CN118307474A - Contracture medicine with synergistic analgesic effect and preparation method and application thereof - Google Patents
Contracture medicine with synergistic analgesic effect and preparation method and application thereof Download PDFInfo
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- CN118307474A CN118307474A CN202410522303.3A CN202410522303A CN118307474A CN 118307474 A CN118307474 A CN 118307474A CN 202410522303 A CN202410522303 A CN 202410522303A CN 118307474 A CN118307474 A CN 118307474A
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- celecoxib
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Abstract
The invention relates to the technical field of medicines, and in particular discloses a contracture drug with a synergistic analgesic effect, a preparation method and application thereof. The invention also comprises a preparation method of the contracture drug, and application of the contracture drug or pharmaceutically acceptable salt and pharmaceutically acceptable carrier and composition thereof in preparing drugs for preventing and treating pains or nervous system diseases. The contracture drug can obviously improve the water solubility of celecoxib, has a dose-dependent analgesic effect on different types of pains such as inflammatory pains, neuralgia, visceral pains and the like, has an analgesic effect obviously superior to that of a drug A or a drug B, and has a good application prospect.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a contracture drug with a synergistic analgesic effect, a preparation method and application thereof.
Background
Pain is the third greatest health problem following cardiovascular and cerebrovascular diseases and tumors, severely affecting the quality of life of the patient, and thus positively and effectively treating pain is of great importance. In recent years, along with the continuous elucidation of the pain mechanism, the occurrence and development of pain are found to have the characteristics of multi-mechanism and multi-factor action, and different mechanisms mutually influence and mutually regulate and control. Based on the above results, researchers have proposed a "multi-mode analgesic" strategy. The contracture drug is one of effective strategies for realizing a multi-mode analgesic scheme, and refers to the fact that two identical or different lead compounds or drugs are connected through covalent bonds in a combined mode and conjugated into a novel multifunctional molecule, and a synergistic effect is generated in vivo, so that the drug effect is enhanced, side effects are reduced, pharmacokinetic properties are improved, or the selectivity of the effect is improved. Compared with the combined application of multiple drugs and compound drugs, the contracture drug can reduce the dosage, improve the treatment effect, has uniform pharmacokinetic characteristics, avoids the interaction between the drugs and the toxic and side effects caused by the interaction, and improves the compliance of patients.
Nonsteroidal anti-inflammatory drugs (NSAIDs) act on Cyclooxygenase (COX) to inhibit the synthesis of prostaglandins from arachidonic acid, and exert anti-inflammatory and analgesic effects. Cyclooxygenase has two subtypes, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Wherein COX-1 is expressed in most tissues such as blood vessels, stomach, kidney, blood platelets and the like, and participates in platelet aggregation, vasomotor and renal blood flow regulation so as to maintain the stability of physiological functions of cells, tissues and organs; COX-2 is usually expressed very poorly and is significantly enhanced when cells are stimulated by inflammation. Blocking COX-1 tends to cause a decrease in platelet and gastrointestinal function, thereby limiting the clinical use of non-selective NSAIDs. The efficacy of selective NSAIDs is largely dependent on the inhibition of COX-2, thus reducing the incidence of adverse effects. Celecoxib is the first COX-2 inhibitor approved by the U.S. food and drug administration and has analgesic and anti-inflammatory activity, and the structure is shown in formula A. Celecoxib has less effect on platelet and gastrointestinal function than traditional nonsteroidal anti-inflammatory drugs, but poor water solubility, and greater individual differences in bioavailability after oral administration to patients. Long-term use of celecoxib has been shown to increase the risk of serious cardiovascular events, such as thrombosis and atherosclerosis.
Gamma-aminobutyric acid is the most important and abundant inhibitory neurotransmitter in the human central nervous system, and has a structure shown in formula B-1. It plays an important role in central hyperexcitations associated with nerve damage, acting on inhibitory synapses in the brain through binding to specific transmembrane receptors in the presynaptic and postsynaptic plasma membranes. An increase in the concentration of gamma-aminobutyric acid in the spinal cord is reported to cause analgesia, while a decrease in the concentration of gamma-aminobutyric acid causes neuropathic pain. Studies show that exogenous gamma-aminobutyric acid can weaken the response of normal rat nucleus accumbens pain excitation neurons to harmful stimulus, has the effect of gamma-aminobutyric acid receptor mediated analgesia, and prompts that the gamma-aminobutyric acid receptor and other transmitters are involved in regulating the activity of nucleus accumbens. Furthermore, in the paclitaxel-induced neuropathic pain model, the excitability of neuropathic pain rats in electrophysiological recordings was reversed following administration of gamma-aminobutyric acid. These results indicate that gamma-aminobutyric acid plays an important role in the regulation of pain response.
Gabapentin and pregabalin, which were originally used for anticonvulsant (structures B-2 and B-3), have now become first-line drugs for the treatment of neuropathic pain. Gabapentin and pregabalin are structurally similar to gamma-aminobutyric acid but are not agonists of gamma-aminobutyric acid receptors. They reduce Ca 2+ influx by acting on the α 2δ1 subunit of presynaptic voltage-gated Ca 2+ channels, thereby inhibiting neurotransmitter (e.g., glutamate, norepinephrine) release, reducing neuronal excitability. However, only 40% -60% of patients obtain pain relief when they are used. In addition, gabapentin and pregabalin also present a range of side effects, such as shortness of breath and ataxia. Studies have shown that the toxic and side effects of the medicaments can be reduced and the curative effect can be improved by combined administration.
According to the invention, celecoxib, gamma-aminobutyric acid, gabapentin or pregabalin are condensed to prepare the contracture drug, so that the water solubility of celecoxib is improved, and meanwhile, a synergistic analgesic effect can be generated through various analgesic mechanisms, so that a better analgesic effect is obtained, the side effect related to dosage is reduced, and the clinical medication requirement is better met. Therefore, the analgesic celecoxib and the gamma-aminobutyric acid, gabapentin or pregabalin contracture drug have wide application prospect for treating pain.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art, and provides a contracture drug with a synergistic analgesic effect, a preparation method and application thereof, so as to solve the problems of the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions: the contracture medicine with synergistic analgesic effect is formed by connecting medicine A and medicine B through chemical bonds, wherein the medicine A is celecoxib, and the medicine B is gamma-aminobutyric acid (GABA), gabapentin or pregabalin, and the structure is as follows:
a preparation method of a contracture drug with synergistic analgesic effect comprises the following specific steps:
Step 1: protecting amino groups in a gamma-aminobutyric acid, gabapentin or pregabalin structure by di-tert-butyl dicarbonate, allyl chloroformate, trimethylsilyl ethoxycarbonyl chloride or 9-fluorenylmethyl chloroformate to obtain an intermediate I;
step 2: carrying out condensation reaction on celecoxib and the intermediate I obtained by the reaction in the step 1 in the presence of EDCI/DMAP/triethylamine to obtain an intermediate II;
step 3: removing protecting groups from the intermediate II obtained in the step 2 in ethyl acetate hydrochloride gas, tetra (triphenylphosphine) palladium/tri-n-butyltin hydride, tetraethylammonium fluoride, tetramethylammonium fluoride, triethylamine or pyridine to obtain the contracture drug as described in claim 1.
As a preferable technical scheme of the invention, the molar ratio of the celecoxib to the intermediate I in the step 2 is 1:1-2.
As a preferable technical scheme of the invention, the molar ratio of the intermediate I in the step 2 to EDCI, DMAP and triethylamine is sequentially 2-3: 0.1 to 0.5:2 to 5.
As a preferred embodiment of the present invention, the reaction of step 2 is carried out in a solvent which is tetrahydrofuran, methylene chloride or N, N-diformylcarboxamide.
A pharmaceutical composition comprises the above contracture drug or its pharmaceutically acceptable salt with synergistic analgesic effect and pharmaceutically acceptable carrier.
An application of a contracture medicine or its pharmaceutical composition with synergistic analgesic effect in preparing medicine for preventing or treating pain or nervous system diseases is provided.
As a preferred embodiment of the invention, the pain is acute pain, chronic pain, neuropathic pain, inflammatory pain, nociceptive pain, cancerous pain, hyperalgesia and visceral pain.
As a preferred embodiment of the present invention, the neurological disorder is pain, anxiety, depression, parkinson's disease, alzheimer's disease, or schizophrenia.
Compared with the prior art, the invention has the beneficial effects that:
According to the invention, celecoxib and gamma-aminobutyric acid, gabapentin or pregabalin are subjected to covalent connection by chemical means, and finally a novel molecular entity with high analgesic activity is obtained.
The invention proves that celecoxib and pregabalin have synergistic analgesic effect through an isoradiometric analysis method, have no obvious toxic or side effect while playing analgesic effect, and have good clinical application prospect.
The in vivo pain model test results show that the analgesic efficacy of the twin drug compound 1, the compound 2 and the compound 3 provided by the invention is superior to that of the combination drug administration and the single drug administration, so that the pain caused by various acute and chronic inflammatory pains can be obviously improved, and the peripheral neuralgia induced by taxol can be relieved. Since these in vivo pharmacological models are closely related to pain, the compounds or compositions thereof provided by the present invention have the potential to treat pain-related diseases.
The in vivo inflammation model test results show that the twin drug compound 1 provided by the invention has higher anti-inflammatory activity than the combination and single drug, and the compound or the composition thereof provided by the invention has the potential of treating inflammation and inflammatory pain as the treatment of inflammation is a feasible method for improving pain.
Drawings
FIG. 1 is a schematic representation of the analgesic effect of celecoxib, gamma-aminobutyric acid and gabapentin in a carrageenan-induced acute inflammatory pain model;
FIG. 2 is a schematic representation of analgesic effects of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and the contracture drug celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in a carrageenan-induced acute inflammatory pain model;
FIG. 3 is a graphical representation of the efficacy of the contracture drugs celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in combination with equimolar amounts of celecoxib, gamma-aminobutyric acid or gabapentin;
FIG. 4 is a schematic representation of analgesic effects of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and the contracture drug celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in a complete Freund's adjuvant-induced model of chronic inflammatory pain;
FIG. 5 is a schematic representation of analgesic effects of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and the contracture drug celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in an iodoacetic acid-induced model of osteoarthritis pain;
FIG. 6 is a schematic representation of analgesic effects of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and the contracture drug celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in a formalin-induced inflammatory pain model;
FIG. 7 is a schematic representation of analgesic effects of celecoxib, pregabalin and the contracture drug celecoxib-pregabalin hydrochloride (Cel-Pre, compound 3) in a paclitaxel-induced neuropathic pain model;
FIG. 8 is a schematic representation of analgesic effect of celecoxib and pregabalin combination in paclitaxel-induced neuropathic pain model;
FIG. 9 is a graph showing the comparison of the effects of celecoxib-pregabalin hydrochloride (Cel-Pre, compound 3) as a contracture drug with single and combinations of equimolar celecoxib and pregabalin in a paclitaxel-induced neuropathic pain model;
FIG. 10 is a graphical representation of changes in toe swelling volume of mice following administration of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) in a carrageenan-induced acute inflammatory pain model;
FIG. 11 is a graph showing changes in toe swelling volume of mice following administration of celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) as a contracture drug in a complete Freund's adjuvant-induced chronic inflammatory pain model;
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the attached drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making clear and defining the scope of the present invention.
The invention provides a contracture drug with synergistic analgesic effect and a composition thereof, wherein the contracture drug is formed by connecting a drug A and a drug B through chemical bonds, the drug A is celecoxib, and the drug B is gamma-aminobutyric acid, gabapentin or pregabalin.
The invention also provides a method of preventing or treating a disease comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention. The diseases include pain and/or nervous system diseases, wherein the pain related diseases are acute pain, chronic pain, neuropathic pain, inflammatory pain, nociceptive pain, cancerous pain, hyperalgesia and visceral pain, and the nervous system diseases are pain disorders, anxiety disorders, depression, parkinson's disease, alzheimer's disease, schizophrenia.
Experimental animals: ICR mice (18-25 g,4-6 weeks), male and female halves, were kept at constant temperature (22.+ -. 3 ℃) and 55.+ -. 5% relative humidity and subjected to light/dark cycles for 12 hours.
Experimental materials: celecoxib (Celecoxib), gamma aminobutyric acid (GABA), gabapentin (Gabapentin) and Pregabalin (Pregabalin) were purchased from Shanghai microphone Biochemical technologies Co.
The structure of the compounds of the invention is determined by Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS). NMR was determined using BrukerAV-500 nuclear magnetic instruments with deuterated chloroform (CDCl 3) and deuterated methanol (MeOD) as the measurement solvents and Tetramethylsilane (TMS) as the internal standard. Chemical shift (δ) units are ppm.
HR-MS was determined using AGLIENT LC-MS/MS QTOF 6530 (manufacturer: aglient, MS model: QTOF 6530).
A. compound synthesis examples;
Example 1:
4-amino-N-((4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)sulfonyl)butanamide hydrochloride (celecoxib-gamma-aminobutyric acid hydrochloride, compound 1)
The reaction formula:
Intermediate (1A) is synthesized by the reaction of gamma-aminobutyric acid and di-tert-butyl dicarbonate, and further the intermediate (1B) is generated by the reaction of celecoxib, and finally, the target product celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) is obtained by deprotection under acidic conditions.
The first step: synthesis of 4- ((tert-butoxycarbonyl) amino) butanoic acid (1A);
To a 100mL single port flask, 4mL of water, 0.50g (4.85 mmol,1.0 eq) of gamma-aminobutyric acid, 0.31g (7.82 mmol,1.5 eq) of sodium hydroxide were added with stirring, and 1.27g (5.82 mmol,1.2 eq) of di-tert-butyl dicarbonate was added dropwise. After stirring at room temperature for 4 hours, TLC monitored complete reaction of gamma-aminobutyric acid. The remaining di-tert-butyl dicarbonate was washed off with 20mL of methylene chloride (repeated three times). The aqueous phase was adjusted to ph=2 with concentrated hydrochloric acid, extracted 3 times with 30ml of dichloromethane, dried over anhydrous sodium sulfate and spun-dried at 35 ℃ to give product 1A as a colorless oil, 7.00g, in yield 50.76％.1H NMR(500MHz,Chloroform-d)δ4.80(s,1H),3.19(q,J＝7.6,6.9Hz,2H),2.39(t,J＝7.3Hz,2H),1.82(p,J＝7.0Hz,2H),1.44(s,9H).HR–MS(ESI)m/z 204.1234([M+H]+,C9H18NO4 +,calculated 204.1230).
And a second step of:
tert-butyl(4-oxo-4-((4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)sulfonamido)butyl)carbamate(1B) Is synthesized by (1);
In a 100mL single port flask, 20mL of methylene chloride, 0.50g (2.4 mmol,1.2 eq) of 1A,0.76g (2 mmol,1.0 eq) of celecoxib, 0.46g (2.4 mmol,1.2 eq) of EDCI,0.03g (0.24 mmol,0.12 eq) of DMAP are added. After stirring at room temperature for 2 hours, TLC monitored 1B reaction was complete. Adding 30mL of water, standing for layering, adding 30mL of dichloromethane into the water layer for extraction, combining the organic layers, washing with 30mL of saturated saline solution, drying with anhydrous sodium sulfate, filtering, spin-drying, and performing column chromatography to obtain a colorless oily substance with the yield of 1.22g of the product 1B 87.53％.1HNMR(500MHz,Chloroform-d)δ8.08(d,J＝8.4Hz,2H),7.53–7.47(m,2H),7.19(d,J＝7.9Hz,2H),7.15–7.10(m,2H),6.76(s,1H),4.85(t,J＝6.6Hz,1H),3.10(q,J＝6.3Hz,2H),2.40(s,3H),2.29(t,J＝6.6Hz,2H),1.74(p,J＝6.3Hz,2H),1.46(s,9H).HR–MS(ESI)m/z 567.1889([M+H]+,C26H30F3N4O5S+,calculated 567.1884).
And a third step of:
4-amino-N-((4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)sulfonyl)butanamide hydrochloride (celecoxib-gamma-aminobutyric acid hydrochloride, synthesis of compound 1);
In a 50mL reaction flask, 10mL of ethyl acetate and 0.40g of 1B were added, and 10mL of HCl/EA solution was added dropwise. After 4 hours at room temperature, TLC monitored 1B reaction was complete. Suction filtration, filter cake washing with 5mL ethyl acetate, and drying to obtain the product compound 1 as white powder of 0.23g, yield 64.83%.
1H NMR(500MHz,Methanol-d4)δ8.09–8.03(m,2H),7.58–7.52(m,2H),7.24(d,J＝8.0Hz,2H),7.19(d,J＝8.2Hz,2H),6.93(s,1H),2.96–2.90(m,2H),2.46(t,J＝7.0Hz,2H),2.38(s,3H),1.88(p,J＝7.2Hz,2H),1.36–1.27(m,1H),0.95–0.85(m,1H).13C NMR(126MHz,MeOD)δ171.04,145.77,144.13,143.83,143.52,143.20,139.78,139.10,129.34,129.03,128.69,125.68,125.35,124.46,122.32,120.19,118.05,105.81,38.49,32.06,26.58,21.64.HR–MS(ESI)m/z 503.1133([M+H]+,C21H23ClF3N4O3S+,calculated 503.1126).
Example 2:
2-(1-(aminomethyl)cyclohexyl)-N-((4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)sulf onyl)acetamide hydrochloride( Synthesis of celecoxib-gabapentin hydrochloride, compound 2);
The reaction formula:
Intermediate (2A) is synthesized by the reaction of gabapentin and di-tert-butyl dicarbonate, and further the intermediate (2B) is generated by the reaction of the gabapentin and the di-tert-butyl dicarbonate and celecoxib, and finally, the target product celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) is obtained by deprotection under acidic conditions.
The first step: synthesis of 2- (1- (((tert-butoxycarbonyl) amino) methyl) cyclohexyl) ACETIC ACID (2A);
in a 100mL single port flask, 20mL of water and 1.7g (10 mmol,1.0 g) of gabapentin were added, and 0.6g (15 mmol,1.5 g) of sodium hydroxide was added with stirring, and 2.2g (12 mmol,1.2 g) of di-tert-butyl dicarbonate was added dropwise. After stirring at room temperature for 4 hours, TLC monitored that 2A was complete. The remaining di-tert-butyl dicarbonate was washed off with 20mL of methylene chloride (repeated three times). The aqueous phase was adjusted to ph=2 with concentrated hydrochloric acid, extracted 3 times with 30ml of dichloromethane, dried over anhydrous sodium sulfate and spun-dried at 35 ℃ to give product 1A as a colorless oil, 1.97g, in yield 73.13％.1H NMR(500MHz,Chloroform-d)δ3.18(t,J＝8.5Hz,2H),2.32(s,2H),1.56–1.49(m,4H),1.46(s,9H),1.45–1.27(m,6H).HR–MS(ESI)m/z 272.1855([M+H]+,C14H26NO4 +,calculated 272.1856).
And a second step of: tert-butyl
((1-(2-oxo-2-((4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)sulfonamido)ethyl)cycloh exyl)methyl)carbamate(2B) Is synthesized by (1);
In a 100mL single port flask, 20mL of methylene chloride, 0.3g (1.2 mmol,1.2 eq) of 2A,0.38g (1 mmol,1.0 eq) of celecoxib, 0.23g (1.2 mmol,1.2 eq) of EDCI,0.02g (0.16 mmol,0.16 eq) of DMAP are added. After stirring at room temperature for 2 hours, TLC monitored that 2A was complete. Adding 30mL of water, standing for layering, adding 30mL of dichloromethane into the water layer for extraction, combining the organic layers, washing with 30mL of saturated saline solution, drying with anhydrous sodium sulfate, filtering, spin-drying, and performing column chromatography to obtain a colorless oily substance with the yield of 0.65g of the product 1B 92.64％.1H NMR(500MHz,Chloroform-d)δ11.99(s,1H),8.13–8.07(m,2H),7.51–7.45(m,2H),7.18(d,J＝7.9Hz,2H),7.15–7.09(m,2H),6.75(s,1H),3.09(d,J＝7.3Hz,2H),2.40(s,3H),2.14(s,2H),1.49(s,9H),1.48–1.38(m,4H),1.38–1.24(m,4H),1.10(ddd,J＝13.5,9.4,3.7Hz,2H).HR–MS(ESI)m/z635.2517([M+H]+,C31H38F3N4O5S+,calculated 635.2510).
And a third step of:
2-(1-(aminomethyl)cyclohexyl)-N-((4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)sulf onyl)acetamide hydrochloride( Synthesis of celecoxib-gabapentin hydrochloride, compound 2);
In a 50mL reaction flask, 10mL of ethyl acetate and 0.40g of 2B were added, and 10mL of HCl/EA solution was added dropwise. After 4 hours at room temperature, TLC monitored that 2B was complete. Suction filtering, washing the filter cake with 5mL ethyl acetate, and drying to obtain white powder of 0.13g of the product compound 2 with the yield of 36.15％.1H NMR(500MHz,Methanol-d4)δ8.10–8.04(m,2H),7.60–7.53(m,2H),7.27–7.17(m,4H),6.94(s,1H),2.99(s,2H),2.49(s,2H),2.38(s,3H),1.54(d,J＝7.0Hz,1H),1.49(dq,J＝8.1,4.6,3.9Hz,4H),1.39(dt,J＝13.3,5.1Hz,3H),1.32(dt,J＝13.4,6.0Hz,2H).13C NMR(126MHz,MeOD)δ171.01,145.78,143.87,143.56,143.36,143.26,139.78,138.86,129.34,129.17,128.70,125.71,125.36,122.31,120.18,118.04,105.89,48.14,47.97,47.91,47.80,47.74,47.63,47.46,47.29,47.12,40.00,36.29,35.73,32.83,25.16,22.53,20.65.HR–MS(ESI)m/z 571.1756([M+H]+,C26H31ClF3N4O3S+,calculated571.1752).
Example 3:
(S)-3-(aminomethyl)-5-methyl-N-((4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)sulfo nyl)hexanamide hydrochloride( Synthesis of celecoxib-pregabalin hydrochloride, compound 3);
Intermediate (3A) is synthesized by the reaction of pregabalin and allyl chloroformate, and further the intermediate (3B) is generated by the reaction of pregabalin and celecoxib, and finally the target product celecoxib-pregabalin hydrochloride (Cel-Pre, compound 3) is obtained under the condition of bis (triphenylphosphine) palladium dichloride.
The first step: synthesis of (S) -3- (((allyloxy) carbonyl) amino) methyl) -5-methylhexanoic acid (3A)
In a 100mL single port flask, 4mL of tetrahydrofuran, 16g (100 mmol,1.0 eq) of pregabalin, 16mL (180 mmol,1.8 eq) of saturated sodium bicarbonate solution, cooling to 10℃and dropwise adding 18g (150 mmol,1.5 eq) of allyl chloroformate. After stirring at room temperature for 4 hours, TLC monitored complete pregabalin reaction. 50mL of water was added, the mixture was extracted once with 20mL of dichloromethane, the pH of the aqueous phase was adjusted to pH=1-2 with concentrated hydrochloric acid, the aqueous phase was extracted 3 times with 50mL of dichloromethane, dried over anhydrous sodium sulfate, and spun-dried at 35℃to give product 3A as a colorless oil with a yield of 4.6g 18.81％.1H NMR(500MHz,Chloroform-d)δ5.94(ddt,J＝16.4,10.9,5.7Hz,1H),5.37–5.29(m,1H),5.27–5.20(m,1H),5.11–4.95(m,1H),4.68–4.56(m,2H),3.32(dt,J＝14.1,5.5Hz,1H),3.14(dt,J＝13.9,6.9Hz,1H),2.34(qd,J＝15.1,6.1Hz,2H),2.25–2.08(m,1H),1.69(dt,J＝13.4,6.7Hz,1H),1.40–1.23(m,1H),1.20(tt,J＝12.2,6.1Hz,2H),0.92(dd,J＝10.0,6.6Hz,6H).HR–MS(ESI)m/z244.1546([M+H]+,C12H22NO4 +,calculated 244.1543).
And a second step of: all l
(S)-(4-methyl-2-(2-oxo-2-((4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)sulfonamido)ethyl)pentyl)carbamate(3B) Is synthesized by (1);
In a 100mL single port flask, 3.09g (13 mmol,1.0 eq) of 3A,4.85g (13 mmol,1.0 eq) of celecoxib, 3.66g (19 mmol,1.5 eq) of EDCI,0.15mg (1.3 mmol,0.1 eq) of DMAP are added. After stirring at room temperature for 2 hours, TLC monitored 3A reaction was complete. Washing with 10mL of water for 3 times, drying with anhydrous sodium sulfate, adding silica gel, stirring, performing column chromatography, eluting with petroleum ether and ethyl acetate=1:1 to obtain colorless oily product with product 3B of 1.06g, and yield of 1.06g 13.75％.1HNMR(500MHz,Chloroform-d)δ10.79(s,1H),8.12–8.06(m,2H),7.50–7.44(m,2H),7.17(d,J＝7.9Hz,2H),7.10(d,J＝8.1Hz,2H),6.73(s,1H),5.92(ddt,J＝17.2,10.4,5.7Hz,1H),5.33(dq,J＝17.2,1.6Hz,1H),5.25(dq,J＝10.2,1.2Hz,1H),4.98(t,J＝6.8Hz,1H),4.62(dt,J＝5.8,1.4Hz,2H),3.21(ddd,J＝14.9,7.2,3.1Hz,1H),3.03(dt,J＝14.8,6.5Hz,1H),2.38(s,3H),2.19–2.07(m,2H),1.98–1.86(m,1H),1.57(dt,J＝13.4,6.7Hz,2H),1.07(tt,J＝13.8,6.6Hz,2H),0.85(dd,J＝6.6,1.0Hz,6H).HR–MS(ESI)m/z 605.2046([M+H]+,C29H32F3N4O5S+,calculated 605.2040).
And a third step of:
(S)-3-(aminomethyl)-5-methyl-N-((4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)sulfo nyl)hexanamide hydrochloride( Synthesis of celecoxib-pregabalin hydrochloride, compound 3);
In a 4mL reaction flask, 2mL of methylene chloride, 400mg (660. Mu. Mol,1.0 eq) of 3B,160mg (28. Mu. Mol,0.3 eq) of bis (triphenylphosphine) palladium dichloride, 800mg (2.75. Mu. Mol,4.1 eq) of tri-n-butyltin hydride were added. After stirring at room temperature for 2 hours, TLC monitored about half of the reaction. Spin-drying at 35deg.C, column chromatography, eluting with dichloromethane (methanol=100:1), and spin-evaporating to obtain colorless oily compound 3 (0.10 g) with yield 29.02％.1H NMR(500MHz,Chloroform-d)δ8.41(s,1H),7.93(dq,J＝8.7,2.2Hz,2H),7.54–7.47(m,1H),7.47–7.42(m,1H),7.23–7.11(m,4H),6.76(d,J＝9.6Hz,1H),2.41(s,1H),2.40(s,2H),1.59(dd,J＝13.5,6.7Hz,2H),1.40–1.30(m,4H),0.97–0.91(m,5H),0.88(tt,J＝11.3,6.5Hz,5H).HR–MS(ESI)m/z 559.1755([M+H]+,C25H31ClF3N4O3S+,calculated 559.1752).
B. Pharmacological examples;
Example 4: evaluation of analgesic effects of celecoxib, gamma-aminobutyric acid and gabapentin in a carrageenan-induced acute inflammatory pain model;
Carrageenan-induced inflammatory pain experiments are an animal model widely used to evaluate acute inflammatory pain. Carrageenan is a sulfated polysaccharide extracted from red seaweed and used in animals to cause local inflammatory reactions such as tissue swelling congestion and hyperalgesia.
Each set of experiments used 6 mice, including 3 male mice and 3 female mice. The baseline mechanical foot-reduction threshold (MECHANICAL WITHDRAWAL thresholds, MWTs, expressed in g) was measured for each mouse prior to carrageenan or saline injection. Mechanical allodynia was assessed using a ZH-ZKL mechanical acupuncture pain apparatus. Briefly, mice were acclimatized in an elevated mesh floor under a clear plastic room (21×27×15 cm) without restraint. After 30 minutes, a progressive force was applied to the left hind paw of the mouse. The device automatically records the mice MWTs when they retract into the hind paw. Each test was repeated three times (test interval: 30 seconds). After 3 hours of carrageenan or saline injection, administration was performed by gavage or saline. MWTs were tested at 0, 30, 60, 90, 120 and 180 minutes post-dose. The maximum possible effect percentage (PERCENTAGE OFMAXIMUM POSSIBLE EFFECT,% MPE) is calculated from the following formula: % mpe= (MWT 1-MWT2)/(MWT3-MWT2) ×100%, where MWT 1 refers to the maximum threshold after drug treatment, MWT 2 refers to the threshold after modeling, and MWT 3 refers to the threshold of normal mice before modeling. The extent and duration of analgesia were estimated by the area under the curve (area under the curve, AUC). AUC describing the change of mechanical foot-reduction threshold over time was calculated by using GRAPHPAD PRISM 9.0.0 software by means of the approximate trapezoidal rule.
Experimental dose grouping settings are as in table 1:
Table 1: experimental grouping of celecoxib, gamma-aminobutyric acid and gabapentin in carrageenan-induced acute inflammatory pain models
Experimental results: the mechanical foot contraction threshold was significantly lower than baseline levels 3 hours after carrageenan injection into the sole of the mice, indicating successful induction of inflammatory pain. Celecoxib (2.28, 15.00, 25.00 mg/kg), gamma aminobutyric acid (0.30, 0.06,1.20 mg/kg) and gabapentin (25.00, 50.00, 75.00, 100.00 mg/kg) both reduced carrageenan-induced inflammatory pain in a dose-dependent manner. Analgesic inhibition rates of celecoxib at 2.28, 15.00 and 25.00mg/kg in inflammatory pain are 12.93%,48.42%,64.51% respectively, and half-dose ED 50 is 14.98±25.72mg/kg; analgesic inhibition rates of 0.30,0.60 and 1.20mg/kg gamma-aminobutyric acid in inflammatory pain are 28.48%,39.12% and 73.02% respectively, and half of the effective dose ED 50 is 0.68+ -0.56 mg/kg;25, 50, 75, 100mg/kg gabapentin has an analgesic inhibition rate of 16.15%,47.20%,67.86%,80.76% respectively in inflammatory pain, and half of the effective dose ED 50 is 52.80+ -1.10 mg/kg. The results are shown in detail in FIG. 1.
Example 5: evaluation of analgesic effects of celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in carrageenan-induced acute inflammatory pain model;
carrageenan-induced inflammatory pain model experimental procedure was the same as in example 1.
Experimental dose grouping settings are as in table 2:
Table 2: experimental grouping of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and the contracture drug celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in carrageenan-induced acute inflammatory pain models
Experimental results: the mechanical foot contraction threshold was significantly lower than baseline levels 3 hours after carrageenan injection into the sole of the mice, indicating successful induction of inflammatory pain. Contracture drug compound 1 (1.00,3.00, 10.00 mg/kg) and contracture drug compound 2 (5.00, 10.00, 20.00 mg/kg) exhibited significant dose-dependent analgesic effects in the inflammatory pain model. In carrageenan-induced acute inflammatory pain: the analgesic inhibition rate of the compound 1 from low to high doses 1.00,3.00, 10.00mg/kg is 47.03%,62.99%,73.13% respectively, and half-effective dose ED 50 is 1.19+ -0.31 mg/kg; the analgesic inhibition rate of compound 2 was 32.76%,41.20%,63.39 and the half-dose ED 50 was 12.23±6.64mg/kg at three doses, 3.00, 10.00, 30.00mg/kg, respectively, from low to high. In this model, the ED 50 value of the contracture drug is lower than the ED 50 value of celecoxib and gabapentin single drug. The results are shown in detail in FIG. 2.
Example 6: in carrageenan-induced inflammatory pain models, the contracture drugs celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) were tested in drug efficacy comparison with equimolar celecoxib, gamma-aminobutyric acid or gabapentin single and combinations;
carrageenan-induced inflammatory pain model experimental procedure was the same as in example 1.
Experimental dose grouping settings are as in table 3:
Table 3: equimolar dose efficacy vs. experimental grouping in carrageenan-induced acute inflammatory pain model
Experimental results: to further evaluate the pharmacodynamic advantage of contracture drug compound 1 and contracture drug compound 2, this example compared the differences in analgesic effects of compound 1 (3.00 mg/kg) with equimolar celecoxib (2.28 mg/kg), gamma-aminobutyric acid (0.62 mg/kg) alone and in combination (celecoxib 2.28mg/kg + gamma-aminobutyric acid 0.62 mg/kg), and of compound 2 (10 mg/kg) with equimolar celecoxib (7.13 mg/kg), gabapentin (3.20 mg/kg) alone and in combination (celecoxib 7.13mg/kg + gabapentin 3.20 mg/kg) in a carrageenan-induced inflammatory pain model. The eight groups of regimens all varied in the extent of treatment of inflammatory pain. In the drug effect comparison experiment of the contracture drug celecoxib-gamma-aminobutyrate hydrochloride group: the analgesic activity of the contracture medicine is highest, and the inhibition rate is 62.99%; the inhibition rates of the combined use and the single gamma-aminobutyric acid and celecoxib are 44.81%, 39.12% and 12.93% respectively. In the efficacy comparison experiment of celecoxib-gabapentin group: the analgesic activity of the same contracture medicine is highest, and the inhibition rate is 41.20%; the inhibition rate of the combination is 29.74 percent; celecoxib and gabapentin show little analgesic activity at this dose. Thus, the analgesic activity of the contracture drug compound 1 and the compound 2 is superior to that of the combination therapy and the single drug therapy at the equimolar dose. The results are shown in detail in FIG. 3.
Example 7: evaluation of analgesic effects of celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in a complete freund adjuvant-induced chronic inflammatory pain model;
The complete Freund's adjuvant induced pain model is a common model of chronic inflammatory pain. The complete Freund's adjuvant was injected subcutaneously into the experimental animals and showed a mechanical pain-sensitive and thermal pain-sensitive behavioral response lasting 1-2 weeks.
Each set of experiments used 6 mice, including 3 male mice and 3 female mice. The baseline MWT of each mouse was measured prior to injection of complete freund's adjuvant or physiological saline. Mechanical allodynia was assessed using a ZH-ZKL mechanical acupuncture pain apparatus. Briefly, mice were acclimatized in an elevated mesh floor under a clear plastic room (21×27×15 cm) without restraint. After 30 minutes, a progressive force was applied to the left hind paw of the mouse. The device automatically records the mice MWTs when they retract into the hind paw. Each test was repeated three times (test interval: 30 seconds). After 3 hours of complete Freund's adjuvant or physiological saline injection, the administration was performed by gastric lavage or physiological saline. MWTs were tested at 0, 30, 60, 90, 120 and 180 minutes post-dose. % MPE is calculated by the following formula: % mpe= (MWT 1-MWT2)/(MWT3-MWT2) ×100%, where MWT 1 refers to the maximum threshold after drug treatment, MWT 2 refers to the threshold after modeling, and MWT 3 refers to the threshold of normal mice before modeling. The extent and duration of analgesia was estimated by AUC values. AUC describing the change of mechanical foot-reduction threshold over time was calculated by using GRAPHPAD PRISM 9.0.0 software by means of the approximate trapezoidal rule.
Experimental dose grouping settings are as in table 4:
table 4: experimental grouping of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and the contracture drug celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in a complete Freund adjuvant-induced chronic inflammatory pain model
Experimental results: after 24 hours, the mechanical foot-shrink threshold was significantly reduced in model mice compared to baseline values, indicating successful establishment of complete freund adjuvant-induced chronic inflammatory pain. Dose-dependent reversal of contracture drug compound 1 (1.00,3.00, 10.00 mg/kg) was complete with freund adjuvant-induced mechanical allodynia and analgesic efficacy peaked at the 90 minute time point post-administration. Contracture drug compound 2 (5.00, 10.00, 20.00 mg/kg) likewise reversed mechanical ectopic pain in a dose-dependent manner, with analgesic efficacy peaking 90 minutes after administration. Half-effective doses ED 50 of Compound 1 and Compound 2 were obtained by dose-response curves of 2.51+ -0.14 mg/kg and 13.31+ -0.73 mg/kg, respectively, and analgesic inhibition rates of the two contractants were 31.74%,54.30%,75.05% and 17.97%,37.38%,66.62%, respectively. The results are shown in detail in FIG. 4.
Example 8: evaluation of analgesic effects of celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in iodoacetic acid-induced osteoarthritis pain model
The iodate-induced osteoarthritis pain model is a classical drug evaluation system. The mice were anesthetized by intravenous administration of etomidate emulsion (2 mg/kg) and then 20 μl of iodoacetic acid (10 mg/mL in physiological saline) was injected into the joint space using a 30 gauge needle. Shortly after the injection of iodoacetic acid, the inflammation induces a change in the histopathological structure of the knee joint, causing the osteoarthritis pain to change from inflammation-related acute pain to spontaneous and induced chronic pain.
Each set of experiments used 6 mice, including 3 male mice and3 female mice. The baseline MWT of each mouse was measured prior to injection of iodoacetic acid or physiological saline. Mechanical allodynia was assessed using a ZH-ZKL mechanical acupuncture pain apparatus. Briefly, mice were acclimatized in an elevated mesh floor under a clear plastic room (21×27×15 cm) without restraint. After 30 minutes, a progressive force was applied to the left hind paw of the mouse. The device automatically records the mice MWTs when they retract into the hind paw. Each test was repeated three times (test interval: 30 seconds). After 24 hours of injection of iodoacetic acid or physiological saline, the administration by gastric lavage or physiological saline is performed. MWTs were tested at 0, 30, 60, 90, 120 and 180 minutes post-dose. % MPE is calculated by the following formula: % mpe= (MWT 1-MWT2)/(MWT3-MWT2) ×100%, where MWT 1 refers to the maximum threshold after drug treatment, MWT 2 refers to the threshold after modeling, and MWT 3 refers to the threshold of normal mice before modeling. The extent and duration of analgesia was estimated by AUC values. AUC describing the change of mechanical foot-reduction threshold over time was calculated by using GRAPHPAD PRISM 9.0.0 software by means of the approximate trapezoidal rule.
Experimental dose grouping settings are as in table 5:
Table 5: experimental grouping of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and the contracture drug celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in an iodoacetic acid-induced osteoarthritis pain model
Experimental results: after 24 hours of injection of 20 μl of 1% iodoacetic acid into the knee joint of the mice, the mechanical foot shrinkage threshold was significantly reduced, suggesting successful establishment of the osteoarthritis pain model. Contracture drug compound 1 (3, 10, 30 mg/kg) and compound 2 (5, 10, 20 mg/kg) exhibited significant dose-dependent analgesic effects in the osteoarthritis pain model, with the result being expressed as AUC. In iodoacetic acid-induced osteoarthritis pain: the analgesic inhibition rate of the compound 1 from low to high doses of 3.00, 10.00 and 30.00mg/kg is 25.73 percent, 43.00 percent and 63.57 percent respectively, and the half effective dose ED 50 is 14.48+/-1.26 mg/kg; the analgesic inhibition rates of the compound 2 from low to high doses of 5.00, 10.00 and 20.00mg/kg are 18.21%,46.26% and 62.02% respectively, and the half-effective dose ED 50 is 12.86+/-4.85 mg/kg. The results are shown in detail in FIG. 5.
Example 9: evaluation of analgesic effects of celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in formalin-induced inflammatory pain model;
Formalin experiments are a common model in the current basic research of pain. Biphasic behavioral withdrawal was induced following formalin injection: the first phase occurs 10 minutes before injection, directly activating the pain receptors; the second phase occurs 10-45 minutes after injection, and is painful due to persistent transmission of nociceptive information due to local chronic inflammation.
Each set of experiments used 6 mice, including 3 male mice and 3 female mice. Pain was assessed using the ZH-PAN801 automated pain analysis system. On the day of the experiment, a small metal ring was placed on the left hind paw of the mouse. Mice were acclimatized in plexiglas containers for at least 30 minutes prior to testing. After 30 minutes of gastric lavage or physiological saline, 1% formalin solution was subcutaneously injected into the left hind paw of the mouse, and then the mouse was immediately placed in an automatic pain analyzer, and the number of foot lifts per minute was recorded within 45 minutes. Phase I was defined as 0-10min after formalin injection, and phase II was defined as 10-45 min. % MPE is calculated by the following formula: % mpe= [ (number of foot lifts in control group-number of foot lifts in drug group)/number of foot lifts in control group ] ×100%.
Experimental dose grouping settings are as in table 6:
Table 6: experimental grouping of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) and the contracture drug celecoxib-gabapentin hydrochloride (Cel-GBP, compound 2) in formalin-induced inflammatory pain models
Experimental results: both contracture drug compound 1 and compound 2 reduced formalin phase II nociceptive behavior, but had no effect on phase I response. In formalin-induced second phase pain: compound 1 was found to have an analgesic inhibition rate of 30.25%,56.89%,67.29% for the low to high doses 1.00,3.00, 10.00mg/kg, respectively, and a half-dose effective ED 50 of 1.19±2.68mg/kg; the analgesic inhibition rate of compound 2 was 15.28%,41.56%,60.28% and half-effective dose ED 50 was 13.69+ -0.22 mg/kg at three doses of 3.00, 10.00, 20.00mg/kg, respectively, from low to high. The results are shown in detail in FIG. 6.
Example 10: evaluation of analgesic effects of celecoxib, pregabalin and the contracture drug celecoxib-pregabalin hydrochloride (Cel-Pre, compound 3) in paclitaxel-induced neuropathic pain model;
paclitaxel is capable of inducing a peripheral neuropathic pain state characterized by allodynia and hyperalgesia caused by noxious or innocuous stimuli.
The baseline mechanical foot-reduction threshold (MWT) was measured for each mouse prior to paclitaxel or saline injection. Mechanical allodynia was assessed using a ZH-ZKL mechanical acupuncture pain apparatus. Briefly, mice were acclimatized in an elevated mesh floor under a clear plastic room (21×27×15 cm) without restraint. After 30 minutes, a progressive force was applied to the left hind paw of the mouse. The device automatically records the mice MWTs when they retract into the hind paw. Each test was repeated three times (test interval: 30 seconds). A paclitaxel stock solution (6.00 mg/mL) was prepared using a mixed solution of polyoxyethylated castor oil (50%) and absolute ethanol (50%), and then diluted 30-fold to 0.20mg/mL with physiological saline. Mice were intraperitoneally injected with paclitaxel 0.20mg/kg (1 time per day for 5 consecutive days) to induce peripheral neuropathic pain. On the sixth day, administration by gastric lavage or physiological saline. MWTs were tested at 0, 30, 60, 90 and 120 minutes post-administration. % MPE is calculated by the following formula: % mpe= (MWT 1-MWT2)/(MWT3-MWT2) ×100%, where MWT 1 refers to the maximum threshold after drug treatment, MWT 2 refers to the threshold after modeling, and MWT 3 refers to the threshold of normal mice before modeling. The extent and duration of analgesia was estimated by AUC values. AUC describing the change of mechanical foot-reduction threshold over time was calculated by using GRAPHPAD PRISM 9.0.0 software by means of the approximate trapezoidal rule.
Experimental dose grouping settings are as follows in table 7:
table 7: experimental grouping of celecoxib, pregabalin and the contracture drug celecoxib-pregabalin hydrochloride (Cel-Pre, compound 3) in paclitaxel-induced neuropathic pain models
Experimental results: on day six, the mechanical foot-shrinking threshold was significantly below baseline levels, indicating successful induction of neuropathic pain. Celecoxib (10.00, 20.00, 30.00 mg/kg), pregabalin (6.00, 12.00, 24.00 mg/kg) and contracture drug compound 3 (3.00,6.00, 12.00 mg/kg) each reduced paclitaxel-induced neuropathic pain in a dose-dependent manner. Analgesic inhibition rates of celecoxib of 10.00, 20.00 and 30.00mg/kg in neuropathic pain are 39.62 percent, 58.20 percent and 83.84 percent respectively, and half-effective dose ED 50 is 17.08+/-0.83 mg/kg; the analgesic inhibition rate of pregabalin in neuropathic pain is 42.40%,65.60%,70.10% respectively, and half-effective dose ED 50 is 7.52+ -4.08 mg/kg;3.00,6.00, 12.00mg/kg of Compound 3 had an analgesic inhibition in neuropathic pain of 36.99%,52.31%,65.66% respectively, and half-dose ED 50 was 4.27+ -1.72 mg/kg. The median effective dose ED 50 value of contracture drug compound 3 was far lower than ED 50 value of celecoxib and pregabalin single drug in neuropathic pain, indicating that contracture drug has analgesic activity higher than both single drugs. The results are shown in detail in FIG. 7.
Example 11: evaluation of analgesic effect of celecoxib and pregabalin combination in paclitaxel-induced neuropathic pain model;
the experimental procedure for paclitaxel-induced neuropathic pain model was the same as in example 9.
Table 8: experimental grouping of celecoxib and pregabalin combinations in paclitaxel-induced neuropathic pain models
Experimental results:
After paclitaxel-induced neuropathic pain of mice is successfully established, the mechanical foot-shrinking threshold of mice in the model group is obviously reduced. Co-administration of celecoxib and pregabalin with ED 50 fixed dose components (1/2, 1/4, 1/8) significantly improved paclitaxel-induced neuropathic pain, with analgesic efficacy exhibiting dose-dependency. The analgesic inhibition rates of 1/2ED 50 celecoxib+1/2 ED 50 pregabalin, 1/4ED 50 celecoxib+1/4 ED 50 pregabalin, 1/8ED 50 celecoxib+1/8 ED 50 pregabalin are 66.47%, 54.03% and 43.00% respectively, the actual ED 50mix is 4.69+ -0.28 mg/kg, and the theoretical ED 50add is 12.44+ -0.66 mg/kg. The actual ED 50mix of the celecoxib and pregabalin drug combination was below the predicted additivity line and the interaction index (gamma) was 0.38, indicating that the drug combination exhibited significant synergistic analgesic effects in paclitaxel-induced neuropathic pain. The results are shown in detail in FIG. 8.
Example 12: in a paclitaxel-induced neuropathic pain model, a drug effect comparison experiment of a contracture drug celecoxib-pregabalin hydrochloride (Cel-Pre, compound 3) and an equimolar celecoxib and pregabalin single drug and combined drug effect is carried out;
the experimental procedure for paclitaxel-induced neuropathic pain model was the same as in example 9.
Experimental dose grouping settings are as follows in table 9:
table 9: equimolar dose efficacy vs. experimental grouping in paclitaxel-induced neuropathic pain models
Experimental results: to further evaluate the pharmacodynamic advantage of the contracture drug celecoxib-pregabalin hydrochloride, this example compares the difference in analgesic effect of compound 3 (12.00 mg/kg) versus equimolar celecoxib (2.28 mg/kg), pregabalin (0.62 mg/kg) alone and in combination (celecoxib 2.28mg/kg + pregabalin 0.62 mg/kg) in a paclitaxel-induced neuropathic pain model. The four groups of regimens all had different degrees of treatment for neuropathic pain. In the comparative experiment, the compound 3 has the highest analgesic activity, and the combination use is inferior, and the single drug effect is poor. Thus, the curative effect of the contracture drug compound 3 is better than that of the combined use and single drug treatment at the equimolar dose. The results are shown in detail in FIG. 9.
Example 13: evaluation of anti-inflammatory effect of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) in a carrageenan-induced acute inflammatory pain model;
Carrageenan can cause biphasic inflammatory responses characterized by mouse toe edema. It has been reported that significant increases in cyclooxygenase 2 and prostaglandin protein expression are observed in patients with inflammation, indicating that treating inflammation is a viable method for ameliorating inflammatory pain.
Each set of experiments used 6 mice, including 3 male mice and 3 female mice. Toe volume was measured for each mouse using a ZH-LUO-G7 toe swelling meter prior to carrageenan or saline injection. Briefly, the toe of the mouse was immersed in a beaker on the instrument itself (the beaker was filled with pure water), and the device automatically recorded the toe volume of the mouse. Each test was repeated three times (test interval: 30 seconds). After 3 hours of carrageenan or saline injection, administration was performed by gavage or saline. Toe volumes of mice were measured at 0, 30, 60, 90, 120 and 180 minutes post-dose.
Experimental dose grouping settings are as in table 10:
table 10: experimental grouping of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) in carrageenan-induced inflammation model
Experimental results: carrageenan-induced models can cause inflammatory pain as well as inflammation and edema, so the invention compares the anti-inflammatory effects of the contracture drugs, single drugs and combinations by measuring the toe swell volume of mice in this model. Before the start of the experiment, edema was caused by injecting 20 μl of 1% carrageenan solution into the plantar tissue of the mice. The peak edema occurred within 3-4 hours after injection, so the toe volume of the mice was measured again after 3 hours using the ZH-LUO-G7 toe swelling meter. The toe swelling volume was increased in each group compared to the blank group. Contracture drug compound 1 (3 mg/kg) and its equimolar amount in combination and monotherapy reduced the extent of carrageenan-induced toe swelling. Notably, the swelling inhibition rates of the contracture drug compound 1, the combination, celecoxib single drug and gamma-aminobutyric acid single drug were 60.54%,64.90%,55.17% and 52.38%, respectively, suggesting that contracture drug compound 1 has comparable anti-inflammatory activity to the combination in the carrageenan model. The results are shown in detail in FIG. 10.
Example 14: evaluation of anti-inflammatory effect of the contracture drug celecoxib-gamma-aminobutyric acid hydrochloride (Cel-GABA, compound 1) in a model of chronic inflammatory pain induced by complete freund's adjuvant;
Complete Freund's adjuvant is a commonly used inflammatory agent that can induce local inflammatory responses in experimental animals.
Each set of experiments used 6 mice, including 3 male mice and 3 female mice. Toe volumes of each mouse were measured using a ZH-LUO-G7 toe swelling meter prior to injection of complete freund's adjuvant or physiological saline. Briefly, the toe of the mouse was immersed in an instrument-equipped beaker (filled with pure water), and the device automatically recorded the toe swell volume of the mouse. Each test was repeated three times (test interval: 30 seconds). After 24 hours from injection of complete Freund's adjuvant or physiological saline, the administration was performed by gastric lavage or physiological saline. Toe volumes of mice were measured at 0, 30, 60, 90, 120 and 180 minutes post-dose.
Experimental dose grouping settings are as in table 11:
Table 11: experimental grouping of the contracture drug celecoxib-gamma-aminobutyrate in carrageenan-induced inflammation models
Experimental results: to further verify whether contracture drugs have an improved anti-inflammatory effect, the present invention also conducted toe swelling experiments in another inflammation model. The toe swelling of the mice is induced by injecting the complete Freund adjuvant, the anti-inflammatory effect of the contracture drug compound 1 (3.00 mg/kg) is optimal, the swelling inhibition rate is 48.33%, the swelling inhibition rate of the combined use is inferior to 40.50%, the swelling inhibition rates of the celecoxib single drug and the gamma-aminobutyric acid single drug are 17.46% and 14.81%, respectively, which indicates that the anti-inflammatory effect of the contracture drug compound 1 in the complete Freund adjuvant model is superior to that of the combined use and single drug treatment at equimolar doses. The results are shown in detail in FIG. 11.
The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (9)
1. A contracture drug with synergistic analgesic effect, which is characterized in that: the contracture medicine is formed by connecting a medicine A and a medicine B through chemical bonds, wherein the medicine A is celecoxib, and the medicine B is gamma-aminobutyric acid (GABA), gabapentin or pregabalin, and the structure is as follows:
2. A method for preparing a contracture drug with synergistic analgesic effect as claimed in claim 1, characterized in that: the method comprises the following specific steps:
Step 1: protecting amino groups in a gamma-aminobutyric acid, gabapentin or pregabalin structure by di-tert-butyl dicarbonate, allyl chloroformate, trimethylsilyl ethoxycarbonyl chloride or 9-fluorenylmethyl chloroformate to obtain an intermediate I;
step 2: carrying out condensation reaction on celecoxib and the intermediate I obtained by the reaction in the step 1 in the presence of EDCI/DMAP/triethylamine to obtain an intermediate II;
step 3: removing protecting groups from the intermediate II obtained in the step 2 in ethyl acetate hydrochloride gas, tetra (triphenylphosphine) palladium/tri-n-butyltin hydride, tetraethylammonium fluoride, tetramethylammonium fluoride, triethylamine or pyridine to obtain the contracture drug as described in claim 1.
3. The method for preparing a contracture drug with synergistic analgesic effect as claimed in claim 2, characterized in that: the molar ratio of celecoxib to intermediate I in step 2 is 1:1-2.
4. The method for preparing a contracture drug with synergistic analgesic effect as claimed in claim 2, characterized in that: the molar ratio of the intermediate I in the step 2 to EDCI, DMAP and triethylamine is sequentially 2-3: 0.1 to 0.5:2 to 5.
5. The method for preparing a contracture drug with synergistic analgesic effect as claimed in claim 2, characterized in that: the reaction of step 2 is carried out in a solvent which is tetrahydrofuran, dichloromethane or N, N-diformylcarboxamide.
6. A pharmaceutical composition characterized by: a pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
7. Use of a contracture drug according to claim 1 or a pharmaceutical composition according to claim 6 for the preparation of a medicament for the prevention or treatment of pain or neurological diseases.
8. The use according to claim 7, characterized in that: the pain is acute pain, chronic pain, neuropathic pain, inflammatory pain, nociceptive pain, cancerous pain, hyperalgesia and visceral pain.
9. The use according to claim 7, characterized in that: the nervous system diseases are pain, anxiety, depression, parkinson's disease, alzheimer's disease and schizophrenia.
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| Publication Number | Publication Date |
|---|---|
| CN118307474A true CN118307474A (en) | 2024-07-09 |
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