HK1172026B - Etomidate analogues that do not inhibit adrenocortical steroid synthesis - Google Patents

Description

Etomidate analogues that do not inhibit adrenocortical steroid synthesis

Cross Reference to Related Applications

Priority of U.S. provisional application No.61/224,751 at 35u.s.c. § 119(e), filed 7, 10, 2009, the content of which is incorporated herein by reference in its entirety.

Government support

The invention was made with government support under grant number P01-58448 by the national institutes of health. The united states government has certain rights in this invention.

Technical Field

The present invention relates to etomidate analogues with improved pharmacokinetic and pharmacodynamic properties and their use as anaesthetics.

Background

In the united states, sepsis has an incidence of approximately 750,000 cases per year, mortality of 30-50%, and annual costs of 170 billion dollars (Hotchkiss, r.s. and i.e. karl, the pathophysiology and treatment of sepsis, N Engl J Med, 2003, 348 (2): p 138-50). In severe cases, sepsis is often associated with extreme reductions in blood pressure, massive vasodilation, shock, and multiple organ failure. Patients with sepsis often require general anesthesia for important interventions (e.g., intubation and surgery). Unfortunately, all general anesthetics produce potentially life-threatening serious side effects, especially in critically ill patients with sepsis. The most feared is that almost all anesthetics produce cardiovascular suppression.

Etomidate is a highly potent IV anesthetic distinct from other general anesthetics due to its ability to maintain cardiovascular stability. Etomidate at a concentration of 2. mu.M induces the disappearance of the tadpole righting reflex (Husain, S.S. et al, 2- (3-Methyl-3H-diaziren-3-y1) ethyl1- (1-phenylethyl) -1H-imidazole-5-carboxylate: a derivative of the stereoselective genetic animal assay for a photonic ligand and gateway channels, J Med Chem, 2003, 46 (7): p1257-65) and the disappearance of human reactivity (artifact, J.R., F.O.Holly and D.R.Stanski, derived sensitivity reactivity in the polypeptide and ligand degradation: repair distribution, reaction distribution, 3627, 19). There is compelling evidence at the molecular level: etomidate through modulation of GABA_AReceptor function produces an anesthetic effect (Jurd et al, Faseeb J (2003), 17 (2): 250-2 and Rusch et al, J Biol Chem (2004), 279 (20): 20982-92). Etomidate low concentration GABA-induced GABA_AThe receptor-mediated current increased but had little effect on the current induced by high concentrations of GABA. This shifts the GABA concentration-response curve to the left,reducing the EC50 of GABA. This receptor mechanism is also believed to be responsible for the anesthetic action of propofol.

Where etomidate acts on GABA_AThe point at which the recipient exerts an anesthetic effect is being increasingly understood. The research and identification of the photoaffinity label_ATwo amino acids in the receptor that contribute to the etomidate binding site: met-236 on the alpha subunit and Met-286 on the beta subunit (Li et al, J Neurosci, (2006), 26 (45): 11599-605). Ray acetylcholine receptor (Torpedo acetylcholine receptor) structure-based GABA_AModeling of receptor structural homology strongly suggests that these two amino acids contribute to the anesthetic binding pocket at the interface between the alpha and beta subunits. This conclusion is supported by mutagenesis studies that show that mutating these amino acids to tryptophan decreases the sensitivity of the receptor to etomidate (Stewart et al, Mol Pharmacol (2008), 74 (6): 1687-95).

Inhibition of steroid synthesis is a potentially fatal side effect of etomidate administration, particularly for critically ill patients (e.g., patients with sepsis) who are most likely to benefit from the use of etomidate. This inhibition is extremely potent and occurs at doses less than etomidate which produces general anesthesia. It is also extremely dangerous because it significantly increases the mortality rate of critically ill patients receiving continuous etomidate infusion. For example, Watt, i, and i.m. leidingham, Anaesthesia (1984), 39 (10): 973-81 found retrospectively: patients with critical trauma are often in need of vasopressors (p < 0.0001); even after matching age, gender and wound severity scores, with benzodiazepineSedation with etomidate had approximately 3-fold higher mortality rates (77% vs 28%; p < 0.0005) compared to sedation. Etomidate, due to its action on steroid synthesis, cannot be safely administered to critically ill patients in long-term continuous infusion and even given as a single administration for the purpose of inducing anesthetic effects in septic patientsIntravenous bolus injections, the fear of which has also recently increased. It has been proposed that morbidity and mortality may be reduced by empirically administering exogenous steroids (Ray, d.c. and d.w.mckeown, CritCare (2007), 11 (3): R56); however, this approach is suboptimal, as the dose, timing and duration of steroid therapy for any given patient will be extrapolated. In addition, administration of exogenous steroids can itself produce serious complications (especially in the case of sepsis), including altered glucose homeostasis, impaired wound healing, and immunosuppression. It has been suggested that these complications explain, at least in part, the results of the CORTICUS study, which suggests that: even in critically ill patients thought to suffer from adrenocortical insufficiency, exogenous steroids do not improve survival, although they reduce the need for vasopressors (squirng et al, N Engl J Med (2008), 358 (2): 111-24).

Etomidate inhibits the synthesis of adrenocortical steroids primarily by binding to and inhibiting 11 β -hydroxylase (i.e., CYP11B1), a cytochrome P450 enzyme essential for the biosynthesis of cortisol, corticosterone, and aldosterone (de Jong et al, J Clin endocrinolometab (1984), 59 (6): 1143-7). The half maximal inhibitory concentration of etomidate (IC50) is in the low nanomolar range (Lamberts et al, J Pharmacol Exp The (1987), 240 (1): 259-64 and Roumen et al, J Comut Aided Mol Des (2007), 21 (8): 455-71), which is a range of concentrations several orders of magnitude below its hypnotic/anesthetic concentration.

Previous crystallographic studies of imidazole-containing drugs (e.g., ketoconazole) with various cytochrome P450 enzymes have shown that high affinity binding of the two requires a strong attractive interaction ("complexation") between the basic nitrogen in the imidazole ring of the drug and the heme iron at the enzyme catalytic site (Zhao et al, J Biol Chem (2006), 281 (9): 5973-81; Podust et al, Proc Natl Acad Sci USA (2001), 98 (6): 3068-73; and Verras et al, Protein Eng Des Sel (2006), 19 (11): 491-6); cytochrome p450 enzymes (including 11 β -hydroxylase) contain a heme prosthetic group at their catalytic site. Although it is not limited toNo 11 β -hydroxylase has been crystallized nor its interaction with etomidate is clear, but homology modeling studies indicate that high affinity binding of etomidate to 11 β -hydroxylase also requires coordination between the basic nitrogen in the imidazole ring of etomidate and the heme iron of the enzyme. This yields the following predictions: by replacing the basic nitrogen with other chemical groups that do not coordinate to heme iron, etomidate (without destroying potent anesthetic and GABA activities) can be designed that does not bind 11 β -hydroxylase with high affinity (and thus no anti-adrenergic activity)_AIn the case of receptor activity). This would be highly desirable as it retains potent anesthetic activity and GABA_AReceptor modulating activity without inhibiting adrenocortical function at clinically relevant doses.

There is a high demand for safer general anesthetics for critically ill patients, especially for patients with sepsis. If (R) -etomidate is not a potent inhibitor of adrenocortical function, it has many properties that make it an ideal general anesthetic (e.g., high anesthetic potency, less effect on cardiovascular function than other anesthetics, and higher therapeutic index).

Therefore, there is a need in the art to develop analogues of (R) -etomidate that retain many of the beneficial properties of (R) -etomidate (e.g., fast onset of action, little effect on blood pressure, high therapeutic index) but do not cause potentially dangerous inhibition of adrenocortical function. Such an analogue would allow the anesthetic effect to be more safely administered to critically ill patients. The present invention addresses the above-mentioned needs.

Disclosure of Invention

The present invention is directed to compounds of formula (I):

wherein the content of the first and second substances,

R₁is L₁C (O) OT or L₁C(O)OL₂C(O)OT；

R₂Is substituted or unsubstituted C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl or C₂-C₁₀Alkynyl, or is R₁；

n is an integer of 0 to 5;

R₃each independently is halogen or R₂；

R₄And R₅Independently hydrogen, halogen, CN or CF₃；

L₁And L₂Each independently is a bond, substituted or unsubstituted C₁-C₁₀Alkylene radical, C₂-C₁₀Alkenylene or C₂-C₁₀Alkynylene, wherein the backbone of the alkylene may contain one or more heteroatoms; and is

T is H, substituted or unsubstituted C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl or C₂-C₁₀Alkynyl, nitrophenol or cyclopropyl, wherein the backbone of the alkyl group may comprise one or more heteroatoms.

The compounds of formula (I) include pharmaceutically acceptable salts thereof, stereoisomeric mixtures thereof, and enantiomers thereof.

The compounds of formula (I) have improved pharmacokinetic and pharmacodynamic properties compared to (R) -etomidate, such that the anesthetic properties are equal or improved while reducing undesired side effects. The compounds of formula (I) are etomidate analogues that retain the favorable anesthetic properties of (R) -etomidate, but do not cause clinically significant inhibition of adrenocortical function.

Another aspect of the invention is directed to an anesthetic pharmaceutical composition comprising an effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier.

Yet another aspect of the present invention is directed to a method of providing an anesthetic effect in a mammal, or a method comprising administering to said mammal an effective anesthetic compound or pharmaceutical composition of formula (I).

Another aspect of the invention is the use of a compound of formula (I) as substantially described herein, as, or in the manufacture of, a formulation for providing an anaesthetic effect in a subject in need thereof.

Drawings

Figure 1 shows the structures of etomidate (3- (1-phenylethyl) imidazole-4-carboxylic acid ethyl ester), carboetomidate (3- (1-phenylethyl) pyrrole-2-carboxylic acid ethyl ester), MOC-etomidate, MOC-carboetomidate, and carboetomidate analogs.

Fig. 2A and 2B show carbon etomidate concentration-response curves for tadpole reversal reflection disappearance (LORR). In fig. 2A, each data point represents the results for a single tadpole. In fig. 2B, each data point represents an average of 5 tadpoles. Using the Waud DR, J Pharmacol ExpTher, (1972), 183 (3): 577 the method in 607 fits the data set to a curve. A total of 40 tadpoles were used to define this concentration-response curve.

Fig. 3 shows an electrophysiological curve indicating: human alpha expressed in Xenopus oocytes by 10. mu.M carboetomidate₁β₂γ_2LGABA_AReceptor (FIG. 3A) and etomidate insensitive alpha₁β₂M286Wγ_2LGABA_AThe current mediated by the mutant (FIG. 3B) was increased. These results indicate that in GABA_ACarboetomidate binds to the same site on the receptor as etomidate. The first and last curves are controls (i.e. no anesthetic). The middle curve shows the effect of increasing carboetomidate. All currents in FIG. 3A were in the same cellInitiating; similarly, all of the currents in fig. 3B were also induced in the same cells.

Figure 4 shows a further example of the modulation of human gamma-aminobutyric acid (GABA) type a receptor function by carboetomidate. Figure 4A is a graph showing that the current mediated by the wild-type receptor reversibly increases under the action of 10 μ M carboetomidate when triggered by γ -aminobutyric acid in EC 5-10. Figure 4B is a graph showing the minimal increase in current mediated by etomidate-insensitive mutant receptors under the action of 10 μ M carboetomidate when triggered by γ -aminobutyric acid in EC 5-10.

FIG. 5 is a graph of the concentration-response curve of an anesthetic agent showing the inhibitory effect of cortisol synthesis by H295R adrenocortical cells. It is noted that carboetomidate is at least 3 orders of magnitude less effective than etomidate in inhibiting cortisol production.

Figure 6A is a graph showing the anesthetic dose-response curve for rat LORR after bolus iv administration. Each data point was from a single rat.

Fig. 6B is a graph showing the relationship between the dose of the anesthetic and the time required for the rat to self-right after the administration of the anesthetic. Each data point was from a single rat.

Figure 7A shows a graph of mean blood pressure in rats as a function of time following administration of equal anesthetic doses of propofol, etomidate and carboetomidate, demonstrating that the inhibitory effect (depress) of carboetomidate on blood pressure is significantly less than that of propofol and etomidate. Each point represents the mean blood pressure over a period of 30 s. Error bars are standard deviation. All anesthetics were administered at a dose that was twice their ED50 for LORR. In the propofol group and etomidate group, n ═ 3 rats. In the carboetomidate group, n ═ 4 rats.

Figure 7B shows the effect of 14mg/kg carboetomidate (n ═ 7), 2mg/kg etomidate (n ═ 6), and Dimethylsulfoxide (DMSO) only adjuvant (n ═ 4) on mean blood pressure in rats. Hypnotics were administered at a dose twice their respective ED50 that turned the normal reflex off in DMSO vehicle. All rats received the same amount of DMSO vehicle (350. mu.L/kg). At time 0, DMSO vehicle alone or hypnotic in DMSO vehicle was injected. Each data point represents the mean (± SD) of the mean blood pressure changes over each 30s time period. P < 0.05 compared to DMSO adjuvant alone.

Figure 8 shows that the serum concentration of corticosterone (adrenocorticosteriod) was relatively unchanged compared to vehicle (control) 15min after carboetomidate administration, but significantly decreased after the same anesthetic dose of etomidate. In these rats, ACTH was administered 15min after the administration of anesthetic or adjuvant_1-24Corticosterone production was stimulated and plasma corticosterone concentrations were then measured after 15 min. Etomidate and carboetomidate were at 2-fold higher doses than their respective ED50 for LORR. Each group had 4 rats. Error bars are standard deviation.

Detailed Description

The present invention relates to safer (R) -etomidate analogs that retain the beneficial characteristics of (R) -etomidate (e.g., potent anesthetic action, fast onset of anesthetic action, little effect on blood pressure), but have a substantially reduced effect on adrenocortical steroid synthesis.

The compounds described herein are understood to be etomidate analogues (R-enantiomer or S-enantiomer) wherein the basic nitrogen has been substituted by a CH group. Without wishing to be bound by theory, substitution of the basic nitrogen with a CH group reduces the binding affinity of these compounds for 11 β -hydroxylase. The compounds of the present invention may be further augmented with one or more additional metabolically labile ester groups, either directly or through various linking groups (e.g., -CH)₂CH₂-) are attached to various positions of the core molecule. At the end of the ester group, there may be a "tail" group (e.g., -CH)₃). Various embodiments of the invention are discussed below.

The present invention is directed to compounds of formula (I):

R₁is L₁C (O) OT or L₁C(O)OL₂C (O) OT. In a preferred embodiment, R₁Is L₁C(O)OT。

R₂Is substituted or unsubstituted C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl or C₂-C₁₀Alkynyl, or is R₁. Preferably, R₂Is alkyl (e.g. CH)₃) Or is an ester R₁(such as CH)₂CH₂C(O)OCH₃). In a most preferred embodiment, R₂Is CH₃Or CH₂CH₃。

R₃Each independently of the other being halogen, halogen radioisotope or R₂. Preferred halogens include fluorine and chlorine. The variable n is an integer from 0 to 5. In a preferred embodiment, n ranges from 0 to 3, and is most preferably 0.

R₄And R₅Independently H, halogen, CN or CF₃. Preferably, R₄And R₅Are not halogen, CN or CF₃Or R is₄And R₅At least one of is halogen, CN or CF₃. More preferably, R₄And R₅Is Br or CN.

Linking group L₁And L₂Each independently is a bond, substituted or unsubstituted C₁-C₁₀Alkylene radical, C₂-C₁₀Alkenylene or C₂-C₁₀Alkynylene radical. The backbone of the alkylene group may contain one or more heteroatoms, such as O, N or S. Preferably, L₁And L₂Each independently is a bond or a straight chain C₁-C₄An alkylene group. Most preferably, L₁Is a bond or CH₂CH₂And L is₂Is CH₂CH₂、CH₂(CH₂)₄CH₂Or CH₂CH₂O(CH₂)₃. In the most preferred embodiment, L₂Is CH₂CH₂。

The tail T can be H, substituted or unsubstituted C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl or C₂-C₁₀Alkynyl. The alkyl backbone may contain one or more heteroatoms, such as O, N or S. The tail may also be cyclopropyl, nitrophenol, or any other suitable electron withdrawing group. Preferably, T is C₁-C₄An alkyl group. Most preferably, T is CH₃、CH₂CH₃、CH₂CH₂CH₂CH₃、CH₂CH(OH)CH₃Or CH₂CH₂OCH₃. In a most preferred embodiment, T is CH₃. In another most preferred embodiment, T is nitrophenol. In another most preferred embodiment, T is H. In yet another preferred embodiment, T is CH₂CH(OH)CH₃。

The compounds of formula (I) include pharmaceutically acceptable salts thereof, stereoisomeric mixtures thereof, and enantiomers thereof. The compounds of the present invention also include physiologically acceptable salts of the compounds of formula (I). Preferred physiologically acceptable salts are the acid addition salts known to those skilled in the art. Common physiologically acceptable acid addition salts include, but are not limited to, hydrochloride, oxalate and tartrate salts.

In certain embodiments of the compound, R₁Is L₁C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, and T is CH₂CH₃。

In other embodiments of the compounds, R₁Is L₁C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, and T is CH₃。

In still other embodiments of the compounds, R₁Is L₁C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, and T is H.

In a preferred embodiment of said compound, R₁Is L₁C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, R₄Is H, R₅Is H, and T is CH₂CH₃。

In another preferred embodiment of said compound, R₁Is L₁C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, R₄Is H, R₅Is Br or CN, and T is CH₂CH₃。

In another preferred embodiment of said compound, R₁Is L₁C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, R₄Is Br or CN, R₅Is H, and T is CH₂CH₃。

In another preferred embodiment of said compound, R₁Is L₁C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, R₄Is H, R₅Is H, and T is CH₂CH(OH)CH₃。

In another preferred embodiment of said compound, R₁Is L₁C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, R₄Is H, R₅Is H, and T is H.

In still other embodiments of the compounds, R₁Is L₁C(O)OT，R₂Is CH₂CH₃N is 0, L₁Is a bond, and T is CH₃。

In other embodiments of the compounds, R₁Is L₁C(O)OT，R₂Is CH₂CH₃N is 0, L₁Is a bond, and T is CH₂CH₃。

In certain embodiments of the compound, R₁Is L₁C(O)OT，R₂Is CH₃N is 1-5, R₃Each independently is halogen, L₁Is a bond, and T is CH₂CH₃。

In other embodiments of the compounds, R₁Is L₁C(O)OT，R₂Is CH₃N is 1-5, R₃Each being fluorine, L₁Is a bond, and T is CH₂CH₃。

In still other embodiments of the compounds, R₁Is L₁C(O)OT，R₂Is CH₂CH₃N is 1, R₃Is fluorine, L₁Is a bond, and T is CH₂CH₃。

In another preferred embodiment of said compound, R₁Is L₁C(O)OL₂C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, L₂Is CH₂CH₂And T is CH₃。

In certain embodiments of the compound, R₁Is L₁C(O)OL₂C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, L₂Is CH₂(CH₂)₄CH₂And T is CH₂CH₂CH₂CH₃。

In other embodiments of the compounds, R₁Is L₁C(O)OL₂C(O)OT，R₂Is CH₃N is 0, L₁Is a bond, L₂Is CH₂CH₂O(CH₂)₃And T is CH₂CH₂OCH₃。

In certain embodiments of the compound, R₁Is L₁C(O)OL₂C(O)OT，R₂Is CH₃，R₃Each independently of the other is halogen, n is 1-5, L₁Is a bond, L₂Is CH₂CH₂And T is CH₃。

In other embodiments of the compounds, R₁Is L₁C(O)OL₂C(O)OT，R₂Is CH₃，R₃Each independently of the other is halogen, n is 1-5, L₁Is a bond, L₂Is CH₂(CH₂)₄CH₂And T is CH₂CH₂CH₂CH₃。

In still other embodiments of the compounds, R₁Is L₁C(O)OL₂C(O)OT，R₂Is CH₃，R₃Each independently of the other being halogen n is 1-5, L₁Is a bond, L₂Is CH₂CH₂O(CH₂)₃And T is CH₂CH₂OCH₃。

In further embodiments of the compounds, R₁Is L₁C(O)OT，R₂Is CH₃At least one R₃Is CH₂CH₂C(O)OCH₃，L₁Is a bond, and T is CH₂CH₃。

In still a further embodiment of the compounds, R₁Is L₁C(O)OL₂C(O)OT，R₂Is CH₃At least one R₃Is CH₂CH₂C(O)OCH₃，L₁Is a bond, L₂Is CH₂CH₂And T is CH₃。

In a preferred embodiment of said compound, R₁Is L₁C(O)OT，R₂Is CH₂CH₂C(O)OCH₃N is 0, L₁Is a bond, and T is CH₂CH₃。

In the process of formationIn another preferred embodiment of the compounds, R₁Is L₁C(O)OT，R₂Is CH₃N is 0, L₁Is CH₂CH₂And T is CH₂CH₃。

In certain embodiments, the compound of formula (I) is selected from the group consisting of:

pharmaceutically acceptable salts of the above compounds, stereoisomeric mixtures of the above compounds, and enantiomers of the above compounds.

The carbon atom bridging the six-and five-membered rings is the chiral center. Thus, the compounds may be in the form of pure enantiomers. In a preferred embodiment, the enantiomer is the R enantiomer. In another embodiment, the enantiomer is the S enantiomer.

The compounds of formula (I) preferably have the same stereochemistry as (R) -etomidate. R₂、R₃、L₁、L₂And T may be a branched hydrocarbon chain, but not to the extent of steric hindrance or conjugation that interferes with the desired activity.

In certain embodiments, the compound comprises two or more ester groups. Suitable ester-containing groups (e.g., linker-ester-tail or ester-tail) can be added to the bridging carbon, or to the benzene ring or various positions of the core molecule.

Also preferred are fast metabolizing etomidate analogs with an ester group on carboetomidate that are free of steric hindrance and/or are electrically independent from pi-electron systems (pi electron systems) in imidazole and benzene rings. Such ester groups, like the ester groups in other drugs with very short duration of action (e.g., remifentanil and esmolol), are believed to be highly sensitive to hydrolysis by esterases. See U.S. patent nos. 3,354,173; U.S. patent nos. 5,466,700; U.S. Pat. No.5,019,583 and U.S. patent publication No. US 2003/0055023.

R₂、T、L₁And L₂The substituents may each independently be substituted with one or more electron withdrawing groups. In certain embodiments, the electron withdrawing group is a halogen, nitrophenol, or cyclopropyl. Other electron withdrawing groups such as hydroxyl, amino, nitro, nitrile, sulfonate, carboxylate, halogen, thiol, and unsaturated hydrocarbon groups may also be used. The presence of the electron-withdrawing group serves to increase the partial positive charge on the ester carbonyl atom, thereby increasing the susceptibility of the esterase to nucleophilic attack and thereby further enhancing the rapid hydrolysis by the esterase.

The compounds of the present invention have shown anesthetic activity and enhanced GABA_AReceptor activity. The compounds of the present invention consistently exhibit potent in vitro and in vivo anesthetic effects and enhanced GABA_AThe effect of the receptor. These results indicate that the compounds of the present invention are highly active agents with potent in vitro and in vivo activity. Importantly, the compounds have reduced inhibitory activity associated with adrenal corticosteroid synthesis in vitro and in vivo and/or short duration of anesthetic action.

The above compounds can be administered alone in a mixture with another of the above compounds or in combination with an acceptable pharmaceutical carrier. The invention therefore also relates to pharmaceutical compositions comprising an effective amount of at least one compound according to the invention, with or without a pharmaceutically or physiologically acceptable carrier. The compounds may, where appropriate, be administered in the form of physiologically acceptable salts, for example acid addition salts.

The invention also includes methods of treating animals or humans. The method comprises administering to an animal or human an effective amount of at least one compound of the invention or a physiologically acceptable salt thereof, with or without a pharmaceutically acceptable carrier. Intravenous administration is preferred. See U.S. patent No.4,289,783, which is incorporated herein by reference in its entirety.

The present invention is a potent sedative hypnotic that does not significantly inhibit adrenocortical function and can be used to produce and/or maintain anesthesia, sedation, or lower central nervous system excitability. It exhibits one or more of the following advantageous properties compared to the alternative agents: higher potency, shorter duration of therapeutic effect, shorter duration of side effects, reduced adrenocortical inhibition, higher therapeutic index, lower toxicity, reduced cardiovascular inhibition and titration to achieve the desired effect are more convenient. The present invention may be administered as a single bolus intravenous injection or as a continuous intravenous infusion. Other routes of delivery may include oral, rectal, transmucosal, subcutaneous, or inhalation.

Pharmaceutical composition

For administration to a subject, the compounds described herein can be provided in a pharmaceutically acceptable composition. Accordingly, another aspect of the present invention is directed to a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier. These pharmaceutically acceptable compositions comprise an effective amount of one or more compounds described herein formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specifically formulated for administration in solid or liquid form, including in administration forms suitable for: (1) oral administration, e.g. drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g. buccal tablets, sublingual tablets and systemically absorbed tablets), boluses (boluses), powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., as a sterile solution or suspension or sustained release formulation, by subcutaneous injection, intramuscular injection, intravenous injection (e.g., bolus injection or infusion) or epidural injection; (3) topical application, for example as a cream (cream), ointment (ointment) or controlled release patch or dermal spray; (4) intravaginal or intrarectal administration, such as pessaries, creams or foams; (5) sublingual administration; (6) ophthalmic administration; (7) transdermal administration; (8) transmucosal administration; or (9) nasal administration.

In certain embodiments, the compounds of the present invention may be used in the form of pharmaceutically acceptable salts. Suitable acids capable of forming salts with the compounds of the present invention include: inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid, trifluoroacetic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Suitable bases capable of forming salts with the compounds of the present invention include: inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as monoalkyl, dialkyl and trialkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine, pyridine, methyl pyridine, dicyclohexylamine, N' -dibenzyl ethylenediamine, etc.) and optionally substituted ethanolamines (e.g., ethanolamine and diethanolamine, triethanolamine, etc.).

The term "pharmaceutically acceptable" or "pharmacologically acceptable" as used herein refers to compounds, materials, compositions and/or dosage forms which meet the following criteria: within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Furthermore, for administration to animals (e.g., humans), it will be understood that the compositions should meet sterility, pyrogenicity, general safety and purity Standards as required by the FDA Office of Biological Standards of biologics Standards.

The term "pharmaceutically acceptable carrier" as used hereinBy "body" is meant a pharmaceutically acceptable material, composition or excipient, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in the transport or transport of a test compound from one organ or portion of an organism to another organ or portion of an organism. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth (powdered tragacanth); (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes (suppository waxes); (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) a pH buffer solution; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents (bulking agents), such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) c₂-C₁₂Alcohols, such as ethanol; and (23) other non-toxic compatible substances for pharmaceutical formulations. Wetting agents, coloring agents, releasing agents, coating agents, disintegrating agents, binding agents, sweetening agents, flavoring agents, perfuming agents, protease inhibitors, plasticizers, emulsifying agents, stabilizing agents, viscosity increasing agents, film forming agents, solubilizing agents, surfactants, preservatives and antioxidants can also be present in the formulation. Terms such as "excipient", "carrier" or "Pharmaceutically acceptable carriers "and the like are used interchangeably herein.

For liquid formulations, the pharmaceutically acceptable carrier may be an aqueous or non-aqueous solution, suspension, emulsion or oil. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and cod liver oil. The solution or suspension may also comprise the following components: sterile diluents such as water for injection, saline solution, fixed oils (fixed oils), polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide).

Liposomes and non-aqueous vehicles (e.g., fixed oils) can also be used. The use of such media and agents as pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use of such media or agent in the compositions is contemplated. Auxiliary active compounds may also be incorporated into the composition.

As noted above, the composition may further comprise binders (e.g., acacia, corn starch, gelatin, carbomer, ethylcellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone), disintegrants (e.g., corn starch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers having various pH and ionic strengths (e.g., tris-HCl, acetate, phosphate), additives to prevent surface adsorption (such as albumin or gelatin), detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), glidants (e.g., colloidal silicon dioxide), antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents (e.g., peppermint, methyl salicylate, or citrus flavor), preservatives (e.g., thimerosal, benzyl alcohol, p-hydroxybenzoic acid), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymeric coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethylcellulose, acrylates, polymethacrylates), and/or adjuvants.

It is particularly advantageous to formulate oral and intravenous compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit is calculated to contain a predetermined amount of active compound to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are directly dependent on and determined by: the unique characteristics of the active compound; the specific therapeutic effect to be achieved; and limitations inherent in the art of compounding active compounds for the treatment of individuals. The pharmaceutical composition may be contained in a container, pack or dispenser together with instructions for administration. Pharmaceutical compositions typically contain the active ingredient in an amount of at least 0.01% by weight (i.e., the weight of the compound of the invention per unit weight of the total pharmaceutical composition). wt% is the weight ratio of active ingredient to total composition. Thus, for example, 0.1 wt% means that 0.1g of the compound is contained per 100g of the total composition.

The manner in which pharmaceutical compositions containing active ingredients are prepared, for example by mixing, granulating, or tableting processes, is well known in the art. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the active agent is mixed with additives customary for this purpose, such as adjuvants, stabilizers or inactive diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, and also aqueous, alcoholic or oily solutions and the like (as described above).

For intravenous administration, glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with appropriate buffering capacity in the pH range acceptable for intravenous administration can be used as a buffering agent. A sodium chloride solution whose pH has been adjusted to a desired range with an acid or a base (e.g., hydrochloric acid or sodium hydroxide) may also be used. Typically, the pH of the intravenous formulation may range from about 5 to about 12.

Subcutaneous formulations comprising suitable buffering agents and isotonic agents (isotonicity agents) may be prepared in accordance with procedures well known in the art at a pH in the range of about 5 to about 12. The formulation may be formulated to deliver a daily dose of the active agent in one or more daily subcutaneous administrations. One of ordinary skill in the art will readily select an appropriate formulation pH and buffer depending on the solubility of the compound or compounds to be administered. Sodium chloride solutions that have been adjusted to a desired range of pH with an acid or base (e.g., hydrochloric acid or sodium hydroxide) may also be used in the subcutaneous formulation. Typically, the pH of the subcutaneous formulation may range from about 5 to about 12.

Method of the invention

Another aspect of the present invention is directed to a method of providing an anesthetic effect in a subject, the method comprising administering to the subject a pharmaceutical composition substantially identical to that described above. Thus, in certain embodiments, the method comprises administering an effective dose of the compound. The effective dose comprises 0.01-100mg/kg of the compound. The term "effective dose" or "effective amount" as used herein means an amount sufficient to elicit the desired pharmacological effect. The actual effective amount will, of course, vary with the particular compound, the technique of administration, the effect desired, the duration of the effect, and the side effects, and can readily be determined by one skilled in the art. Thus, an effective dose of a compound described herein is an amount sufficient to induce and maintain general anesthesia or conscious sedation (conscoussedation) in a subject.

Data from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

The effective dose can be estimated initially from cell culture assays. The dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 determined in cell culture (i.e., the concentration of the therapeutic agent that achieves half-maximal inhibition of symptoms). Levels in plasma can be determined by, for example, high performance liquid chromatography. The effect of any particular dose can be monitored by suitable biological analysis.

Typically, administration in such compositions results in administration of the compounds of the invention in the following dosage ranges: 1. mu.g/kg to 150mg/kg, 1. mu.g/kg to 100mg/kg, 1. mu.g/kg to 50mg/kg, 1. mu.g/kg to 20mg/kg, 1. mu.g/kg to 10mg/kg, 1. mu.g/kg to 1mg/kg, 100. mu.g/kg to 100mg/kg, 100. mu.g/kg to 50mg/kg, 100. mu.g/kg to 20mg/kg, 100 μ g/kg to 10mg/kg, 100 μ g/kg to 1mg/kg, 1mg/kg to 100mg/kg, 1mg/kg to 50mg/kg, 1mg/kg to 20mg/kg, 1mg/kg to 10mg/kg, 10mg/kg to 100mg/kg, 10mg/kg to 50mg/kg, or 10mg/kg to 20 mg/kg. It is to be understood that the ranges given herein include all intermediate ranges, for example, the range of 1mg/kg to 10mg/kg includes 1mg/kg to 2mg/kg, 1mg/kg to 3mg/kg, 1mg/kg to 4mg/kg, 1mg/kg to 5mg/kg, 1mg/kg to 6mg/kg, 1mg/kg to 7mg/kg, 1mg/kg to 8mg/kg, 1mg/kg to 9mg/kg, 2mg/kg to 10mg/kg, 3mg/kg to 10mg/kg, 4mg/kg to 10mg/kg, 5mg/kg to 10mg/kg, 6mg/kg to 10mg/kg, 7mg/kg to 10mg/kg, 8mg/kg to 10mg/kg, and 9mg/kg to 10mg/kg, and the like. It will be further understood that ranges within the ranges given above are also within the scope of the invention, e.g., within the range of 1mg/kg to 10mg/kg, dosage ranges such as 2mg/kg to 8mg/kg, 3mg/kg to 7mg/kg, and 4mg/kg to 6mg/kg, etc.

The term "administering" and/or "administering" a compound shall be understood to mean providing a compound or composition of the invention to a subject in need of induction of an anesthetic effect. Thus, the term "administering" refers to placing a compound or composition of the invention into a subject by a method or route such that the compound or composition is at least partially localized at a desired site, thereby inducing and/or maintaining general anesthesia or conscious sedation in the subject.

The compounds described herein can be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.

Exemplary modes of administration include, but are not limited to: injection, infusion, instillation, inhalation, or ingestion. "injection" includes, but is not limited to: intravenous injection and infusion, intramuscular injection and infusion, arterial injection and infusion, intrathecal injection and infusion, intraventricular injection and infusion, intravesicular injection and infusion, intraocular injection and infusion, intracardiac injection and infusion, intradermal injection and infusion, intraperitoneal injection and infusion, transtracheal injection and infusion, subcutaneous injection and infusion, subcuticular injection and infusion, intraarticular injection and infusion, subcapsular injection and infusion, subarachnoid injection and infusion, intraspinal injection and infusion, intracerebrospinal injection and infusion, and intrasternal injection and infusion. In some embodiments, the composition is administered by intravenous infusion or injection.

In preferred embodiments, the method comprises administering a single effective dose of an injection of the compound, followed by continuous or discontinuous infusion of the compound.

In certain embodiments, the method comprises administering an effective dose of a compound of formula (I) by continuous infusion.

The compounds described herein may be administered to a subject in conjunction with additional pharmaceutically active agents or treatment modalities (therapeutic modalities) for specific indications. Exemplary pharmaceutically active compounds include, but are not limited to, the compounds described in: harrison's Principles of InternalMedicine, 13 th edition, authors T.R.Harrison et al, McGraw-Hill N.Y., NY; physicians Desk Reference, 50 th edition, 1997, Oradell New Jersey, medical Economics Co.; pharmacological Basis of Therapeutics, 8 th edition, Goodmanand Gilman, 1990; united States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990; the current edition of Goodman and Oilman's the pharmacological Basis of Therapeutics; and The current version of The Merck Index; the contents of all of these materials are incorporated by reference herein in their entirety.

Thus, in certain embodiments, the method further comprises administering to the subject an effective amount of a therapeutic agent selected from the group consisting of an analgesic, a paralytic agent (paralytic agent), and another sedative hypnotic agent. Non-limiting examples of sedative hypnotic agents include benzodiazepinesClasses, barbiturates, ketamine, propofol, isoflurane and desflurane. Non-limiting examples of analgesics include non-steroidal anti-inflammatory drugs (NSAIDs), paracetamol/acetaminophen, COX-2 inhibitors, and opioids (opioids). Non-limiting examples of paralytic agents include rocuronium bromide (rapacuuronium), micracuronium chloride (mivacurium), succinylcholine, vecuronium bromide (vecuronium), and cisacammonium atracurium (cisat)racurium)。

The term "subject" as used herein means a mammal, such as a human or an animal. Typically the animal is a vertebrate such as a primate, rodent, domestic animal or hunting animal. Primates include chimpanzees, cynomologous monkeys (cynomologous monkeys), spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), avian species (e.g., chickens, emus, ostriches), and fish species (e.g., trout, catfish, and salmon). Patients or subjects include any subset of the foregoing, e.g., one or more groups of species (e.g., human, primate, or rodent) are excluded from all of the foregoing subjects. In certain embodiments, the subject is a mammal, such as a primate (e.g., a human). The terms "patient" and "subject" are used interchangeably herein. In certain embodiments, the subject is a mammal.

Synthesis of Compounds of the invention

The compounds of the present invention may be prepared by synthetic methods known to those skilled in the art, especially in conjunction with the prior art and the specific preparation examples provided below. Suitable modifications of the starting materials can also be made by methods well known in the art.

The compounds of the present invention may be prepared by a process comprising the steps of: coupling a phenyl group of formula (II) with a pyrrole of formula (III):

wherein the content of the first and second substances,

R₂is substituted or unsubstituted C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl or C₂-C₁₀Alkynyl, or is R₁；

n is an integer of 0 to 5;

R₃each independently is halogen or R₂；

Wherein the content of the first and second substances,

R₁is L₁C (O) OT or L₁C(O)OL₂C(O)OT；

R₄And R₅Independently H, halogen, CN or CF₃；

L₁And L₂Each independently is a bond, substituted or unsubstituted C₁-C₁₀Alkylene radical, C₂-C₁₀Alkenylene or C₂-C₁₀Alkynylene, wherein the backbone of the alkylene may contain one or more heteroatoms; and is

T is H, substituted or unsubstituted C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl radical, C₂-C₁₀Alkynyl, nitrophenol or cyclopropyl, wherein the backbone of the alkyl group may comprise one or more heteroatoms.

The reaction between the phenyl of formula (II) and the pyrrole of formula (III) is accompanied by a conformational inversion, resulting in the compound of formula (I). Phenyl groups of formula (II) are readily prepared by reduction of phenylalkylketones and derivatives thereof.

Definition of

Unless otherwise indicated, or otherwise evident from the context, the following terms and phrases include the meanings provided below. Unless otherwise expressly stated, or apparent from the context, the terms and phrases below do not exclude the meaning of those terms and phrases as they may have in the art to which they pertain. The definitions are provided to assist in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is defined only by the claims. Further, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

As used herein, the terms "comprises" or "comprising" are used to indicate that the essential compositions, methods, and individual components thereof, of the present invention, still openly contain the unspecified elements, regardless of whether such elements are essential.

The singular terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprising" means "including". The abbreviation "e.g. (e.g.)" is derived from latin e.g. (exempli gratia) and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g. (e.g.)" is synonymous with the term "e.g. (for example)".

The terms "reduce," "reduced," "reducing," "decrease," or "inhibit" as used herein generally mean a statistically significant amount of reduction. However, for the avoidance of doubt, "reduced", "reduction" or "reduces" or "inhibits" means a reduction by at least 10% compared to a reference level, such as a reduction by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% reduction (e.g., a level in the absence compared to a reference sample), or any value between 10-100% reduction compared to a reference level.

The terms "increased", "increase" or "enhancement" or "activation" as used herein generally mean an increase in a statistically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhancing" or "activating" mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including an increase of 100%, or any value between 10-100% compared to a reference level; or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold, or any value between 2-fold and 10-fold or more, as compared to a reference level.

The term "statistically significant" or "significantly" means statistically significant, typically meaning outside of at least two standard deviations (2SD) of a reference level. The term refers to the statistical evidence of differences. It is defined as the possibility of making a decision to reject an invalid hypothesis when it is actually true.

The term "alkyl" as used herein refers to a saturated straight, branched or cyclic hydrocarbon group. Examples of alkyl groups include, but are not limited to: methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, cyclohexyl, n-octyl, n-decyl, n-dodecyl and n-hexadecyl groups.

The term "alkenyl" as used herein refers to a straight, branched, or cyclic unsaturated hydrocarbon group having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to: allyl, butenyl, hexenyl, and cyclohexenyl groups.

The term "alkynyl" as used herein refers to an unsaturated hydrocarbon group having at least one carbon-carbon triple bond. Representative alkynyl groups include, but are not limited to: ethynyl, 1-propynyl, 1-butynyl, isopentynyl, 1, 3-hexadiynyl, n-hexynyl, 3-pentynyl, 1-hexen-3-ynyl, and the like.

The term "halogen" as used herein refers to an atom selected from fluorine, chlorine, bromine and iodine. The term "halogen radioisotope" refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.

The term "substituted" as used herein refers to the independent substitution of one or more hydrogen atoms on the substituted moiety with substituents independently selected from, but not limited to: alkyl, alkenyl, heterocycloalkyl, alkoxy, aryloxy, hydroxy, amino, amido, alkylamino, arylamino, cyano, halogen, mercapto, nitro, carbonyl, acyl, aryl and heteroaryl groups.

The invention may be defined in any of the following numbered paragraphs:

1. a compound of formula (I):

and pharmaceutically acceptable salts thereof, stereoisomeric mixtures thereof, and enantiomers thereof;

wherein the content of the first and second substances,

R₁is L₁C (O) OT or L₁C(O)OL₂C(O)OT；

R₂Is substituted or unsubstituted C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl or C₂-C₁₀Alkynyl, or is R₁；

n is an integer of 0 to 5;

R₃each independently is halogen or R₂；

R₄And R₅Independently H, halogen, CN or CF₃；

L₁And L₂Each independently is a bond, substituted or unsubstituted C₁-C₁₀Alkylene radical, C₂-C₁₀Alkenylene or C₂-C₁₀Alkynylene, wherein the backbone of the alkylene may contain one or more heteroatoms;

t is H, substituted or unsubstituted C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl radical, C₂-C₁₀Alkynyl, nitrophenol or cyclopropyl, wherein the backbone of the alkyl group may comprise one or more heteroatoms.

2. The compound of paragraph 1 wherein the compound is present as a pure enantiomer.

3. The compound of paragraph 2 wherein the enantiomer is the R enantiomer.

4. The compound according to any of paragraphs 1-3, wherein R₁Is L₁C(O)OT。

5. The compound according to any of paragraphs 1-3, wherein R₁Is L₁C(O)OL₂C(O)OT。

6. The compound of any of paragraphs 1-5, wherein R₂Is selected from CH₃、CH₂CH₃And CH₂CH₂CH₃The group consisting of.

7. The compound of any of paragraphs 1-6 wherein T is selected from the group consisting of H, CH₃、CH₂CH₃、CH₂CH(OH)CH₃And CH₂CH₂CH₃The group consisting of.

8. The compound of any one of paragraphs 1-7, wherein n is 0 or 1.

9. The compound of any of paragraphs 1-8, wherein R₂Is CH₃N is 0, L₁Is a bond, T is H, CH₃、CH₂CH₃Or CH₂CH(OH)CH₃。

10. The compound of any of paragraphs 1-9, wherein R₄And R₅Both are H.

11. The compound of any of paragraphs 1-9, wherein R₄And R₅At least one of which is H and the other is Br or CN.

12. The compound of paragraph 11 wherein R₄Is H, R₅Is Br or CN.

13. The compound of paragraph 11 wherein R₄Is Br or CN, R₅Is H.

14. The compound of paragraph 1 wherein the compound of formula (I) is selected from the group consisting of:

pharmaceutically acceptable salts of the above compounds, stereoisomeric mixtures of the above compounds, and enantiomers of the above compounds.

15. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of any one of paragraphs 1-14 and a pharmaceutically acceptable carrier.

16. A method of providing anesthesia to a subject, said method comprising administering to said subject the pharmaceutical composition of paragraph 15.

17. A method of providing anesthesia to a subject, the method comprising administering to the subject a compound of formula (I) as described in paragraphs 1-14.

18. Use of a compound of any of paragraphs 1-14 for providing anesthesia to a subject.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the following claims.

Those skilled in the art will also readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The molecular complexes and the methods, processes, treatment modalities, molecules, specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Variations and other applications of the present invention will occur to those skilled in the art, which are encompassed within the spirit of the invention and are defined by the scope of the claims.

It will be readily apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention illustratively disclosed herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each of the examples herein, any of the terms "comprising," "consisting essentially of … …," and "consisting of … …" can be substituted by either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood by those skilled in the art that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Examples

The invention is further illustrated by the following examples, which should not be construed as limiting. These examples are illustrative only and are not intended to limit the claimed invention in any way.

Materials and methods

Animals: all animal studies were conducted according to the rules and regulations of the institutional animal care committee of massachusetts general hospital (boston, massachusetts). Xenopus laevis (Xenopus laevis) tadpoles and adult female Xenopus laevis frogs at the early acrobud stage were purchased from Xenopus1 (annorbor, MI). Tadpoles were bred in our laboratory, and frogs were bred at the center of the comparative medical animal management facility in massachusetts general hospital. Adult male Sprague-Dawley rats (300-. A lateral tail vein IV catheter (24gauge, 19mm) was placed under temporary (approximately 1-5min) sevoflurane or isoflurane anesthesia and used for blood draw and IV administration, wherein sevoflurane or isoflurane was delivered using a reagent-specific variable bypass evaporator with continuous gas monitoring. Animals were weighed immediately prior to IV catheter placement and allowed complete recovery from inhalation anesthetic exposure prior to study. In all studies, rats were placed on a heat-preservation table (Kent Scientific, Torrington, CT) as indicated in previous studies to maintain the rectal temperature of the rats between 36-38 ℃. See, for example, Cotton et al, Anesthesiology, (2009), 111: 240-249, the contents of which are incorporated herein by reference.

Disappearance of righting reflections

Tadpole: several groups containing 5 early premolar xenopus laevis tadpoles were placed in 100ml oxygenated water buffered with 2.5mM Tris HCl buffer (pH 7.4) and containing carboetomidate at a concentration of 1-40 μ M. Tadpoles were manually tipped with a fire-polished pipette (flame-polished pipette) every 5min until the response stabilized. If the tadpole can not be righted in 5s after turning to lie on the back, the tadpole righting reflection disappearance (LORR) can be judged. At the end of each study, tadpoles were returned to fresh water to ensure reversibility of hypnotic effect. The use of the Waud DR, J PharmacolExp Ther (1972), 183: 577-607, which is incorporated herein by reference in its entirety, the EC50 of the LORR is determined from the carboetomidate concentration dependence of the LORR.

Rat: rats were temporarily restrained (restrain) in a3 inch diameter, 9 inch long acrylic chamber with a posterior exit. A desired dose of carboetomidate (typically 40mg/ml) in dimethyl sulfoxide (DMSO) was injected via a lateral tail intravenous catheter, followed by a rinse with approximately 1ml of saline. After injection, the rats were removed from the restraint device and turned supine. If the rat is not able to right itself after dosing (all four paws are grounded), the rat can be judged to have LORR. The duration of LORR was determined on a stopwatch and was defined as the time from carboetomidate injection until the animal spontaneously righted itself. ED50 of LORR administered as a bolus was determined from the dose dependence of LORR using the method of wagd. The time of onset (onset time) of LORR was determined by injecting rats with 28mg/kg carboetomidate (in DMSO; 40mg/ml) or 4mg/kg etomidate (in DMSO; 5.7mg/ml) via a lateral tail vein catheter, followed by rinsing with approximately 1ml of physiological saline, respectively. Immediately after injection, the rats were removed from the restraint device and the turning of the rats to the supine position was repeated until they no longer spontaneously turned up. The onset time is defined as the time from injection until LORR occurs.

GABA_AReceptor electrophysiology

Adult female xenopus laevis frogs were anesthetized with 0.2% tricaine (ethyl meta-aminobenzoate) and hypothermic. Ovarian leaves were then excised from the small laparotomy incision and placed into an OR-2 solution (82mM NaCl, 2mM KCl, 2mM MgCl) containing collagenase 1A (1mg/ml)₂5mM HEPES, pH 7.5) for 3h, the oocytes were isolated from the connective tissue.

Injecting oocytes at phases 4 and 5 encoding human GABA_AReceptor alpha₁、β₂(or beta)₂M286W) and γ_2LMessenger RNAs for the subunits (total messenger RNA: about 40 ng; ratio of subunits: 1: 2). Coding for GABA using the mMESSAGE mMACHINE High Yield tagged RNA transcription kit (Ambion, Austin, TX)_AReceptor alpha₁、β₂(or beta)₂M286W) and γ_2LThe complementary DNA of the subunit transcribes this messenger RNA. Prior to electrophysiological experiments, the injected oocytes were placed in ND-96 buffer solution (96mM NaCl, 2mM KCl, 1mM CaCl) containing 50U/ml penicillin and 50. mu.g/ml streptomycin₂，0.8mM MgCl₂10mM HEPES, pH 7.5) at 17 ℃ for at least 18 h.

All electrophysiological recordings were done using a whole-cell two-electrode voltage clamp technique. The oocyte was placed in a 0.04ml recording chamber and pierced with a capillary glass electrode filled with 3M KCl and having an open end resistance of less than 5M Ω. The oocyte was then voltage clamped at-50 mV using a Gene-Clamp 500B amplifier (Axon Instruments, Union City, Calif.) and ND-96 buffer perfused at a rate of 4-6 ml/min. The buffer perfusion was controlled using a 6-channel valve controller (Warner Instruments, Hamden, CT) interfaced with a digitdata 1322A data acquisition system (Axon Instruments) and driven by a Dell personal computer (Round Rock, TX). The current response was recorded using a claudex 9.2 software (Axon Instruments) and processed by a claudft 9.2 software (Axon Instruments) with a Bessel (8 pole) low pass filter with a cut-off frequency of 50 Hz.

For each oocyte, the GABA concentration (EC) giving a maximum current response of 5-10% was determined by measuring the peak current response elicited by a range of concentrations of GABA (in ND-96 buffer) and comparing it to the maximum peak current response elicited by 1mM GABA_5-10GABA). Then, by first using EC_5-10GABA perfuses oocytes for 90s, and then the control peak induced current is measured to evaluate carboetomidate on EC_5-10Influence of GABA-induced current. After a 5min recovery period, oocytes were perfused with carboetomidate for 90s, followed by EC_5-10GABA and carboetomidate perfusion was performed for 90s, and re-measurements were performedThe peak-fixed induced current. After a 15min recovery period, the control experiment (i.e. carbonless etomidate) was repeated to test reversibility. After exposure to carboetomidate, a longer recovery period was used to facilitate removal of the drug. The peak current response in the presence of carboetomidate was then normalized using the average peak current response of the two control experiments. The synergy induced by carboetomidate was quantified by normalized current response in the presence and current response in the absence of carboetomidate.

Hemodynamics in rats

As in previous Cotton et al, Anesthesiology, (2009), 111: 240-249, the effect of hypnotics on rat hemodynamics is defined. The femoral catheter communicating with the scapula was pre-implanted by the supplier (Charles River Laboratories). The animals had completely recovered from the implantation procedure by the time of arrival. During the feeding period and between studies, catheters were maintained open with heparin (500U/ml) and hypertonic (25%) dextrose lock solution, withdrawn prior to each use, and replaced immediately after use.

On the day of the study, after weighing and placing the side tail vein IV catheter, the rats were restrained in an acrylic tubing with a posterior exit and allowed to habituate (occlusion) for approximately 10-20min prior to data collection. The signal from the pressure sensor (TruWave, edwards life sciences, Irvine, CA) was amplified with a custom amplifier (AD620 operational amplifier, JamecoElectronics, Belmont, CA) and digitized (1kHz) with a USB-6009 data acquisition panel (National Instruments, Austin, TX) without an additional filter. All data were collected and analyzed using LabView software (version 8.5 for Macintosh OS X; national instruments).

Data for blood pressure analysis were recorded for 5min immediately before administration of hypnotic and 15min after administration. Carboetomidate dissolved in DMSO (40mg/ml), etomidate dissolved in DMSO (5.7mg/ml), or controls containing DMSO vehicle alone were administered via tail vein catheter, followed by a rinse with approximately 1ml of saline.

Inhibition of cortisol synthesis in vitro

Cortisol synthesis in vitro was determined using the human adrenal cortex cell line H295R (NCI-H295R; ATCC CRL 2128). Make 10⁵An aliquot of cells/well was grown in 12-well plates containing 2ml growth Medium (Dulbecco's Modified Eagle Medium/F12 supplemented with 1% insulin, iron transporter, selenium and linoleic acid; 2.5% NuSerum; and penicillin/streptomycin). When the cells reached near confluence (typically 48-72h), the growth Medium was replaced with an assay Medium containing etomidate or carboetomidate (Dulbecco's Modified Eagle Medium/F12 supplemented with 0.1% insulin, iron transporter, selenium, and containing antibiotics and 20 μ M forskolin). After 48h, 1.2ml of assay medium was collected from each well, centrifuged to pellet any cells or debris, using a commercially available 96-well kit based on horseradish peroxidase conjugated cortisol in a competitive antibody binding assay (R)&D Systems, Minneapolis, MN, KGE008), the cortisol concentration in the supernatant was quantified by enzyme-linked immunosorbent assay.

Inhibition of rat adrenal cortex

Immediately after weighing and placement of the IV catheters, dexamethasone (0.2mg/kg IV; American Regent, Shirley, NY) was administered to each rat to inhibit the release of endogenous adrenocorticotropic hormone (ACTH), inhibit baseline corticosterone production, and inhibit variable stress response to restraint and handling. After 2h of dexamethasone treatment, blood was drawn (for baseline determination of serum corticosterone concentration) and dexamethasone (0.2mg/kg) was administered again, together with intravenous carboetomidate, etomidate, or DMSO adjuvant as a control. The concentrations of carboetomidate and etomidate in DMSO were 40mg/ml and 5.7mg/ml, respectively. ACTH was administered intravenously immediately after hypnotic or adjuvant administration_1-24(25. mu.g/kg; Sigma-Aldrich Chemical Co, St. Louis, Mo.) to stimulate corticosterone production. After 15min, a second blood sample was taken for determination of ACTH_1-24Stimulated serum corticosterone concentration. ACTH_1-24With 1The concentration of mg/ml was dissolved in deoxygenated water as a mother liquor, aliquoted and frozen (-20 ℃); fresh aliquots of the solution were thawed just before each use. Rats in all 3 groups (carboetomidate, etomidate and adjuvant groups) received the same volume of DMSO (350 μ L/kg).

According to previous Cotton et al, Anesthesiology, (2009) 111: 240-249, the concentration of corticosterone in serum was determined. Blood samples were allowed to clot at room temperature (10-60min) and subsequently centrifuged at 3,500g for 5 min. Serum was carefully expressed from the resulting surface fibrin clot using a clean pipette tip, followed by a second centrifugation at 3,500g for 5 min. After the second centrifugation, the resulting clot-free serum layer was transferred to a new vial for final high-speed centrifugation (16,000g, 5min) to pellet any contaminating red blood cells or particles. Serum was transferred to a clean vial and rapidly frozen (-20 ℃) until corticosterone assay. After thawing and heat inactivation of corticosterone-binding globulin (65 ℃, 20min), serum baseline corticosterone concentrations and ACTH were determined using enzyme-linked immunosorbent assay (Diagnostic Systems Laboratories, Webster, TX) and 96-well plate reader (molecular devices, Sunnyvale, Calif.)_1-24Stimulated corticosterone concentrations were quantified.

Statistical analysis

All data are expressed as mean ± SD. Statistical analysis and curve fitting (using linear OR non-linear least squares regression) were performed using Prism v4.0(graphpad software, inc., LaJolla, CA) OR Igor Pro 4.01(Wavemetrics, Lake Oswego, OR) from Macintosh. Unless otherwise indicated, P < 0.05 indicates statistical significance. For multiple comparisons of physiological data derived from rats, we performed either one-way ANOVA or two-way ANOVA followed by Bonferroni post-hoc tests (which relied on unpaired t-tests with Bonferroni correction).

Example 1: synthesis of (R) -1- (1-phenylethyl) -1H-pyrrole-2-carboxylic ester (carboetomidate)

To a stirred solution of ethyl 1H-pyrrole-2-carboxylate (140mg, 1.00mmol) and triphenylphosphine (340mg, 1.30mmol) in dry THF (3mL) at room temperature under an argon atmosphere was added dropwise a solution of (S) -1-phenylethanol (135mg, 1.10mmol) in dry THF (2 mL). A solution (2mL) of di-tert-butyl azodicarboxylate (304mg, 1.32mmol) in dry THF was then added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure. The residue was mixed with ether (5mL) and stirred for 2 h. The residue (Ph) was collected₃PO and hydrazono ester) and washed with diethyl ether (3 × 2 mL). The filtrate was evaporated under reduced pressure to give a residue which was purified by flash chromatography on silica gel (hexane/CH)₂Cl₂7: 3) to yield a colorless viscous liquid: IR (KBr, cm)^-1)：737，1106，1231，1700，2980，3328；¹H NMR(500MHz，CDCl₃)：δ1.30(t，J＝7.5Hz，3H)，1.80(d，J＝7.0Hz，3H)，4.17-4.28(m，2H)，6.17(dd，J＝4.0，3.0Hz，1H)，6.60(q，J＝7.0Hz，1H)，6.98-7.01(m，2H)，7.12-7.14(m，2H)，7.20-7.25(m，1H)，7.28-7.32(m，2H)；¹³C NMR(500MHz，CDCl₃)：δ14.6，22.3，55.5，60.0，108.5，118.5，122.6，125.5，126.4，127.5，128.7，143.3，161.4；C₁₅H₁₈NO₂LC-MS found 244.10, calculated 244.10 for (M + H); analytically calculated value is C, 74.05; h, 7.04; and N, 5.76. Found C, 74.25; h, 6.94; and N, 5.66. The final product was essentially enantiomerically pure (R-enantiomer) as determined by chiral column chromatography. See scheme 1.

Scheme 1

Example 2: carboetomidate is a potent general anesthetic for tadpoles and rats

Tadpole: and testing the anesthetic activity by tadpole flip reflection disappearance analysis. Several groups of xenopus tadpoles, including 5 early premolar tadpoles, were placed in 100ml oxygenated water buffered with 2.5mM Tris HCl buffer (pH 7) and containing carboetomidate at a concentration of 1-40 μ M. See scheme 1 above for the structure of carboetomidate. The tadpoles were tipped over manually with a fire polished pipette every 5 min. If tadpoles are unable to rightize themselves within 5s, the tadpoles are considered to have been anesthetized (with righting reflex disappearance (LORR)). At all concentrations, the righting reflex vanishing response stabilized within 30min of exposure to carboetomidate. At the end of each study, tadpoles were placed back in fresh water to ensure reversibility of hypnotic effect. No evidence of toxicity was observed; when placed back into fresh oxygenated water, the righting reflex of all anesthetized tadpoles is restored.

Figure 2 shows the carboetomidate concentration response curve for anesthesia. The specific gravity of the tadpoles anesthetized in each group increased with increasing carbon etomidate concentration, and at the highest concentration of carbon etomidate (10-40 μ M), all tadpoles were anesthetized. Using a model of Waud, D.R., J Pharmacol Exp Ther (1972), 183 (3): 577-method 607, from which the anaesthetic EC50 (i.e. the concentration at which 50% of the tadpoles were anaesthetic) of carboetomidate was determined to be 5.4 ± 0.5 μ M.

Example 3: carboetomidate versus wild type alpha₁β₂γ_2LGABA_AReceptor and etomidate insensitive alpha₁β₂M286Wγ_2LGABA_AModulation of receptors

Carboetomidate is designed to pass through the same molecular mechanism as (R) -etomidate, i.e. by enhancing GABA_AReceptor function produces anesthesia. From alpha₁β₂γ_2LHuman GABA composed of subunits_AThe receptor is expressed in Xenopus oocytes and is used to study the response of carboetomidate to GABA_AThe effect of receptor-mediated currents, using the current regulation in Raines et al, Anesth Analg (2003), 96 (1): 112-8. TheThe subunit combination forms the most prevalent GABA in the brain_AThe receptor subtype and known to be an etomidate-sensitive receptor, so this subunit combination was chosen.

In each oocyte, GABA was used at a concentration of 3 μ M, which elicited about 10-20% of the maximal response elicited by 1mM GABA (receptor saturating GABA concentration) in the wild type receptor. To evaluate the effect of carboetomidate on GABA-induced currents, a "control" current induced only by GABA was determined. After a recovery period of 5min, the "test" peak current was determined by exposing the oocytes to anesthetic and GABA. After another 5min recovery period, the control experiment was repeated to ensure reversibility. Figure 3A shows representative control and test traces (tracks) obtained in the same oocyte, in the absence and presence of anesthetic, respectively. It was found that carboetomidate enhanced the amplitude of GABA-induced currents by 4-fold at approximately 2-fold (i.e., 10 μ M) compared to carboetomidate anesthesia EC 50.

Expression of alpha in Xenopus oocytes₁β₂M286Wγ_2LGABA_AMutant receptors and for studying carboetomidate on GABA_AMutating the receptor-mediated current, the study used a dual microelectrode voltage clamp technique. In each oocyte, GABA was used at a concentration of 0.3. mu.M, which elicited about 10-20% of the maximal response elicited by 1mM GABA (receptor saturating GABA concentration) in the wild type receptor. Due to these GABA_AThe mutant receptor was 10 times more sensitive to GABA than the wild-type receptor used in example 2, so a lower concentration of GABA was used. Figure 3B shows representative control and test traces obtained in the same oocyte, in the absence and presence of anesthetic, respectively. It was found that carboetomidate had little effect on the amplitude of GABA-induced currents at about 2 times (i.e., 10 μ M) the dose of EC50 under carboetomidate anesthesia. The mutation of the etomidate binding site weakens GABA_AReceptor sensitivity to both etomidate and carboetomidate, so it can be concluded that both etomidate and carboetomidate are likely to bind to GABA_AOn the receptorThe same site of (a).

FIG. 4A (wild type. alpha.)₁β₂γ_2LGABA_AReceptor) and figure 4B (etomidate insensitive alpha)₁β₂M286Wγ_2LGABA_AMutant receptor) showed a change in EC in the absence or presence of 10 μ M carboetomidate_5-10Other representative electrophysiological traces induced by GABA. Carboetomidate significantly enhanced the current mediated by the wild-type receptor (390 ± 80%), but not the current mediated by the etomidate insensitive mutant receptor (-9 ± 16%).

Example 4: carboetomidate is a less potent inhibitor of cortisol synthesis in human adrenocortical cells than etomidate

Next, carboetomidate was tested for its ability to inhibit cortisol synthesis by human adrenocortical cells. The human adrenocortical cell line H295R (NCI-H295R; ATCC # CRL-2128) was used as an in vitro system to evaluate and compare the inhibitory effects of etomidate and carboetomidate on cortisol synthesis. H295R cells expressed most of the key enzymes necessary for steroid production (including all enzymes required for cortisol biosynthesis, e.g. 11 β -hydroxylase) when stimulated with forskolin, these cells produced cortisol and secreted it into the culture medium where cortisol could be readily determined. Inhibition of 11 β -hydroxylase blocks cortisol synthesis and reduces cortisol concentration in the cell culture medium, which is the basis of this assay.

H295R cells were grown to near confluence in growth medium (DMEM/F12 supplemented with 1% ITS containing insulin, iron transporter, selenium and linoleic acid; 2.5% NuSerum; and penicillin/streptomycin). Growth medium was then replaced with cortisol synthesis promoting assay medium (DMEM/F12 supplemented with 0.1% ITS and 20 μ M forskolin) along with etomidate or carboetomidate, or only with the assay medium as a control. After 48h of forskolin stimulation of cortisol synthesis, 1.2ml of assay medium was collected, centrifuged (to remove cells and debris) and the cortisol concentration in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA).

Figure 5 shows that etomidate and carboetomidate both reduced cortisol concentrations in assay media in a concentration-dependent manner. Although both hypnotics inhibit cortisol synthesis in a concentration-dependent manner, the inhibition occurs at concentrations that differ by 3 orders of magnitude. Half maximal inhibitory concentration (IC50) was 1.3 + -0.02 nM for etomidate, and 2000-fold higher (2.6 + -1.5 μ M) for carboetomidate.

Example 5: carboetomidate is a potent and transiently acting general anesthetic in rats

Etomidate or carboetomidate was administered as an intravenous bolus into the tail vein of Sprague Dawley rats. If the rats were not able to right themselves after the administration (all four paws were grounded), the rats were judged to have LORR. LORR is defined as the time from drug injection until the animal spontaneously rights itself. The dose dependence of the anesthetic from the LORR determines the ED50 of the LORR given as a bolus anesthetic.

Figure 6A shows etomidate and carboetomidate dose-response relationships of LORR in rats. The specific gravity of rats with LORR increases with the anesthetic dose. At the highest dose, all rats were anesthetized and there was no significant anesthetic toxicity. From these data, the ED50 for LORR following bolus administration of etomidate and carboetomidate was determined to be 1.00 ± 0.03mg/kg (n ═ 18) and 7 ± 2mg/kg (n ═ 16), respectively. At a dose sufficient to cause LORR in rats, both anesthetics produce LORR within seconds of bolus i.v. administration.

The present inventors have found that the LORR onset time of carboetomidate is greater than previously observed methods such as Cotton et al, Anesthesiology, (2009) 111: this time using etomidate is slow as described in 240-249. The inventors quantified the difference using equal hypnotic doses of carboetomidate and etomidate (28 mg/kg and 4mg/kg, respectively; 4 times the ED50 of LORR) (Cotton et al, Anesthesiology, (2009) 111: 240-. The LORR onset time for carboetomidate was 33 ± 22s (n ═ 10; between 10-63 s) compared to 4.5 ± 0.6s (n ═ 4; between 4-5 s) for etomidate.

Figure 6B shows that the duration of anesthesia (i.e., the time until spontaneous reversal) for the two anesthetics increases approximately linearly with the logarithm of the anesthetic dose. Etomidate (27 ± 7) and carboetomidate (16 ± 4) were similar for the slope of the relationship. Since the slope of this relationship depends on the half-life of the anesthetic in the brain, the results indicate that etomidate and carboetomidate are cleared from the brain at similar rates.

Example 6: the carboetomidate has excellent hemodynamic stability

Etomidate is often selected for inducing anesthesia in critically ill patients relative to other agents because etomidate maintains hemodynamic stability better. To determine whether carboetomidate similarly maintained hemodynamic stability, we measured and compared the effects of propofol, etomidate and carboetomidate on rat heart rate and blood pressure. To compare these drugs at equal anesthetic doses, each drug was administered intravenously at 2-fold the ED50 of its LORR (i.e., 8mg/kg propofol, 2mg/kg etomidate, 14mg/kg carboetomidate). After the animal was habituated, data was recorded 5min before anesthetic injection (baseline) and 15min after anesthetic injection. Mean blood pressure means were calculated every 30s during the study period.

Fig. 7A shows that rats in each group had similar baseline mean heart rate and blood pressure within the first 5 min. When rats were anesthetized (i.e., the first 5-10min after anesthetic administration), the mean blood pressure of rats given all 3 anesthetics decreased. However, at almost all time points during anesthesia carboetomidate and etomidate decreased by a smaller magnitude compared to propofol.

Similarly, figure 7B shows the effect of 14mg/kg carboetomidate (n-7), 2mg/kg etomidate (n-6) and DMSO vehicle (n-4) on mean arterial blood pressure in rats. For carboetomidate and etomidate, the above doses are equivalent hypnotic doses. During the study period, the effect of carboetomidate on mean blood pressure was not significantly greater than that of DMSO adjuvant alone (P > 0.05, two-way ANOVA). However, etomidate significantly reduced mean blood pressure over a period of 30-210s post-administration relative to vehicle. Baseline mean blood pressures were similar for the adjuvant, carboetomidate and etomidate groups, 114 ± 5mmHg, 116 ± 9mmHg and 127 ± 17mmHg (P ═ 0.15, ANOVA), respectively.

Example 7: unlike (R) -etomidate, carboetomidate does not inhibit adrenocortical function 15min after administration

Male Sprague Dawley rats were pretreated with dexamethasone to suppress endogenous ACTH production and minimize baseline serum corticosterone concentrations. DMSO vehicle (control), 2mg/kg etomidate or 14mg/kg carboetomidate was injected intravenously to each rat. For etomidate and carboetomidate, the above doses were equivalent bolus doses of anesthesia (i.e., 2 times the ED50 of LORR). Cortrosyn (i.e., ACTH) was injected immediately thereafter_1-24) To stimulate steroid production. At ACTH_1-24About 0.3ml of blood was taken 15min after dosing for determination of ACTH_1-24Stimulated serum corticosterone concentration. Baseline serum corticosterone concentrations averaged 39 ± 49ng/ml in rats (n-12) and were not significantly different in these 3 groups (carboetomidate, etomidate and control). In all 3 groups, ACTH_1-24The administration of (a) stimulates the production of adrenocortical steroids.

FIG. 8 is a graph showing ACTH_1-24All rats had significantly higher serum corticosterone concentrations 15min after dosing, thus ACTH_1-24The injection of (a) stimulates the production of adrenal corticosteroids. However, with ACTH_1-24Rats that had received adjuvant or equivalent anesthetic doses of carboetomidate before stimulation were compared to ACTH_1-24Has been stimulated beforeRats receiving (R) -etomidate had significantly reduced serum corticosterone concentrations (67% reduction). In contrast, rats that have received carboetomidate have the same serum corticosterone concentration as rats that have received vehicle alone.

Discussion of the related Art

Carboetomidate is a pyrrole analogue of etomidate that retains its hypnotic effect, GABA_AReceptor modulating activity and hemodynamic stability. However, it is a cortisol synthesis inhibitor that is 3 orders of magnitude less potent than etomidate, and unlike etomidate, it does not inhibit adrenocortical function in rats at hypnotic doses.

Etomidate inhibits adrenocortical function primarily by inhibiting the member of the cytochrome P450 superfamily of enzymes, 11 β -hydroxylase (CYP11B 1). 11 β -hydroxylase is required for the synthesis of cortisol, corticosterone and aldosterone. This inhibition occurs at very low etomidate concentrations, which is believed to reflect the high affinity of etomidate for the active site of the enzyme. See, e.g., Zolle et al, J Med Chem (2008) 51: 2244-: 455-471, which is incorporated herein by reference in its entirety. The present inventors have designed carboetomidate to inhibit and/or reduce etomidate's high affinity for 11 β -hydroxylase, resulting from the basic nitrogen in its imidazole ring. Without wishing to be bound by theory, the basic nitrogen in the imidazole ring of etomidate and the heme iron of the active site result in high affinity of etomidate for 11 β -hydroxylase. This interaction in the binding of other imidazole-containing drugs to various cytochrome P450 enzymes has been observed using crystallography techniques. For example, the inhibitor 4- (4-chlorophenyl) imidazole binds in a single orientation to the active site of enzyme 2B4, wherein the basic nitrogen of the imidazole ring of the inhibitor is atThe bond length of (B) coordinates to the heme iron of the enzyme (Scott et al, J Biol Chem (2004) 279: 27294-27301, which is incorporated in its entirety by referenceIncorporated herein in its used form). This binding triggers a conformational transition in which the enzyme closes tightly around the bound ligand. Similarly, imidazole-containing antifungal agents bind within the active sites of CYP130 and CYP121, where the basic nitrogen forms a coordinate bond with the heme iron of the enzyme. See, e.g., Ouellett et al, J Biol Chem (2008) 283: 5069-5080 and Seward et al, J Biol Chem (2006) 281: 39437-. Evidence of such coordination was also found using spectroscopy when a coordination bond was formed with an imidazole-containing inhibitor due to a characteristic spectral shift of the heme group as chromophore. See, e.g., Ouellette et al, J BiolChem (2008) 283: 5069-5080; yano et al, J Med Chem (2006) 49: 6987 7001; locuson et al, Drug Metab Dispos (2007), 35: 614-622; and Hutzler et al, Chem Res Toxicol (2006) 19: 1650 1659, all of which are incorporated herein by reference in their entirety. Although the interaction of etomidate with 11 β -hydroxylase has not been experimentally clarified using crystallography or spectroscopy techniques, computer homology modeling suggests that coordination between the basic nitrogen of the hypnotic and the heme iron of the enzyme also contributes to high affinity binding (Roumen et al, JComputaided Mol Des (2007) 21: 455-471).

The inventors compared the inhibitory potency of carboetomidate and etomidate using an adrenocortical carcinoma cell assay. This assay has previously been used to compare the efficacy of drugs to inhibit the synthesis of adrenocortical steroids. See, for example, Fallo et al, Endocr Res (1996) 22: 709-; fallo et al, Chemotherapy (1998) 44: 129-134; and Fassacht et al, Eur J Clin Invest (2000)30(suppl 3): 76-82, all of which are incorporated herein by reference in their entirety. The results show that carboetomidate is a cortisol synthesis inhibitor that is 3 orders of magnitude less potent than etomidate. This is consistent with the important role of the basic nitrogen of hypnotics in stabilizing binding to enzymes, which also provides strong evidence: the high affinity binding of etomidate to 11 β -hydroxylase can be eliminated by replacing the nitrogen with another chemical group (CH in the present invention) that does not coordinate to heme iron. Due to its renal functionGlandular cortex inhibition efficacy was low and carboetomidate was unable to inhibit ACTH in rats when given as a bolus injection at hypnotic doses_1-24Stimulating the production of corticosterone.

Although carboetomidate is an in vitro cortisol synthesis inhibitor that is 3 orders of magnitude less potent than etomidate, it is only moderately less potent as a hypnotic. Its hypnotic potency against tadpoles (Husain et al, J Med Chem (2003) 46: 1257-. These results show that the efficacy of producing undesirable side effects can be significantly reduced without greatly affecting the hypnotic efficacy by altering the anesthetic structure.

Like etomidate, carboetomidate significantly enhanced wild-type alpha₁β₂γ_2LGABA_AThe function of the receptor. For GABA_ADirect activation of receptors and agonist modulation are described in Rusch et al, J Biol Chem (2004) 279: 20982-20992, which are incorporated herein by reference in their entirety. Without wishing to be bound by theory, carboetomidate also acts by acting on GABA_AThe action of the receptors produces hypnosis. Heretofore on GABA_AElectrophysiological studies of the receptor have shown that mutations at the putative etomidate binding site in the β subunit (M286W) almost completely abolished the potentiating effect of etomidate (Siegwart et al, J Neurochem (2002) 80: 140-148, which is incorporated herein by reference in its entirety). The results herein show that this mutation also abrogated the potentiating effect of carboetomidate, demonstrating that carboetomidate can be obtained by binding GABA_AGABA modulation at the same binding site as etomidate on receptor_AReceptor function.

The magnitude of the synergy (390 ± 80%) observed by the inventors in this study with 10 μ M carboetomidate was not greater than the magnitude of the synergy (660 ± 240%) observed by the inventors in the previous study with 4 μ M etomidate. This may suggest that carboetomidate versus etomidate is acting on GABA_AThe receptor is less potent and/or less efficient. Without wishing to be bound by theory, this mayExplaining why carboetomidate requires higher concentrations (in tadpole trials) and higher doses (in rat trials) to produce LORR compared to etomidate. It also shows that the moderate alkaline nitrogen in the imidazole ring of etomidate is helpful for the etomidate to play a role in GABA_AThe action of the receptor.

LORR is slower in onset with carboetomidate than with etomidate. The reason for this is not clear. However, since both hypnotics potentiate GABA_AThe receptor, apparently delayed in onset, is probably due to the slower arrival of carboetomidate at the site of action in the brain than etomidate.

Reference to the literature

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All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

Claims (30)

1. A compound of formula (I):

as well as pharmaceutically acceptable salts of said compounds, stereoisomeric mixtures of said compounds and enantiomers of said compounds,

wherein:

R₁is L₁C(O)OT；

R₂Is C₁-C₁₀An alkyl group;

n is an integer of 0 to 5;

R₃each independently is halogen;

R₄and R₅Independently H, halogen, CN or CF₃；

L₁Is a bond;

t is ethyl, propyl, isopropyl, cyclopropyl, n-butyl, neopentyl, n-hexyl, cyclohexyl, n-octyl, n-decyl, CH₂CH(OH)CH₃、C₂-C₁₀Alkenyl radical, C₂-C₁₀Alkynyl, or nitrophenol.

2. The compound of claim 1, wherein the compound is present as a pure enantiomer.

3. The compound of claim 2, wherein the enantiomer is the R enantiomer.

4. The compound of claim 1, wherein R₂Is selected from CH₃、CH₂CH₃And CH₂CH₂CH₃The group consisting of.

5. The compound of claim 1, wherein T is selected from the group consisting of CH₂CH₃、CH₂CH(OH)CH₃And CH₂CH₂CH₃The group consisting of.

6. The compound of claim 1, wherein n is 0 or 1.

7. The compound of claim 1, wherein R₂Is CH₃N is 0, L₁Is a bond, T is CH₂CH₃Or CH₂CH(OH)CH₃。

8. The compound of claim 1, wherein R₄And R₅Both are H.

9. The compound of claim 1, wherein R₄And R₅At least one of which is H and the other is Br or CN.

10. The compound of claim 9, wherein R₄Is H, R₅Is Br or CN.

11. The compound of claim 9, wherein R₄Is Br or CN, R₅Is H.

12. The compound of claim 1, wherein the compound of formula (I) is selected from the group consisting of:

etomidate, etomidate,

Pharmaceutically acceptable salts of the above compounds, stereoisomeric mixtures of the above compounds, and enantiomers of the above compounds.

13. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of any one of claims 1-12 and a pharmaceutically acceptable carrier.

14. Use of a pharmaceutical composition according to claim 13 in the manufacture of a formulation providing an anaesthetic effect.

15. Use of a compound of formula (I) as defined in any one of claims 1 to 12 in the manufacture of a formulation providing an anaesthetic effect.

16. A compound of formula (I):

as well as pharmaceutically acceptable salts of said compounds, stereoisomeric mixtures of said compounds and enantiomers of said compounds,

wherein:

R₁is L₁C(O)OL₂C(O)OT；

R₂Is C₁-C₁₀An alkyl group;

n is an integer of 0 to 5;

R₃each independently is halogen;

R₄and R₅Independently H, halogen, CN or CF₃；

L₁Is a bond;

L₂is C₁-C₁₀An alkylene group;

t is C₁-C₁₀An alkyl group.

17. The compound of claim 16, wherein the compound is present as a pure enantiomer.

18. The compound of claim 17, wherein the enantiomer is the R enantiomer.

19. The compound of claim 16, wherein R₂Is selected from CH₃、CH₂CH₃And CH₂CH₂CH₃The group consisting of.

20. The compound of claim 16, wherein T is selected from the group consisting of CH₃、CH₂CH₃And CH₂CH₂CH₃The group consisting of.

21. The compound of claim 16, wherein n is 0 or 1.

22. The compound of claim 16, wherein R₂Is CH₃N is 0, L₁Is a bond, T is CH₃Or CH₂CH₃。

23. The compound of claim 16, wherein R₄And R₅Both are H.

24. The compound of claim 16, wherein R₄And R₅At least one of which is H and the other is Br or CN.

25. The compound of claim 16, wherein R₄Is H, R₅Is Br or CN.

26. The compound of claim 24, wherein R₄Is Br or CN, R₅Is H.

27. The compound of claim 16, wherein the compound of formula (I) is:

MOC-carboetomidate,

Pharmaceutically acceptable salts thereof, stereoisomeric mixtures thereof and enantiomers thereof.

28. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of any one of claims 16-27 and a pharmaceutically acceptable carrier.

29. Use of a pharmaceutical composition according to claim 28 in the manufacture of a formulation providing an anaesthetic effect.

30. Use of a compound of formula (I) as defined in any one of claims 16 to 27 in the manufacture of a formulation which provides an anaesthetic effect.