031 [M + H] . HPLC purity 99%.
2
9
39
6
4
4
.1.12.
methyl
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
9
-((4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)
1
phenyl)amino)-9-oxononanoate (5f). White solid; 73% yield; mp 139.8 °C; H NMR (400 MHz,
Chloroformꢀd) δ 8.58 (s, 1H), 7.68 (s, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.54 (s, 1H), 7.40 (d, J = 8.4 Hz,
2
H), 6.37 (s, 1H), 4.71 (t, J = 8.9 Hz, 1H), 3.65 (s, 3H), 3.14 (s, 6H), 2.69 – 2.41 (m, 2H), 2.40 – 2.24
1
3
(m, 4H), 2.11 – 1.95 (m, 4H), 1.80 – 1.51 (m, 6H), 1.42 – 1.24 (m, 6H). C NMR (101 MHz, CDCl ) δ
3
1
5
74.34, 171.30, 164.30, 155.44, 152.09, 151.49, 136.57, 132.22, 131.70, 120.75, 119.05, 112.18, 100.99,
7.99, 51.51, 37.54, 34.07, 30.16, 29.09, 29.00, 28.96, 25.61, 24.89, 24.67. ESIꢀHRMS m/z calcd for
+
+
C H N O 549.3184, found 549.3179 [M + H] . HPLC purity 99%.
30
41
6
4
General Procedure for the Synthesis of 6a-f. To a stirred solution of the compound 5a-f (1 mmol,)
in methanol (20 mL) was added a solution of hydroxylamine (50% in water, 5.5 mL). The resulting
solution was stirred for 18 h under reflux. Then the solvent was removed under vacuum and the crude
residue purified by chromatography on a silica gel column (dichloromethane – methanol, gradient 100:0
→
90:10) to obtain the desired hydroxamic acids 6a-f.
4.1.13. N1-(2-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)phenyl) -N8-hydroxyoctanediamide (6a). White solid; 62% yield; mp
1
1
66.9 °C (decompose); H NMR (400 MHz, DMSOꢀd ) δ 9.72 (s, 1H), 8.71 (s, 1H), 8.37 (s, 1H), 7.99
6
(d, J = 8.1 Hz, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.23 – 7.12 (m, 1H), 7.04 (t, J = 7.6 Hz, 1H), 6.56 (s, 1H),
4
4
1
1
.67 (p, J = 8.7 Hz, 1H), 3.04 (s, 6H), 2.50 (t, J = 2.0 Hz, 1H), 2.41 – 2.22 (m, 4H), 1.92 (t, J = 7.3 Hz,
13
H), 1.86 – 1.78 (m, 2H), 1.64 – 1.40 (m, 6H), 1.34 – 1.20 (m, 4H). C NMR (101 MHz, DMSOꢀd ) δ
6
72.44, 169.48, 163.33, 156.20, 152.67, 151.74, 133.77, 132.12, 129.65, 125.37, 123.31, 123.17, 112.33,
00.97, 57.11, 36.30, 35.05, 32.72, 30.53, 28.89, 28.79, 25.70, 25.51, 24.95. ESIꢀHRMS m/z calcd for
2
2
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Journal of Medicinal Chemistry
+
+
C H N O 536.2980, found 536.2985 [M + H] . HPLC purity 98%.
28
38
7
4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
.1.14.
N1-(3-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)phenyl) -N8-hydroxyoctanediamide (6b). White solid; 49% yield; mp
1
1
76.2 °C (decompose); H NMR (400 MHz, DMSOꢀd ) δ 10.36 (s, 1H), 9.78 (s, 1H), 9.48 (s, 1H), 8.73
6
(
s, 1H), 8.69 (s, 1H), 8.08 (s, 1H), 7.54 – 7.43 (m, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H),
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
6
1
1
.57 (s, 1H), 4.86 (p, J = 8.9 Hz, 1H), 3.14 – 2.96 (m, 6H), 2.41 – 2.24 (m, 4H), 2.01 – 1.81 (m, 6H),
1
3
.67 – 1.53 (m, 4H), 1.48 (q, J = 7.1 Hz, 2H), 1.35 – 1.19 (m, 4H). C NMR (101 MHz, DMSOꢀd ) δ
6
71.41, 169.54, 163.53, 156.14, 152.38, 151.85, 141.60, 139.81, 131.86, 128.70, 114.17, 113.01, 111.81,
1
10.66, 101.13, 56.88, 36.79, 32.72, 30.38, 28.95, 28.93, 25.53, 24.67. ESIꢀHRMS m/z calcd for
+
+
C H N O 536.2980, found 536.2988 [M + H] . HPLC purity 97%.
28
38
7
4
4
.1.15.
N -(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo
1
[
2,3-d]pyrimidin-2-yl)amino)phenyl) -N6-hydroxyadipamide (6c). White solid; 48% yield; mp
1
211.9 °C (decompose); H NMR (400 MHz, DMSOꢀd ) δ 10.38 (s, 1H), 9.77 (s, 1H), 9.45 (s, 1H), 8.78
6
–
3
1
1
8.63 (m, 2H), 7.74 (d, J = 8.5 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 6.57 (s, 1H), 4.72 (t, J = 8.8 Hz, 1H),
.05 (s, 6H), 2.49 – 2.40 (m, 2H), 2.33 – 2.22 (m, 2H), 2.04 – 1.89 (m, 6H), 1.72 – 1.61 (m, 2H), 1.60 –
13
.46 (m, 4H). C NMR (101 MHz, DMSOꢀd ) δ 170.66, 169.11, 163.06, 155.71, 152.24, 151.41,
6
36.47, 133.05, 131.51, 119.49, 118.61, 111.42, 100.85, 57.07, 36.25, 34.75, 32.34, 29.71, 25.07, 24.33.
+
+
ESIꢀHRMS m/z calcd for C H N O 508.2667, found 508.2671 [M + H] . HPLC purity 96%.
26
34
7
4
4
.1.16.
N -(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo
1
[2,3-d]pyrimidin-2-yl)amino)phenyl) -N7-hydroxyheptanediamide (6d). White solid; 57% yield; mp
1
1
80.7 °C (decompose); H NMR (400 MHz, DMSOꢀd ) δ 10.36 (s, 1H), 9.76 (s, 1H), 9.46 (s, 1H), 8.72
6
(
s, 1H), 8.71 – 8.67 (m, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 6.56 (s, 1H), 4.72 (p, J =
8.9 Hz, 1H), 3.16 – 2.95 (m, 6H), 2.49 – 2.39 (m, 2H), 2.27 (t, J = 7.3 Hz, 2H), 2.03 – 1.88 (m, 6H),
13
1
1
3
.72 – 1.46 (m, 6H), 1.34 – 1.21 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ 171.08, 169.51, 163.37,
6
56.02, 152.56, 151.72, 136.76, 133.39, 131.81, 119.78, 118.91, 111.73, 101.19, 57.39, 36.63, 32.63,
+
0.03, 28.76, 25.43, 25.39, 24.65. ESIꢀHRMS m/z calcd for C H N O 522.2823, found 522.2825 [M
27 36
7
4
2
3
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Page 24 of 78
+
+
H] . HPLC purity 98%.
.1.17.
[2,3-d]pyrimidin-2-yl)amino)phenyl) -N8-hydroxyoctanediamide (6e). White solid; 76% yield; mp
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
N -(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo
1
1
1
1
9
1
6
1
3
82.7 °C (decompose); H NMR (400 MHz, DMSOꢀd ) H NMR (400 MHz, DMSOꢀd ) δ 10.33 (s, 1H),
6
6
.73 (s, 1H), 9.42 (s, 1H), 8.71 (s, 1H), 8.66 (s, 1H), 7.79 – 7.65 (m, 2H), 7.58 – 7.46 (m, 2H), 6.56 (s,
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
H), 4.80 – 4.62 (m, 1H), 3.05 (s, 6H), 2.50 – 2.39 (m, 2H), 2.27 (t, J = 7.4 Hz, 2H), 2.03 – 1.89 (m,
1
3
H), 1.70 – 1.45 (m, 6H), 1.35 – 1.22 (m, 4H). C NMR (101 MHz, DMSOꢀd ) δ 171.15, 169.57,
6
63.42, 156.05, 152.53, 151.76, 136.77, 133.41, 131.86, 119.83, 118.96, 111.75, 101.15, 57.40, 36.75,
+
2.73, 30.05, 28.94, 28.89, 25.57, 25.52, 24.65. ESIꢀHRMS m/z calcd for C H N O 536.2980, found
28
38
7
4
+
536.2985 [M + H] . HPLC purity 98%.
.1.18.
4
N -(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo
1
[2,3-d]pyrimidin-2-yl)amino)phenyl) -N9-hydroxynonanediamide (6f). White solid; 49% yield; mp
1
185.5 °C (decompose); H NMR (400 MHz, DMSOꢀd ) δ 10.36 (s, 1H), 9.75 (s, 1H), 9.46 (s, 1H), 8.71
6
(s, 1H), 8.69 (s, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 6.56 (s, 1H), 4.72 (p, J = 8.9 Hz,
1
1
1
3
5
H), 3.05 (t, J = 6.8 Hz, 6H), 2.49 – 2.40 (m, 2H), 2.27 (t, J = 7.4 Hz, 2H), 2.05 – 1.87 (m, 6H), 1.69 –
13
.42 (m, 6H), 1.33 – 1.18 (m, 6H). C NMR (101 MHz, DMSOꢀd ) δ 171.18, 169.58, 163.38, 156.02,
6
52.55, 151.72, 136.76, 133.40, 131.81, 119.80, 118.93, 111.73, 101.19, 65.38, 57.39, 36.78, 32.72,
+
0.03, 29.10, 29.02, 28.95, 25.64, 25.58, 24.65. ESIꢀHRMS m/z calcd for C H N O 550.3136, found
29
40
7
4
+
50.3141 [M + H] . HPLC purity 97%.
General Procedure for the Synthesis of 9a-9d.
The title compound was prepared from 7a-7d (14.6 mmol, 1 equiv), 4ꢀaminobenzoic acid (8) (2 g,
1
4.6 mmol, 1 equiv), EDCI (4.2 g, 21.9 mmol, 1.5 equiv), HOBt (2.37 g, 17.52 mmol, 1.2 equiv), DIEA
(5.06 g, 43.8 mmol, 3 equiv) and DMF (30 mL) using the procedure described for compound 3a-3f to
obtain the desired product 9a-9d.
1
4
.1.19. methyl 5-(4-aminobenzamido)pentanoate (9a). Lightꢀbrown oil; 63% yield; H NMR (400
MHz, Chloroformꢀd) δ 7.66 – 7.56 (m, 2H), 6.69 – 6.61 (m, 2H), 6.21 (s, 1H), 3.66 (s, 3H), 3.41 (q, J =
2
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Journal of Medicinal Chemistry
1
3
6
.6 Hz, 2H), 2.36 (t, J = 7.1 Hz, 2H), 1.76 – 1.52 (m, 4H). C NMR (101 MHz, CDCl ) δ 174.10,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
67.28, 149.44, 128.60, 124.22, 114.17, 51.63, 39.34, 33.53, 29.15, 22.12. ESIꢀHRMS m/z calcd for
+
+
C H N O 251.1390, found 251.1395 [M + H] . HPLC purity 99%.
13
19
2
3
4
.1.20. methyl 6-(4-aminobenzamido)hexanoate (9b). Lightꢀbrown solid; 66% yield; mp 118.3°C;
1
H NMR (400 MHz, DMSOꢀd ) δ 7.97 (t, J = 5.5 Hz, 1H), 7.67 – 7.48 (m, 2H), 6.62 – 6.47 (m, 2H),
6
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
5
4
1
2
.57 (s, 2H), 3.57 (s, 3H), 3.18 (q, J = 6.6 Hz, 2H), 2.29 (t, J = 7.2 Hz, 2H), 1.50 (dp, J = 23.1, 7.5 Hz,
13
H), 1.28 (td, J = 8.6, 4.1 Hz, 2H). C NMR (101 MHz, DMSOꢀd ) δ 173.30, 166.08, 151.39, 128.57,
6
+
21.40, 112.46, 51.11, 38.71, 33.20, 29.03, 25.94, 24.20. ESIꢀHRMS m/z calcd for C H N O
3
14
21
2
+
65.1547, found 265.1550 [M + H] . HPLC purity 99%.
4.1.21. methyl 7-(4-aminobenzamido)heptanoate (9c). Lightꢀbrown solid; 87% yield; mp 95.8 °C;
1
H NMR (400 MHz, Chloroformꢀd) δ 7.57 (dd, J = 8.5, 2.4 Hz, 2H), 6.72 – 6.49 (m, 2H), 6.40 – 6.05
m, 1H), 4.03 (s, 2H), 3.63 (s, 3H), 3.40 – 3.27 (m, 2H), 2.35 – 2.22 (m, 2H), 1.56 (dp, J = 15.5, 7.4 Hz,
(
13
4H), 1.41 – 1.24 (m, 4H). C NMR (101 MHz, CDCl ) δ 174.27, 167.38, 149.62, 128.59, 124.09,
3
+
114.09, 51.50, 39.79, 33.96, 29.58, 28.78, 26.61, 24.79. ESIꢀHRMS m/z calcd for C H N O
15 23 2 3
+
2
79.1703, found 279.1704 [M + H] . HPLC purity 99%.
.1.22. methyl 8-(4-aminobenzamido)octanoate (9d). Lightꢀbrown solid; 73% yield; mp 59.6 °C;
H NMR (400 MHz, Chloroformꢀd) δ 7.62 – 7.55 (m, 2H), 6.63 (dd, J = 8.7, 2.4 Hz, 2H), 3.64 (s, 3H),
.37 (td, J = 7.2, 5.8 Hz, 2H), 2.28 (t, J = 7.5 Hz, 2H), 1.58 (dt, J = 14.2, 7.2 Hz, 4H), 1.37 – 1.27 (m,
4
1
3
1
3
6
H). C NMR (101 MHz, CDCl ) δ 174.31, 167.26, 149.36, 128.56, 124.36, 114.18, 51.49, 39.87,
3
+
34.03, 29.73, 29.01, 28.95, 26.80, 24.83. ESIꢀHRMS m/z calcd for C H N O 293.1860, found
16
25
2
3
+
2
93.1856 [M + H] . HPLC purity 95%.
General Procedure for the Synthesis of 10a-10d.
The title compound was prepared from 9a-9d (2 mmol, 1 equiv), compound 4 (586 mg, 2 mmol, 1
equiv), Pd(OAc) (11 mg, 0.05 mmol, 0.025 equiv), BINAP (62 mg, 0.1 mmol, 0.05 equiv) and Cs CO
3
2
2
(1.37 g, 4.2 mmol, 2.1 equiv) and 1,4ꢀdioxane (20 mL) using the procedure described for compound
5
a-5f to obtain the desired product 10a-10d.
2
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4
.1.23.
methyl
5-(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
[
2,3-d]pyrimidin-2-yl)amino) benzamido)pentanoate (10a). White solid; 77% yield; mp 195.0 °C; H
NMR (400 MHz, DMSOꢀd ) δ 9.81 (s, 1H), 8.79 (s, 1H), 8.29 (t, J = 5.7 Hz, 1H), 7.97 – 7.88 (m, 2H),
6
7
.84 – 7.73 (m, 2H), 6.61 (s, 1H), 4.86 – 4.66 (m, 1H), 3.58 (s, 3H), 3.25 (q, J = 6.2 Hz, 2H), 3.06 (s,
H), 2.50 – 2.42 (m, 2H), 2.35 (t, J = 6.9 Hz, 2H), 2.07 – 1.95 (m, 4H), 1.74 – 1.63 (m, 2H), 1.62 – 1.48
6
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
3
(
m, 4H). C NMR (101 MHz, DMSOꢀd ) δ 173.75, 166.17, 163.32, 155.52, 152.53, 151.49, 144.04,
6
1
2
9
32.53, 128.23, 126.89, 117.36, 112.41, 101.05, 57.51, 51.63, 40.01, 39.10, 33.45, 30.39, 30.11, 29.22,
+
+
4.66, 22.48. ESIꢀHRMS m/z calcd for C H N O 507.2714, found 507.2720 [M + H] . HPLC purity
2
7
35
6
4
9%.
4.1.24.
methyl
6-(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo
1
[
2,3-d]pyrimidin-2-yl)amino) benzamido)hexanoate (10b). White solid; 84% yield; mp 190.1 °C; H
NMR (400 MHz, Chloroformꢀd) δ 8.66 (s, 1H), 7.79 – 7.73 (m, 4H), 7.71 (s, br, 1H), 6.44 (s, 1H), 6.30
– 6.22 (m, 1H), 4.85 – 4.67 (m, 1H), 3.65 (s, 3H), 3.46 (q, J = 6.7 Hz, 2H), 3.15 (s, 6H), 2.68 – 2.49 (m,
1
3
2
H), 2.33 (t, J = 7.4 Hz, 2H), 2.15 – 1.98 (m, 4H), 1.80 – 1.58 (m, 6H), 1.50 – 1.35 (m, 2H). C NMR
(
101 MHz, CDCl ) δ 174.14, 167.07, 163.94, 154.73, 151.78, 151.49, 143.09, 132.40, 127.87, 127.27,
3
1
17.45, 112.79, 100.89, 58.05, 51.56, 39.71, 33.88, 30.14, 29.42, 26.45, 24.66, 24.49. ESIꢀHRMS m/z
+
+
calcd for C H N O 521.2871, found 521.2873 [M + H] . HPLC purity 99%.
2
8
37
6
4
4
.1.25.
methyl
7-(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]
1
pyrimidin-2-yl)amino) benzamido)heptanoate (10c). White solid; 76% yield; mp 87.2 °C; H NMR
(400 MHz, Chloroformꢀd) δ 8.65 (s, 1H), 7.90 (d, J = 9.5 Hz, 1H), 7.74 (s, 4H), 6.42 (s, 1H), 6.29 (q, J
=
5.4 Hz, 1H), 4.83 – 4.68 (m, 1H), 3.64 (s, 3H), 3.42 (q, J = 6.8 Hz, 2H), 3.14 (s, 6H), 2.64 – 2.51 (m,
1
3
2
H), 2.29 (t, J = 7.5 Hz, 2H), 2.10 – 1.99 (m, 4H), 1.73 – 1.57 (m, 6H), 1.43 – 1.29 (m, 4H). C NMR
(101 MHz, CDCl ) δ 174.23, 167.11, 163.96, 154.80, 151.76, 151.52, 143.12, 132.33, 127.84, 127.29,
3
1
17.46, 112.75, 100.89, 58.04, 51.52, 39.90, 33.96, 30.13, 29.59, 28.79, 26.63, 24.80, 24.65. ESIꢀHRMS
+
+
m/z calcd for C H N O 535.3027, found 535.3029 [M + H] . HPLC purity 99%.
2
9
39
6
4
4
.1.26.
methyl
8-(4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo
2
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Journal of Medicinal Chemistry
1
[
2,3-d]pyrimidin-2-yl)amino) benzamido)octanoate (10d). White solid; 78% yield; mp 166.2 °C; H
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NMR (400 MHz, Chloroformꢀd) δ 8.65 (d, J = 3.2 Hz, 1H), 7.82 – 7.65 (m, 4H), 6.43 (d, J = 4.8 Hz,
1H), 4.91 – 4.53 (m, 1H), 3.65 (s, 3H), 3.52 – 3.32 (m, 2H), 3.15 (s, 6H), 2.67 – 2.50 (m, 2H), 2.36 –
2
.24 (m, 2H), 2.15 – 1.98 (m, 4H), 1.70 (t, J = 6.1 Hz, 2H), 1.67 – 1.54 (m, 4H), 1.41 – 1.25 (m, 6H).
13
C NMR (101 MHz, CDCl ) δ 174.30, 167.09, 163.96, 154.74, 151.78, 151.52, 143.05, 132.40, 127.85,
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
1
2
27.36, 117.46, 112.79, 100.89, 58.04, 51.51, 40.00, 34.03, 30.14, 29.73, 29.03, 28.96, 26.82, 24.83,
+
4
+
4.66. ESIꢀHRMS m/z calcd for C H N O 549.3184, found 549.3189 [M + H] . HPLC purity 99%.
3
0
41
6
General Procedure for the Synthesis of 11a-11d.
The title compound was prepared from 10a-10d (2 mmol, 1 equiv), solution of hydroxylamine (50%
in water, 10 mL). and CH OH (20 mL) using the procedure described for compound 6a-6f to obtain the
3
desired product 11a-11d.
4
.1.27.
7-cyclopentyl-2-((4-((5-(hydroxyamino)-5-oxopentyl)
carbamoyl)phenyl)amino)-N,N-dimethyl -7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (11a).
1
White solid; 62% yield; mp 189.0 °C (decompose); H NMR (400 MHz, DMSOꢀd ) δ 10.38 (s, 1H),
6
9
.83 (s, 1H), 8.79 (s, 1H), 8.70 (s, 1H), 8.31 (t, J = 5.7 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.5
Hz, 2H), 6.60 (s, 1H), 5.75 (s, 1H), 4.83 – 4.63 (m, 1H), 3.25 (q, J = 6.2 Hz, 2H), 3.12 – 2.95 (m, 6H),
13
2
.51 – 2.43 (m, 2H), 2.10 – 1.92 (m, 6H), 1.76 – 1.60 (m, 2H), 1.62 – 1.43 (m, 4H). C NMR (101 MHz,
DMSOꢀd ) δ 169.51, 166.16, 163.31, 155.51, 152.54, 151.48, 144.03, 132.51, 128.25, 126.89, 117.35,
6
112.40, 101.07, 57.51, 55.37, 35.09, 32.54, 30.10, 29.46, 24.66, 23.28. ESIꢀHRMS m/z calcd for
+
+
C H N O 508.2667, found 508.2671 [M + H] . HPLC purity 98%.
26
34
7
4
4
.1.28.
7-cyclopentyl-2-((4-((6-(hydroxyamino)-6-oxohexyl)
carbamoyl)phenyl)amino)-N,N-dimethyl -7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (11b).
1
White solid; 62% yield; mp 94.5 °C (decompose); H NMR (400 MHz, DMSOꢀd ) δ 10.36 (s, 1H), 9.84
6
(s, 1H), 8.79 (s, 1H), 8.69 (s, 1H), 8.30 (t, J = 5.8 Hz, 1H), 7.99 – 7.76 (m, 4H), 6.61 (s, 1H), 4.75 (p, J
=
8.9 Hz, 1H), 3.23 (q, J = 6.7 Hz, 2H), 3.06 (s, 6H), 2.50 – 2.40 (m, 2H), 2.09 – 1.88 (m, 6H), 1.77 –
13
1
.63 (m, 2H), 1.56 – 1.42 (m, 4H), 1.37 – 1.23 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ 169.52,
6
2
7
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Journal of Medicinal Chemistry
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1
3
66.10, 163.29, 155.50, 152.56, 151.46, 144.01, 132.49, 128.24, 126.89, 117.32, 112.38, 101.09, 57.50,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
4
5.11, 32.72, 30.09, 29.58, 27.85, 26.64, 25.43, 24.66. ESIꢀHRMS m/z calcd for C H N O 522.2823,
2
7
36
7
+
found 522.2831 [M + H] . HPLC purity 98%.
.1.29.
4
7-cyclopentyl-2-((4-((7-(hydroxyamino)-7-oxoheptyl)
carbamoyl)phenyl)amino)-N,N-dimethyl -7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (11c).
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
White solid; 58% yield; mp 112.2°C (decompose); H NMR (400 MHz, DMSOꢀd ) δ 10.36 (s, 1H), 9.85
6
(s, 1H), 8.79 (s, 1H), 8.29 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 8.5 Hz, 2H), 6.61 (s,
1
H), 4.75 (p, 1H), 3.24 (q, J = 6.7 Hz, 2H), 3.05 (s, 6H), 2.49 – 2.40 (m, 2H), 2.07 – 1.97 (m, 4H), 1.94
1
3
(t, J = 7.3 Hz, 2H), 1.76 – 1.60 (m, 2H), 1.57 – 1.43 (m, 4H), 1.38 – 1.22 (m, 4H). C NMR (101 MHz,
DMSOꢀd ) δ 169.55, 166.12, 163.29, 155.50, 152.56, 151.45, 144.00, 132.48, 128.24, 126.91, 117.33,
6
112.38, 101.09, 57.50, 35.06, 32.71, 30.08, 29.71, 28.85, 26.75, 25.59, 24.98, 24.66. ESIꢀHRMS m/z
+
+
calcd for C H N O 536.2980, found 536.2985 [M + H] . HPLC purity 98%.
2
8
38
7
4
4.1.30.
-cyclopentyl-2-((4-((8-(hydroxyamino)-8-oxooctyl)carbamoyl)phenyl)amino)-N,N-dimethyl
7
-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (11d). White solid; 71% yield; mp 105.9 °C
1
(decompose); H NMR (400 MHz, DMSOꢀd ) δ 10.35 (s, 1H), 9.85 (s, 1H), 8.79 (s, 1H), 8.30 (t, J = 5.7
6
Hz, 1H), 7.91 (d, J = 8.5 Hz, 2H), 7.81 (d, J = 8.5 Hz, 2H), 6.61 (s, 1H), 4.75 (p, 1H), 3.24 (q, J = 6.7
Hz, 2H), 3.05 (s, 6H), 2.52 – 2.39 (m, 2H), 2.07 – 1.96 (m, 4H), 1.93 (t, J = 7.4 Hz, 2H), 1.74 – 1.63 (m,
1
3
2
H), 1.55 – 1.42 (m, 4H), 1.33 – 1.18 (m, 6H). C NMR (101 MHz, DMSOꢀd ) δ 169.56, 166.12,
6
163.29, 155.50, 152.56, 151.45, 144.00, 132.48, 128.24, 126.91, 117.33, 112.38, 101.08, 57.50, 35.06,
+
3
2.72, 30.09, 29.78, 29.04, 29.00, 26.93, 25.57, 24.66. ESIꢀHRMS m/z calcd for C H N O 550.3136,
2
9
40
7
4
+
found 550.3141 [M + H] . HPLC purity 99%.
General Procedure for the Synthesis of 13a-13d.
The title compound was prepared from 12a-12d (60 mmol, 1 equiv), pꢀPhenylenediamine (2c) (6.49 g,
6
0 mmol, 2 equiv), EDCI (8.27 g, 45 mmol, 1.5 equiv), HOBt (4.87 g, 36 mmol, 1.2 equiv), DIEA (7.76
g, 60 mmol, 2 equiv) and DMF (60 mL) using the procedure described for compound 3a-3f to obtain the
2
8
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Journal of Medicinal Chemistry
desired product 13a-13d.
.1.31. N-(4-aminophenyl)hexanamide (13a). White solid; 60% yield; mp 87.8 °C; H NMR (400
MHz, Chloroformꢀd) δ 7.78 (s, 1H), 7.26 – 7.21 (m, 2H), 6.58 – 6.53 (m, 2H), 3.57 (s, 2H), 2.31 – 2.19
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
4
1
3
(
m, 2H), 1.75 – 1.59 (m, 2H), 1.36 – 1.26 (m, 4H), 0.90 – 0.84 (m, 3H). C NMR (101 MHz, CDCl ) δ
3
1
71.74, 143.17, 129.50, 122.16, 115.33, 37.41, 31.48, 25.53, 22.47, 14.00. ESIꢀHRMS m/z calcd for
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
+
+
C H N O 207.1492, found 207.1494 [M + H] . HPLC purity 99%.
12 19
2
1
4
.1.32. N-(4-aminophenyl)heptanamide (13b). White solid; 40% yield; mp 89.2 °C; H NMR (400
MHz, Chloroformꢀd) δ 7.31 – 7.23 (m, 2H), 7.15 (s, 1H), 6.62 (d, J = 8.6 Hz, 2H), 3.67 – 3.37 (m, 2H),
13
2
.29 (t, J = 7.6 Hz, 2H), 1.69 (p, J = 7.5 Hz, 2H), 1.42 – 1.22 (m, 6H), 0.91 – 0.86 (m, 3H). C NMR
(101 MHz, CDCl ) δ 171.25, 143.19, 129.39, 122.01, 115.39, 37.63, 31.59, 28.99, 25.75, 22.53, 14.06.
3
+
+
ESIꢀHRMS m/z calcd for C H N O 221.1648, found 221.1650 [M + H] . HPLC purity 99%.
13
21
2
1
4
.1.33. N-(4-aminophenyl)octanamide (13c). White solid; 82% yield; mp 98.8 °C; H NMR (400
MHz, Chloroformꢀd) δ 7.63 (s, 1H), 7.26 – 7.21 (m, 2H), 6.60 – 6.54 (m, 2H), 3.57 (br, 2H), 2.26 (t, J =
13
7
.6 Hz, 2H), 1.66 (t, J = 7.3 Hz, 2H), 1.37 – 1.18 (m, 8H), 0.86 (t, J = 6.6 Hz, 3H). C NMR (101 MHz,
CDCl ) δ 171.63, 143.17, 129.47, 122.13, 115.34, 37.51, 31.73, 29.30, 29.11, 25.85, 22.64, 14.12.
3
+
+
ESIꢀHRMS m/z calcd for C H N O 235.1805, found 235.1807 [M + H] . HPLC purity 99%.
14 23
2
1
4
.1.34. N-(4-aminophenyl)nonanamide (13d). White solid; 66% yield; mp 99.2 °C; H NMR (400
MHz, Chloroformꢀd) δ 7.28 (d, J = 3.6 Hz, 2H), 7.10 (s, 1H), 6.64 (d, J = 8.4 Hz, 2H), 3.59 (s, 2H), 2.31
1
3
(
t, J = 7.6 Hz, 2H), 1.71 (p, J = 7.4 Hz, 2H), 1.41 – 1.21 (m, 10H), 0.89 (t, J = 6.5 Hz, 3H). C NMR
(101 MHz, CDCl ) δ 171.22, 143.19, 129.38, 122.00, 115.39, 37.65, 31.84, 29.37, 29.32, 29.17, 25.78,
3
+
+
2
9
2.66, 14.11. ESIꢀHRMS m/z calcd for C H N O 249.1961, found 249.1963 [M + H] . HPLC purity
15 25 2
9%.
General Procedure for the Synthesis of 14a-14d.
The title compound was prepared from 13a-13d (2 mmol, 1 equiv), compound 4 (586 mg, 2 mmol, 1
equiv), Pd(OAc) (11 mg, 0.05 mmol, 0.025 equiv), BINAP (62 mg, 0.1 mmol, 0.05 equiv) and Cs CO
3
2
2
(1.37 g, 4.2 mmol, 2.1 equiv) and 1,4ꢀdioxane (20 mL) using the procedure described for compound
2
9
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