CN107903246A - A kind of sulfamide derivative, preparation method and its it is antitumor in application - Google Patents

Abstract

The invention discloses a kind of sulfamide derivative formula（Ⅰ）, formula（Ⅰ）For new construction, formula is also disclosed（Ⅰ）Preparation method and its application as Nampt inhibitor in antitumor drug.Suppressing the activity experiment of Nampt and mtt assay measure formula（Ⅰ）Good activity is all shown in experiment to the inhibitory action of different tumour cells.Formula（Ⅰ）Structure is

Description

A kind of sulfamide derivative, preparation method and its it is antitumor in application

Technical field

The present invention relates to a kind of sulfamide derivative, preparation method and its it is antitumor in application.

Background technology

Nampt（Nicotinamide phosphoribosyl transferase, NAMPT) again Name Nampt visfatin or pre-B cell clone enhancer PBEF, regulates and controls required energy matter in mammalian cell The level of NAD.Cancer cell has very high NAD consumption and metabolic rate, thus the rate-limiting enzyme NAMPT of NAD route of synthesis becomes The new target drone for the treatment of of cancer, its enzyme inhibitor FK866 and CHS-828 are typical NAMPT inhibitor, field of cancer treatment into Extensive clinical research is gone.

FK866 ((E)-N- [4- (1- benzoyl piperidine -4- bases) butyl] -3- (pyridin-3-yl) acrylamide) exists Inducing cell apoptosis in HepG2 cells, but do not have initial action to cellular energy metabolism.FK866 can be used for treatment and involve The disease such as cancer of Apoptosis imbalance.The prior art is it was demonstrated that the biology of FK866 interference nicotinamide adenine dinucleotide Synthesize and apoptosis-induced cell death and without any DNA damage effect.FK866 suppresses NAMPT and exhausts the cell of NAD but do not draw Play cytotoxicity at once, this hint, synthesize the cancer cell of NAD for anti-dependence niacinamide for, FK866 is promising medicine Thing.In mouse mammary cancer model, FK866 also induces tumor growth delay and tumor radiosensitivity to strengthen, with NAD water The dose dependent of flat, pH and energy state reduces.In THP-1 and K562 Leukemia Cell Lines, it has been observed that FK866 is resisted The chemical sensitization effect of the cell death of tumour medicine 1- methyl-3-nitro -1- nitrosoguanidines (MNNG)-induction.Moved in xenogenesis The effect of GMX1777 is evaluated in plant model and the medicine generation distribution of GMX1778 is measured and its to nicotinoyl by liquid chromatography/mass spectrometry The effect of amine adenine-dinucleotide cellular level.

The above shows that NAMPT inhibitor has good druggability, available for preparation by suppressing niacinamide phosphoric acid Phosphoribosynltransferase is come the medicine of disease that prevents and/or treat, it can also be used to the disease such as anti-curing cancers.New NAMPT suppresses The exploitation of agent is also the hot spot of study of pharmacy.

The content of the invention

It is an object of the present invention to provide a kind of sulfamide derivative, preparation method and its it is antitumor in application. Its structural formula（Ⅰ）For

。

Another object of the present invention is to provide the formula（Ⅰ）Synthetic route：

。

Another object of the present invention is to provide the formula（Ⅰ）Synthesis step：

（1）Pyrrolidines -3- phosgenes and 4,5- dihydro -1H- imidazoles -1- sulfonic acid chlorides react generation 1- ((4,5- bis- in toluene Hydrogen -1H- imidazoles -1- bases) sulfonyl) pyrrolidines -3- dicarbonyl chlorides, post-process and washed respectively with water and hydrochloric acid solution, then column color Compose isolated product；

（2）1- ((4,5- dihydro -1H- imidazoles -1- bases) sulfonyl) pyrrolidines -3- dicarbonyl chlorides and 4- amino -2,6- dichloropyridines Reacted under low temperature, washed by the aqueous solution of water and salt, be finally recrystallized to give formula（Ⅰ）.

Further, the concentration of hydrochloric acid solution used in the post-reaction treatment of sulfonic acid chloride is 1-5 mol/L, preferably 2-3 mol/ L, most preferably 2 mol/L.

Further, formula（Ⅰ）Reaction of formation in, reaction temperature be -30-0 DEG C, preferably 0 DEG C；Wash the salt bag of organic phase The aqueous solution of the salt such as sodium acid carbonate, sodium carbonate, saleratus, disodium hydrogen phosphate, preferably sodium acid carbonate and sodium carbonate are included, most preferably Sodium acid carbonate；Recrystallization solvent used is ethyl acetate/n-hexane（1:5）.

Another object of the present invention is to provide the formula（Ⅰ）Purposes, as Nampt suppress The application of agent.

Further, the formula（Ⅰ）Application in Nampt inhibitor medicaments are prepared.

Another object of the present invention is to provide the formula（Ⅰ）Another aspect purposes, in antitumor drug should With.

Further, the formula（Ⅰ）Application in antitumor drug is prepared.The tumor type includes lung cancer, liver Cancer, stomach cancer, breast cancer, chronic myelogenous leukemia.

Formula according to the present invention（Ⅰ）It is real in the activity for suppressing Nampt for new construction Test and measure formula with mtt assay（Ⅰ）Good activity is all shown in experiment to the inhibitory action of different tumour cells.

Obviously, the above according to the present invention, according to the ordinary technical knowledge and means of this area, is not departing from this hair Under the premise of bright above-mentioned basic fundamental thought, the modification, replacement or change of other diversified forms can also be made.

Embodiment

Embodiment 1：1-（（4,5- dihydro -1H- imidazoles -1- bases）Sulfonyl）The synthesis of pyrrolidines -3- dicarbonyl chlorides

By pyrrolidines -3- phosgenes（16.8ml 0.2mol）In dry toluene（40ml）In solution be added drop-wise to 4,5- dihydros- 1H- imidazoles -1- sulfonic acid chlorides（33.72g 0.2mol）In toluene（30ml）In it is cold（-10℃）In solution so that internal temperature is not More than -5 °C.Once addition is completed, mixture is stirred 30 minutes at -5 DEG C, then adds water（50ml）.Layering, organic layer Use HCl（2mol/l, 50ml）Washing, with anhydrous MgSO₄Dry, vacuum evaporation obtains clear oily matter（9.6g）.The oil is institute Need sulfonamides chlorine and 1-（（4,5- dihydro -1H- imidazoles -1- bases）Sulfonyl）The 2 of pyrrolidines -3- dicarbonyl chlorides:1 mixture.Column color Compose isolated 1-（（4,5- dihydro -1H- imidazoles -1- bases）Sulfonyl）Pyrrolidines -3- dicarbonyl chloride 42.4g, yield 80%.¹H- NMR (400 MHz, CDCl3) δ:1.77(m,1H), 2.07(m, 1H), 2.80(m, 1H), 3.55-3.64(m, 4H), 3.73(m, 1H), 3.84(m, 1H), 3.95-3.98(t, 2H), 7.98(s, 1H).¹³C-NMR (125 MHz, CDCl3) δ: 31.92,43.81, 45.34, 46.45, 50.02, 55.87, 136.36, 174.48.LC-MS(ESI, pos, ion) m/z: 266[M+1].

Embodiment 2：N-（2,6- dichloropyridine -4- bases）-1-（（4,5- dihydro -1H- imidazoles -1- bases）Sulfonyl）Pyrrolidines -3- The synthesis of formamide

By 1-（（4,5- dihydro -1H- imidazoles -1- bases）Sulfonyl）Pyrrolidines -3- dicarbonyl chlorides（53.14g 0.2mol）Anhydrous CH₂Cl₂（36mL）In solution bis- chloro- 4-aminopyridines of 2,6- are carefully added at 0 DEG C（32.60g 0.2mol）With three second Amine（27mL）Anhydrous CH₂Cl₂（90mL）Solution）.Reaction mixture is stirred in ice bath 1 it is small when, then warm to room temperature simultaneously It is stirred overnight.Add cold water（100mL）, and CH is separated from aqueous solution₂Cl₂Layer.Organic phase saturation NaHCO₃Solution（2× 40mL）And water washing, with anhydrous MgSO₄It is dried, filtered and concentrated, obtains crude product, by it from ethyl acetate/n-hexane（1:5） Middle recrystallization, obtains white spicule N-（2,6- dichloropyridine -4- bases）-1-（（4,5- dihydro -1H- imidazoles -1- bases）Sulphonyl Base）Pyrrolidines -3- formamide 66.68g, yield 85%.¹H-NMR (400 MHz, CDCl3) δ: 1.79(m,1H), 2.06(m, 1H), 2.77(m, 1H), 3.17(m, 1H), 3.57-4.06(m, 7H), 7.79(s, 1H), 8.01(s, 2H), 8.84(s, 1H). ¹³C-NMR (125 MHz, CDCl3) δ: 31.21, 43.24, 43.81, 45.14, 45.34, 55.87, 117.50, 136.36, 148.76, 149.0, 1173.83. LC-MS(ESI, pos, ion) m/ z: 392[M+1].

Effect example 1：Formula（Ⅰ）Suppress the activity of Nampt

1st, the preparation of enzyme：Conversion there are into BL21 (DE3) plysS cell inoculations of recombinant plasmid (Nampt-pET28a+) in 2 × YT In culture medium (37ug/ml chloramphenicol and 75ug/ml kanamycins), 37 DEG C of shakings are stayed overnight, with 20 times of substances after collection thalline Long-pending fresh culture is resuspended, and 37 DEG C of cultures induce 5h to OD600 about 0.6 under the conditions of 0.3mM IPTG, 28 DEG C.Centrifugation is received Collect thalline, and be resuspended in lysis buffer (20mM Tris-HCl pH8.0,300mM NaCl), 200W ultrasonic degradations are thin Born of the same parents, ultrasonic 1s gaps 9s, carry out 30min altogether.By lysate in 12500rpm, 4 DEG C of centrifugation 50min Aspirate supernatants.The supernatant Liquid is incubated 1h with Ni-NTA columns (being purchased from QIAGEN companies) in shaking on ice, then uses binding buffer (5mM successively Imidazole, 0.5M NaCl, 20mMTris-Hcl, pH=7.5), wash buffer (40mM imidazole, 0.5M NaCl, 20mM Tris-HCl, pH=7.5) foreign protein is washed away successively, finally with Elution buffer (200mM Imidazole, 0.5MNaCl, 20mM Tris-Hcl, pH=7.5) elution destination protein, and carry out SDS-PAGE detections.It will wash De- destination protein is transferred in bag filter, with Tris-HCl (20mM pH=7.5) dialysis 4~5 of sterilizing in 4 DEG C of refrigerators It is secondary, after 20%PEG20000 concentrations, protein concentration is measured with Bradford methods.

2nd, enzyme reaction system is 25 μ l, wherein the concentration of various components is：50mM Tris-HCl (pH7.5), 0.02% BSA, 12mM MgCl2,2mM ATP, 0.4mM PRPP, 2mM DTT, 2 μ g/mlNampt, 0.2 μM of NAM, 2%DMSO and multiple proportions Diluted compound.The DMSO solution of the various concentrations of 0.5 μ l compounds is first added on 96 orifice plates, 20 μ l enzyme reactions is added and mixes Solution (the enzyme reaction component in addition to substrate) is closed, after 37 DEG C of preincubate 5min, it is anti-to start to add 4.5 μ l substrate NAM solution Should, 37 DEG C of reaction 15min heat 1min after 95 DEG C and terminate enzyme reaction.

3rd, 10 μ l, 20% acetophenones and 2M KOH are sequentially added after enzyme reaction solution after cooled on ice, on vortex mixed instrument Mix and act on 2min after 0 DEG C, add 45 μ l88% formic acid, 70 DEG C of heating 5min, cooled on ice.

4th, using the fluorescent value at microplate reader measure excitation wavelength 382nm, launch wavelength 445nm.

5th, according to formula：E=R/ (1+ (C/IC₅₀)^S) (wherein E is enzymatic activity to+B, and C is compound concentration, R, IC₅₀、S、B For parameter to be fitted), enzymatic activity is fitted the curve of compound concentration in origin softwares, obtains formula（Ⅰ）'s IC₅₀For 3.34 ± 0.27 nM.

Therefore deduce that, formula disclosed in this invention（Ⅰ）, can with the activity for suppressing Nampt As Nampt inhibitor, can be used for the treatment of Nampt relevant disease.

Effect example 2：MTT（Tetrazolium bromide）Method measures formula（Ⅰ）To the inhibitory action of different tumour cells

1st, used tumour cell is：Human lung cancer cell A549, human liver cancer cells Hep G2, human liver cancer cell Bel- 7402, human gastric adenocarcinoma SGC-7901, human breast cancer cell line Bcap-37, people's chronic myelogenous leukemia cell K562.

2nd, experimental procedure

1. the cell in growth period of taking the logarithm, Trypsin Induced, 1640 cell culture fluid tune concentration of cell suspension of RPMI for 6 × 10⁴A/mL.Add 100 μ L of cell suspension per hole in 96 well culture plates, put 37 DEG C, 5% CO₂24h, cell patch are cultivated in incubator Wall.

2. removing 1640 cell culture fluids of RPMI, 1640 cell culture fluids of RPMI of the medicine to be measured of concentration gradient are added 100 μ L, each concentration set 6 parallel holes.96 orifice plates after dosing are placed in 37 DEG C, 5% CO₂48h is cultivated in incubator, is inverted The action effect of micro- Microscopic observation medicine.

Nutrient solution is discarded after the centrifugation of 3.96 orifice plates, after carefully being rushed 2 ~ 3 times with PBS, adds the RPMI containing 0.5% MTT 1640 cell culture fluid, 100 μ L, continue to cultivate 4h.

4. removing supernatant, 150 μ L dimethyl sulfoxide (DMSO)s are added per hole, low-speed oscillation 10min on shaking table is put, ties formazan Brilliant fully dissolving.

5. the optical density in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm（OD values）.

6. parallel hole OD values are represented with mean ± SD, inhibiting rate formula is calculated：[(OD_{Control group}-OD_{Blank group})-(OD_{Drug study group}- OD_{Blank group})]/(OD_{Control group}-OD_{Blank group})*100%。

7. using 5 data processing softwares of GraphPad Prism, by drawing amount effect curve calculation of half inhibitory concentration （IC₅₀）.

1 formula of table（Ⅰ）Human tumor cells IC₅₀Value

Therefore deduce that, formula disclosed in this invention（Ⅰ）It is inhibited to six kinds of human tumor cells, cancer can be used as The drug candidate of prevention and/or treatment carries out more deep research and development, has prompted formula disclosed in this invention（Ⅰ）It can be used for making Standby antitumor drug.

Claims (5)

1. a kind of sulfamide derivative, preparation method and its it is antitumor in application, its structural formula（Ⅰ）For

。

2. formula as claimed in claim 1（Ⅰ）, synthetic route is

。

3. formula as claimed in claim 2（Ⅰ）Synthetic route, its synthesis step is：

（1）Pyrrolidines -3- phosgenes and 4,5- dihydro -1H- imidazoles -1- sulfonic acid chlorides react generation 1- ((4,5- bis- in toluene Hydrogen -1H- imidazoles -1- bases) sulfonyl) pyrrolidines -3- dicarbonyl chlorides, post-process and washed respectively with water and hydrochloric acid solution, then column color Compose isolated product；

（2）1- ((4,5- dihydro -1H- imidazoles -1- bases) sulfonyl) pyrrolidines -3- dicarbonyl chlorides and 4- amino -2,6- dichloropyridines Reacted under low temperature, washed by the aqueous solution of water and salt, be finally recrystallized to give formula（Ⅰ）.

4. formula as claimed in claim 1（Ⅰ）, the application as Nampt inhibitor.

5. formula as claimed in claim 1（Ⅰ）, the application in antitumor drug.