HK1079194B - Dipyridodiazepinones as reverse transcriptase inhibitors - Google Patents

Description

Bipyridinodiazepine * ketone reverse transcriptase inhibitors

Technical Field

The present invention relates to novel compounds and their pharmaceutically acceptable salts, their use in the treatment or prevention of HIV infection (alone or in combination with other therapeutic agents), and pharmaceutical compositions comprising these compounds.

Background

The disease known as acquired immunodeficiency syndrome (AIDS) is caused by the Human Immunodeficiency Virus (HIV), particularly the so-called HIV-1 strain. In order for HIV to be replicable by host cells, the information of the viral genome must be integrated into the DNA of the host cell. However, HIV is a retrovirus, meaning that the genetic information is in the form of RNA. The HIV replication cycle therefore requires the step of transcribing the viral genome (RNA) into DNA, a process that is the reversal of the normal event strand. The enzyme, which has been properly designated Reverse Transcriptase (RT), can transcribe viral RNA into DNA. HIV viral particles include RT copies and viral RNA.

Reverse transcriptase is known to have three enzymatic functions: it can be used as RNA-dependent DNA polymerase, ribonuclease and DNA-dependent DNA polymerase. As an RNA-dependent DNA polymerase, RT transcribes a single-stranded DNA copy of the viral RNA. Acting as a ribonuclease, RT destroys the original viral RNA and frees DNA that has just been produced from the original RNA. Finally, when used as a DNA-dependent DNA polymerase, RT produces a second complementary DNA strand using the first DNA strand as a template. The two strands will form double-stranded DNA that is integrated into the host cell genome by another enzyme called integrase.

Compounds that inhibit the enzymatic function of HIV-1 reverse transcriptase will inhibit the replication of HIV-1 in infected cells. These compounds are useful for the prevention or treatment of HIV-1 infection in humans with a variety of RT inhibitors whose preventive or therapeutic effects are known, such as those demonstrated by 3 '-azido-3' -deoxythymidine (AZT), 2 ', 3' -dideoxyinosine (ddI), 2 ', 3' -dideoxycytidine (ddC), d4T, 3TC, nevirapine, delavirdine, efavirenz and abacavir, which are the primary drugs approved so far for the treatment of AIDS.

As with any antiviral treatment, treatment of AIDS with RT inhibitors ultimately results in a virus that is less sensitive to the administered drug. Resistance to these drugs (reduced sensitivity) results from mutations in the reverse transcriptase segment of the pol gene. Several HIV mutants have been characterized and resistance to known therapeutic agents is due to mutations in the RT gene. Some of the most common mutants in the clinic are: Y181C mutant, wherein the tyrosine (Y) at codon 181 is mutated to a cysteine (C) residue; and K103N, wherein lysine (K) at position 103 is replaced by asparagine (N). Other mutants that occur more and more frequently during treatment with known antiviral drugs include: single mutants V106A, G190A, Y188C and P236L; and double mutants K103N/Y181C, K103N/P225H, K103N/V108I and K103N/L100I.

The continued use of antiviral compounds to prevent HIV infection will necessarily lead to the emergence of more new HIV resistant strains. Thus, there is a need for novel RT inhibitors with different modes of action against various mutants.

Compounds having tricyclic structures as HIV-1 inhibitors are described in U.S. Pat. No. 5,366,972. Other HIV-1 reverse transcriptase inhibitors are described in Hargrave et al, j.med.chem., 34, 2231 (1991).

U.S. Pat. No. 5,705,499 proposes 8-aralkyl-and 8-heteroaralkyl-5, 11-dihydro-6H-bipyridino [3, 2-B: 2 ', 3' -E ] [1, 4] diazepine * is an RT inhibitor. These exemplified compounds were shown to have some activity against wild-type HIV-1RT and mutant HIV-1RT (especially Y181C) and also against other single mutants (although less effective), such as K103N.

WO01/96338A1 and U.S. Pat. No. 6,420,359 disclose diaza * compounds with quinoline and quinoline-N-oxide substituents as RT inhibitors. The exemplified compounds have activity against HIVWT, single and double mutants.

Summary of The Invention

The invention provides a compound containing substituted benzoic acid, which is an effective inhibitor of Wild Type (WT) HIV-1RT and double mutant strains of HIV-1RT (especially double mutant strains K103N/Y181C).

In a first aspect the present invention provides a compound of formula I:

wherein the content of the first and second substances,

R²is H, halogen, (C)_1-4) Alkyl, O (C)_1-4) Alkyl, HN (C)_1-4Alkyl) or N (C)_1-4Alkyl radical)₂；

R⁴Is H or CH₃；

R⁵Is H or CH₃；

R¹²Is H, halogen, (C)_1-4) Alkyl, CF₃Or NO₂；

R¹³Is H, (C)_1-4)Alkyl, halogen, OH or NH₂Provided that R is¹²And R¹³Not all are H; and

R¹⁴is COOR^14aWherein R is^14aIs H or (C)_1-6) An alkyl group; or R¹⁴Is (C)_2-4) alkenyl-COOR^14aWherein R is^14aAs defined herein; or R¹⁴Is (C)_1-4) alkyl-COOR^14aWherein R is^14aAs defined herein;

or a salt or prodrug thereof.

A second aspect of the present invention provides a pharmaceutical composition for the treatment or prophylaxis of HIV infection, comprising a compound of formula I, as described in the specification, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In a third aspect the present invention provides a method of treatment or prophylaxis of HIV infection comprising administering to a patient an HIV inhibiting amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (both as defined herein).

In a fourth aspect the present invention provides a method of treatment or prophylaxis of HIV infection which comprises administering a pharmaceutical composition comprising a compound of formula I as defined herein in combination with an antiretroviral drug.

In a fifth aspect of the invention there is provided a method of preventing perinatal infection with HIV-1 from the mother to the infant, comprising administering to the mother prior to delivery a compound of formula I or a pharmaceutical composition as described herein.

Detailed Description

Definition of

Unless otherwise indicated, the following definitions apply:

the term "(C) as used herein_1-2) Alkyl group "," (C)_1-4) Alkyl and (C)_1-6) Alkyl "(whether occurring alone or in combination with another group) means an acyclic alkyl group containing up to two, four, or six carbon atoms, respectively. Examples of such groups include methyl, ethyl, propyl, butyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl and 1, 1-dimethylethyl.

The term "(C) as used herein_2-4) Alkenyl "(whether it occurs alone or in combination with another group) means an unsaturated acyclic group containing two to four carbon atoms.

The term "halogen" as used herein means a halogen atom and includes fluorine, chlorine, bromine and iodine.

The term "pharmaceutically acceptable salt" as used herein includes derivatives from pharmaceutically acceptable saltsBasic and non-toxic salts. Examples of suitable bases include choline, ethanolamine, and ethylenediamine. Na (Na)⁺、K⁺And Ca⁺⁺Salts are also included within the scope of the present invention (see also Pharmaceutical salts, Birge, s.m. et al, j.pharm.sci., (1977),661-19, incorporated herein by reference).

The term "prodrug" as used herein refers to a pharmaceutically acceptable derivative from which the resulting biotransformation product is the active drug as defined for the compound of formula I. Examples of such derivatives include, but are not limited to, esters and amides (see Goodman and Gilman, The pharmaceutical Basis of therapeutics, 9 th, McGraw-Hill, int. Ed.1995, "Biotransformation of drugs", p11-16, incorporated herein by reference).

Detailed description of the preferred embodiments

Preferably, R²Is H, halogen, (C)_1-4) Alkyl, O (C)_1-4) Alkyl or N (C)_1-4) Alkyl radical)₂. More preferably, R²Is H, Cl, F, (C)_1-4) Alkyl, O (C)_1-4) Alkyl or N (C)_1-4) Alkyl radical)₂. More preferably, R²Is H, Cl, F, CH₃OMe or OEt. Most preferably, R²Is H.

Preferably, R⁴And R⁵Are not identical.

More preferably, R⁴Is H.

More preferably, R⁵Is CH₃。

Preferably, R¹²Is halogen, (C)_1-4) Alkyl, CF₃Or NO₂. More preferably, R¹²Is Br, Cl, CH₃Or CH₃CH₂. Most preferably, R¹²Is CH₃Or CH₃CH₂。

Preferably, R¹³Is H, CH₃Halogen, bitternElement, OH or NH₂. More preferably, R¹³Is H, CH₃Or OH. Most preferably, R¹³Is H.

Preferably, R¹⁴Is COOH, COOMe, (C)_2-4) Alkenyl group COOH or (C)_1-4) Alkyl group COOH. More preferably, R¹⁴Is COOH, CH ═ CH-COOH, CH₂COOH or CH₂CH₂COOH. Most preferably, R¹⁴Is COOH.

The compound of the formula I is a potent inhibitor of wild type HIV and inhibits the double mutant enzyme K103N/Y181C.

The compounds of formula I have inhibitory activity against HIV-1 reverse transcriptase. When administered in an appropriate dosage form, these compounds are useful in the treatment of AIDS, ARC and other conditions associated with HIV-1 infection. Thus another aspect of the present invention is a method of treating HIV-1 infection comprising administering to a human infected with HIV-1 a therapeutically effective amount of a novel compound of formula I as described above. Whether referred to as treatment or prevention, the compounds may also be administered to the mother prior to delivery to prevent perinatal HIV-1 transmission from the mother to the infant.

These compounds of formula I can be administered once or in portions by oral or parenteral administration. Suitable oral dosages of the compounds of formula I are between about 0.5 mg/day and 3 g/day. For a patient weighing 70 kg, the oral dosage of a compound of formula I is preferably between about 100 mg/day and 800 mg/day. In formulations for parenteral administration, a suitable dosage unit may contain from 0.1 to 250 mg of the compound, preferably from 1 mg to 200 mg. It will be appreciated that the dosage administered will vary from patient to patient and that the dosage administered to any particular patient will depend on the diagnosis of the clinician who will determine the appropriate dosage to administer depending on the weight and condition of the patient and its response to the drug.

If the compounds of the present invention are administered orally, they may be administered as medicaments in the form of pharmaceutical preparations comprising these compounds together with a pharmaceutically acceptable carrier compatible therewith. These carrier materials may be inert organic or inorganic carrier materials suitable for oral administration. Examples of such carrier materials are water, gelatin, talc, starch, magnesium stearate, acacia, vegetable oils, polyalkylene glycols, vaseline, etc.

These compounds of formula I may be used in combination with antiretroviral drugs familiar to those of ordinary skill in the art, in combination preparations for simultaneous, separate or sequential administration for the treatment or prevention of HIV infection in a subject. Examples of antiretroviral drugs that may be used in combination therapy with the compounds of formula I include (but are not limited to): nucleoside/nucleotide reverse transcriptase inhibitors (e.g., AZT and tenofovir), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine), protease inhibitors (e.g., Ritanovir)), viral fusion inhibitors (e.g., T-20), CCR5 antagonists (e.g., SCH-351125), CXCR4 antagonists (e.g., AMD03100), integrase inhibitors (e.g., L-870, 810), TAT inhibitors, other in-development drugs (e.g., PRO-542, BMS-806, TMC-114, or AI-183), antifungal or antibacterial agents (e.g., fluconazole), and immunomodulators (e.g., levamisole). Furthermore, one compound of formula I may be used in combination with another compound of formula I.

These pharmaceutical preparations can be prepared in a conventional manner, and can be prepared in solid dosage forms such as tablets, dragees, capsules and the like; or liquid dosage forms such as solutions, suspensions, emulsions, and the like. These pharmaceutical preparations may be subjected to conventional pharmaceutical operations, such as sterilization. In addition, these pharmaceutical preparations may contain conventional adjuvants, such as preserving agents, stabilizing agents, emulsifying agents, flavoring agents, wetting agents, buffering agents, salts for varying the osmotic pressure, and the like. Solid carrier materials which may be used include, for example, starch, lactose, mannitol, methyl cellulose, microcrystalline cellulose, talc, silicon dioxide, dibasic calcium phosphate and high molecular weight polymers (e.g., polyethylene glycol).

For parenteral use, the compounds of formula I may be administered as an aqueous solution, or as a non-aqueous solution, suspension or emulsion in a pharmaceutically acceptable oil, or as a mixed liquid which may contain bacteriostats, antioxidants, preservatives, buffers or other solutes which render the solution isotonic with the blood, thickening agents, suspending agents or other pharmaceutically acceptable additives. Additives of this type include, for example, tartrate, citrate and acetate buffers, ethanol, propylene glycol, polyethylene glycol, chelate-forming agents (e.g., EDTA), antioxidants (e.g., sodium bisulfite, sodium metabisulfite and ascorbic acid), high molecular weight polymers for adjusting viscosity (e.g., liquid polyethylene oxide) and polyethylene derivatives of sorbitan. Preservatives, such as benzoic acid, methyl or propyl p-hydroxybenzoate, benzalkonium chloride and other quaternary ammonium compounds, may also be added if desired.

The compounds of the present invention may also be administered as nasal solutions, comprising in addition to the compounds of the present invention, suitable buffers in aqueous vehicle, tonicity adjusting agents, antimicrobial preservatives, antioxidants and viscosity increasing agents. Examples of agents for increasing the viscosity are polyvinyl alcohol, cellulose derivatives, polyvinylpyrrolidone, polysorbates or glycerol. The added antimicrobial preservative may include benzalkonium chloride, thimerosal, chlorobutanol, or phenylethyl alcohol.

In addition, the compounds provided herein may be administered in the form of suppositories.

Method and synthesis

The compounds of the invention may be prepared using techniques well known to organic synthetic chemists. Exemplary reaction schemes are shown in figures 1 to 4 below. Substituent R²、R⁴、R⁵、R¹²、R¹³And R¹⁴As defined herein.

Scheme 1: intermediate (wherein R⁴Is methyl) preparation

Briefly, aromatic ring substitution of 1(i) (S) with ethylamine_NAR) to intermediate 1 (ii). Then, benefit fromHalogenation of position 5 with a brominating agent (e.g., NBS or bromine) yields 1 (iii). By S being contributed to by a base_nThe AR reaction closes 1(iii) to give tricyclic intermediate 1 (iv). Introduction of R by substitution of the C-2 chloro aromatic ring in 1(iv)²Substituents to give intermediate compound 1 (v).

And (2) a flow scheme: intermediate (wherein R⁵Is methyl) preparation

The reaction sequence of scheme 2 is similar to the reaction sequence described in J.Med.chem.1998, 41, 2960-71 to J.M.Klunder et al and J.Med.chem.1998, 41, 2972-84 to C.L Cywin et al. Briefly, 2(i) aromatic ring substitution (S) with ethylamine_NAR) to intermediate 2 (ii). The ring-opening reaction of the nitro group (e.g., by catalytic hydrogenation) produces 2 (iii). Base-mediated condensation of 2(iii) with, for example, 5-bromo-2-chloro-3-pyridylcarbonyl chloride gives 2 (iv). S by base participation_nThe AR reaction closes 2(iv) to form tricyclic intermediate 2 (v). R in 2(vi)⁵Methyl groups may be introduced using alkylation reactions known in the art, such as methyl iodide.

And (3) a flow path: intermediate (wherein R²Is C_1-4Alkyl) preparation

Briefly, a base-mediated condensation reaction between 3(i) and 3(ii) will yield intermediate 3 (iii). Aromatic ring substitution of 3(iii) with ethylamine (S)_NAR) to intermediate 3 (iv). S with base_nThe AR reaction closes 3(iv) to form a tricyclic intermediate, which is alkylated to produce intermediate compound 3 (v).

And (4) a flow chart: compound (wherein R⁴Methyl) is another preparation method

Briefly, a base-mediated condensation reaction between 4(i) and 3(ii) produces intermediate 4 (ii). Aromatic ring substitution of 4(ii) with ethylamine ((S)_NAR) to intermediate 4 (iii). S with base_nThe AR reaction closes 4(iii) to form the tricyclic intermediate compound 1 (v).

And (5) a flow chart: introduction of benzoic acid derivatives

Briefly, the cross-linking coupling reaction of the bromo derivative 5(i) synthesized as described herein with an allyltin reagent in an aprotic solvent (e.g., DMF) and in the presence of a catalyst will form 5(ii) with a C-8 substituent. Double bonds are oxidized (e.g., ozonide formation by ozonolysis) and then subjected to a reduction reaction to form 5(iii) containing a C-8 hydroxyethyl substituent. When Y is R¹⁴When (except COOH), naphthalene derivatives 5(iv), 5(v) or 5(vi) and 5(iii) are condensed by a Mitsunobu type reaction to produce the compound of formula I. Or, when Y is R¹⁴Radical precursors such as COOCH₃When 5(iv) or 5(v) is condensed with 5(iii) by a Mitsunobu type reaction, Y can then be chemically converted to R¹⁴Substituents, e.g. by COOCH₃To form COOH, thereby obtaining the compound of formula I.

As previously mentioned, the compounds provided by the present invention inhibit the enzymatic activity of HIV-1 RT. Based on the following tests for these compounds, it was found that these compounds inhibit the RNA-dependent DNA polymerase activity of HIV-1 RT. It is known (data not shown) that these compounds also inhibit the DNA-dependent DNA polymerase activity of HIV-1 RT. Inhibition of RNA-dependent DNA polymerization of HIV-1RT by Compounds detected Using the Reverse Transcriptase (RT) assayThe ability of an enzyme to activate. Certain specific compounds described in the examples below were tested in this manner. The results of this assay are given in Table 2 as IC₅₀And EC₅₀The format is listed.

Examples

The invention is illustrated in detail by the following non-limiting examples. All reactions were carried out under nitrogen or argon atmosphere unless otherwise indicated. Temperatures are all expressed in degrees celsius. Unless otherwise indicated, the solution percentages or ratios are expressed in terms of volume-to-volume relationship.

Abbreviations or symbols as used herein include:

DEAD: diethyl azodicarboxylate;

the DIAD: diisopropyl azodicarboxylate;

DIEA: diisopropylethylamine;

DMAP: 4- (dimethylamino) pyridine;

DMSO, DMSO: dimethyl sulfoxide;

DMF: dimethylformamide;

DCC: dicyclohexylcarbodiimide;

ESMS: electrospray mass spectrometry;

et: an ethyl group;

EtOH: ethanol;

EtOAc: acetic acid ethyl ester;

Et₂o: diethyl ether;

HPLC: high performance liquid chromatography;

iPr: isopropyl group;

me: a methyl group;

MeOH: methanol;

MeCN: acetonitrile;

NaHMDS: sodium hexamethyldisilylamino;

NBS: n-bromosuccinimide;

ph: a phenyl group;

TBE: triborate-EDTA;

TBTU: 2- (1H-benzotriazol-1-yl) -N, N' -tetramethyluronium tetrafluoroborate;

TFA: trifluoroacetic acid;

THF: tetrahydrofuran;

PFU: a plaque forming unit;

DEPC: diethylpyrocarbonate;

DTT: dithiothreitol;

EDTA: ethylene diamine tetraacetic acid salt;

UMP: uridine 5' -monophosphate;

UTP: uridine 5' -triphosphate;

MES: 2- (n-morpholino) ethanesulfonic acid;

SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis;

MWCO: molecular weight cutoff;

Bis-Tris Propane: 1, 3-bis { tris (hydroxymethyl) -methylamino } propane;

GSH: reducing glutathione;

OBG: n-octyl-beta-D-glucoside.

Synthesis method

The following examples illustrate the preparation of the compounds of the present invention.

Example 1:

step a:

a THF solution of 2M ethylamine (365 ml, 731 mmol) was added to a solution of 2-chloro-3-nitropyridine 1a (51 g, 325 mmol) in THF (650 ml). The reaction was stirred at room temperature overnight. The resulting reaction mixture was poured into water (about 1.5 l), and the resulting solid was filtered off and dried under reduced pressure to obtain compound 1b (52 g).

Step b:

at 20% Pd (OH)₂A solution of 2- (ethylamino) -3-nitropyridine 1b (52 g) in MeOH (600 ml) in the presence of/C (10.4 g) was stirred under hydrogen (1 atm) at room temperature overnight. The catalyst was removed by filtration through celite. The filtrate was concentrated under reduced pressure to give compound 1c as a black solid (39 g, 88% total yield of steps a and b).

Step c:

solid NaHCO₃(56.3 g, 669 mmol) was added to a cooled solution of 3-amino-2- (ethylamino) pyridine 1c (30.6 g, 223 mmol) in MeCN (740 mL). After 5 minutes, crude 5-bromo-2-chloro-3-pyridylcarbonyl chloride (from 5-bromo-2-hydroxy-3-pyridylcarboxylic acid and SOCl) was added₂Prepared) (1 equivalent, 223 mmol) [ as described in Synth. Commun.1989, 19, 553-A559 to T.W.Gero et al, which is incorporated by reference), but omitting the work-up of the aqueous solution]. After 2 hours, the reaction mixture was poured into ice/water (1.5 l), and the resulting solid was filtered off and washed with water and hexane in that order. After drying overnight under reduced pressure, compound 1d (54.9 g, 69% yield) was obtained as a black solid.

Step d:

to a solution of 2-chloro-N- {2- (ethylamino) -3-pyridinyl } -5-bromo-3-pyridylcarboxamide 1d (54.9 g, 154.4 mmol) in pyridine (308 ml) at 50 ℃, a solution of 1m nahmds in THF (355 ml, 355 mmol) is added dropwise. After 10 minutes, the reaction solution was cooled to room temperature and then poured into ice water (2 liters). The resulting solid was filtered off and washed with water and then hexane. After drying overnight under reduced pressure, compound 1e was obtained as a dark green solid (36 g, yield 75%).

Step e:

NaH (3.5 g, 138 mmol) was added to 8-bromo-5, 11-dihydro-11-ethyl-6H-bipyrido [3, 2-b: 2 ', 3' -e ] [1, 4] diazepin * -6-one 1e (36.7 g, 115 mmol) in DMF (380 ml) and the mixture heated at 50 ℃ for 30 min. The reaction mixture was cooled to room temperature and treated with MeI (14.3 ml, 230 mmol). After 1.5 hours, the reaction mixture was poured into ice water. The resulting solid was filtered and water was then washed with hexane to give compound 1f as a dark gray solid after drying (37.9 g, 99% yield).

Step f:

allyl tributyltin (30.7 ml, 99.0 mmol) and Pd (Ph)₃P)4(5.20 g, 4.50 mmol) was added to degassed 8-bromo-5, 11-dihydro-11-ethyl-5-methyl-6H-bipyrido [3, 2-b: 2 ', 3' -e][1，4]Diaza * -6-one 1f (30.0 g, 90.0 mmol) in DMF (450 ml) (30 min nitrogen sparge to solution). The mixture was stirred at 90 ℃ for 1.5 h, then cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (hexane to EtOAc ratios between 8: 2 and 7: 3) to provide 1g (22.19 g, 84% yield) of compound.

Step g:

bubbling an ozonated oxygen stream through a mixture of 5, 11-dihydro-11-ethyl-5-methyl-8- (2-propenyl) -6H-bipyridino [3, 2-b: 2 ', 3' -e][1，4]Diaza * -6-one 1g (22.19 g, 75.4 mmol) in CH₂Cl₂(150 ml) and MeOH (150 ml) in a cold (-78 ℃ C.) solution over a period of 2.5 hours. Then a nitrogen stream was passed through the solution for 15 minutes, followed by the addition of solid NaBH₄(4.99 g, 132 mmol). The reaction mixture was allowed to warm to room temperature. After 1 hour, add saturated NH₄Aqueous Cl (200 ml) and the mixture was stirred at room temperature for 2 hours. The organic solvent was removed under reduced pressure. Water (300 ml) and CHCl₃(300 ml) was added to the residue. The phases were separated and the aqueous layer was washed with CHCl₃(3X 300 ml) was extracted. The combined organic layers were dried (MgSO)₄) Filtered and concentrated under reduced pressure. By flash chromatography (EtOAc: CHCl)₃4: 1) the resulting residue was purified to give compound 1h (16.1 g, 72% yield) as a white solid.

Example 2:

step a:

EtNH was added over 15 minutes₂An ice-cold solution (49.8 g, 1.10 mol) in toluene (200 ml) was added to an ice-cold solution of 2, 6-dichloro-3-nitropyridine 2a (100.0 g, 0.52 mol) in toluene (225 ml). The mixture was stirred at 0 ℃ for 45 minutes. Water (500 ml) and EtOAc (500 ml) were added and the phases were separated. The organic layer was washed with water (200 ml) and brine (200 ml) in sequence and dried (MgSO)₄) Filtered and concentrated under reduced pressure. The resulting residual solid was recrystallized from MeOH to give compound 2b as yellow needles (83.7 g, 80% yield).

Step b:

compound 2c was prepared in a similar manner to step b of example 1.

Step c:

at room temperature, a solution of 5-bromo-2-chloro-3-pyridylcarbonyl chloride (30.0 g, 97.0 mmol) in MeCN (100 ml) was added via cannula to 3-amino-6-chloro-2- (ethylamino) pyridine 2c (16.6 g, 97.0 mmol) in solid NaHCO₃(14.2 g, 169 mmol) of MeCN (180 mL)) In the solution of (1). The mixture was stirred at room temperature for 1 hour. Water (200 ml) was added and the mixture was stirred for 10 minutes. The resulting suspension was filtered. The resulting solid was washed with water then hexanes and dried under reduced pressure to give compound 2d (28.4 g, 75% yield).

Step d:

a THF solution of 1M NaHMDS (167.5 ml, 167.5 mmol) was slowly added to a solution of 5-bromo-2-chloro-N- {2- (ethylamino) -6-chloro-3-pyridinyl } -3-pyridylcarboxamide 2d (28.4 g, 72.8 mmol) in pyridine (146 ml) and heated to 50 ℃. The reaction mixture was stirred at 50 ℃ for 1.5 hours. The mixture was then poured into an ice-water mixture (1 l) and after 1 hour the resulting suspension was filtered. The resulting solid was washed with water and dried under reduced pressure to give compound 2e (23.4 g, yield 91%).

Step e:

solid NaH (60% oil dispersion, 3.46 g, 86.1 mmol) was added to 8-bromo-2-chloro-5, 11-dihydro-11-ethyl-6H-bipyrido [3, 2-b: 2 ', 3' -e ] [1, 4] diazepin * -6-one 2e (23.4 g, 66.3 mmol) was dissolved in a solution of DMF (220 mL). The mixture was stirred at 50 ℃ for 1 hour and then cooled to room temperature. The mixture was poured into water (1 l) and the resulting suspension was filtered. The resulting solid was washed with water and hexane in this order and dried under reduced pressure to give compound 2f (23.0 g, yield 94%).

Step f:

allyl tributyltin (21.3 ml, 68.7 mmol) and Pd (Ph)₃P)₄(3.61 g, 3.12 mmol) was added to degassed 8-bromo-2-chloro-5, 11-dihydro-11-ethyl-5-methyl-6H-bipyridino [3, 2-b: 2 ', 3' -e][1，4]Diaza * -6-one 2f (23.0 g, 62.5 mmol) was dissolved in DMF (312 ml) (30 min nitrogen was bubbled through the solution). The mixture was heated to 90 ℃ and held for 2 hours. It was concentrated under reduced pressure. The resulting residue was purified by flash chromatography (Hexane: EtOAc 7: 3)The residue was purified to obtain 2g of a compound (13.4 g, yield 65%).

Step g:

ozonized oxygen was bubbled through a cold (-78 ℃) solution of 2-chloro-5, 11-dihydro-11-ethyl-5-methyl-8- (2-propenyl) -6H-bipyridino [3, 2-b: 2 ', 3' -e][1，4]Diaza * -6-one 2g (13.4 g, 40.7 mmol) was dissolved in MeOH (102 mL) and CH₂Cl₂(102 ml) until complete disappearance of the alkene. Nitrogen was bubbled through the solution to remove excess ozone. Then, solid NaBH is added₄(2.69 g, 71.1 mmol) was added in small portions and the solution was slowly warmed to room temperature. After 1 hour, add saturated NH₄Aqueous Cl (150 ml) and the mixture was stirred for 20 min. The organic solvent was removed under reduced pressure. Water (100 ml) was added to this aqueous solution. With CHCl₃The solution was extracted (3X 200 ml). The combined organic layers were dried (MgSO)₄) Filtered and concentrated under reduced pressure. By flash chromatography (EtOAc: CHCl)₃4: 1) to give compound 2h (10.4 g, 77% yield).

Example 3:

step a:

2, 6-difluoropyridine 3a (200 g, 1.74 mol) was added dropwise to a mixture of concentrated sulfuric acid (600 ml) and fuming nitric acid (90%, 400 ml) in an ice bath (the internal temperature was maintained at 5-10 ℃). The resulting mixture was stirred at room temperature overnight. The mixture was poured slowly into 3 kg of ice and Et₂O (2X 2L) extraction. The combined organic layers were washed with 1.5N NaOH (2X 1L) and then saturated NHCO₃The aqueous solution (400 ml) was washed, or until the pH was between 8-9. The organic layer was washed with MgSO₄Drying, filtering and concentrating under reduced pressure until constant weight (to remove unreacted 2, 6-difluoropyridine: 10-12%) is obtained. Compound 3b was obtained as a yellow liquid (207.3 g, 74% yield).

Step b:

a solution of ethylamine (25.7 g, 570 mmol) in THF (250 ml) was added dropwise to a solution of 2, 6-difluoro-3-nitropyridine 3b (45.7 g, 285 mmol) in THF (500 ml) at-40 ℃. After 30 min, the reaction mixture was concentrated under reduced pressure and the resulting residue was dissolved in EtOAc. The organic phase was washed with brine and dried (MgSO₄) Filtering and concentrating. The resulting yellow solid was purified by flash chromatography (15% EtOAc/hexanes) to afford compound 3c as a yellow solid (43.2 g, 82% yield).

Step c:

at 20% Pd (OH)₂A solution of 2-ethylamino-6-fluoro-3-nitropyridine 3C (43.2 g, 230 mmol) in THF (1 l) in the presence of/C (4.35 g) was stirred overnight at room temperature under hydrogen (1 atm). The catalyst was removed by filtration through celite. The filtrate was concentrated under reduced pressure to give compound 3d (36.3 g, yield 95%) as a black solid.

Step d:

solid NaHCO₃(50.4 g, 600 mmol) was added to a cold solution (4 ℃ C.) of 3-amino-2-ethylamino-6-fluoropyridine 3d (31.0 g, 200 mmol) in MeCN (160 mL). After 15 min, a solution of 5-bromo-2-chloro-3-pyridinecarbonyl chloride (1 eq, 200 mmol) in MeCN (155 ml) was added. After 60 minutes of reaction at room temperature, the reaction mixture was poured into water (1.2 l) and stirred for 30 minutes. The resulting solid was filtered off and dried overnight at 50 ℃ under reduced pressure. Compound 3e was obtained as a black solid (73.7 g, 99% yield).

Step e:

a 1M solution of NaHMDS in THF (520 ml, 520 mmol) was added dropwise to a solution of 2-chloro-N- {2- (ethylamino) -6-fluoro-3-pyridinyl } -5-bromo-3-pyridinecarboxamide 3e (73.5 g, 216 mmol) in pyridine (435 ml) at 50 ℃. After 10 minutes, the reaction solution was cooled to room temperature, and then poured into ice water (2 liters). The resulting solid was filtered and washed with water and then hexane. The resulting solid was dried under reduced pressure to give compound 3f (50.6 g, yield 69%) as a dark green solid.

Step f:

NaH (4.28 g, 178 mmol) was added to 8-bromo-5, 11-dihydro-11-ethyl-2-fluoro-6H-bipyrido [3, 2-b: 2 ', 3' -e ] [1, 4] diazepin * -6-one 3f (44 g, 130.5 mmol) was dissolved in a solution of DMF (520 mL) and the mixture was heated at 50 ℃ for 30 minutes. The reaction mixture was then cooled to room temperature and treated with MeI (24.4 ml, 522 mmol). After 1.5 hours, the reaction mixture was poured into ice water. The resulting solid was filtered, washed with water and then hexane, and dried under reduced pressure to give compound 3g (43.2 g, yield 94%) as a dark gray solid.

Step g:

allyl tributyltin (32.0 ml, 103.4 mmol) and Pd (Ph)₃P)₄(5.43 g, 4.70 mmol) to 8-bromo-5, 11-dihydro-11-ethyl-2-fluoro-5-methyl-6H-bipyridino [3, 2-b: 2 ', 3' -e][1，4]Diaza * -6-one 3g (33.0 g, 94.0 mmol) was dissolved in DMF (470 ml) and degassed (45 min nitrogen sparged into the solution). Further Pd (Ph) was added after 1, 2, 3, 4 and 5 hours, respectively₃P)₄(1.09 g, 0.94 mmol) to complete the reaction. The mixture was heated to 90 ℃ and held for 6 hours. The mixture was concentrated under reduced pressure. The resulting residue was purified by flash chromatography (hexane: EtOAc 8: 2 to 7: 3) to provide compound 3h (22.4 g, 76% yield).

Step h:

the ozonated oxygen stream is passed into a 5, 11-dihydro-11-ethyl-2-fluoro-5-methyl-8- (2-propenyl) -6H-bipyridino [3, 2-b: 2 ', 3' -e][1，4]Diaza * -6-one 3h (22.38 g, 71.6 mmol) in CH₂Cl₂(100 ml) and MeOH (100 ml) in a cold (-78 ℃ C.) solution for a period of timeWas 3 hours. Then a stream of nitrogen was passed through the solution for 15 minutes, followed by the addition of solid NaBH₄(5.05 g, 133 mmol). The reaction mixture was warmed to room temperature. After 1 hour, add NaBH to the reaction mixture₄(1.62 g, 43.0 mmol). After a further 1 hour, saturated NH was added thereto₄Aqueous Cl (150 ml) and the mixture was stirred at room temperature for 30 min. The organic solvent was removed under reduced pressure. Water (200 mL) was added and CHCl was used₃The mixture was extracted (3X 300 ml). The combined organic layers were dried (MgSO)₄) Filtered and concentrated under reduced pressure. By flash chromatography (EtOAc: CHCl)₃4: 1) to yield compound 3I as a white solid (19.7 g, 72% yield).

Example 4 (compound numbers 1003 and 1031):

step a:

A1.6M solution of n-butyllithium in hexane (6.22 mL, 9.95 mmol) was added quickly to a cold (-78 ℃ C.) solution of 4a (0.87 g, 4.33 mmol) in THF (20 mL). The mixture was stirred at-78 ℃ for 10 minutes, then warmed to 0 ℃ and maintained at this temperature for 1 hour. Introducing CO₂A gas stream was passed through the reaction mixture for 10 minutes and the solution was made acidic by the addition of 1.0N HCl solution. The solution was extracted with EtOAc. The organic layer was dried (MgSO₄) Filtered and concentrated under reduced pressure. The residue obtained is dissolved in Et₂O (20 ml) with excess CH₂N₂Dissolved in Et₂A solution of O (about 0.6M, 10 ml) was treated for 10 minutes. The mixture was concentrated under reduced pressure and the resulting residue was purified by flash chromatography (hexane: EtOAc 4: 1 to 7: 3) to give 4b (0.13 g, 17% yield).

Step b:

DIAD (86. mu.L, 0.44 mmole) was added over 30 min at 25 deg.CMol) solution in THF (2.0 ml) was added to 1h (100 mg, 0.33 mmol), 4b (60.0 mg, 0.33 mmol) and PPh₃(114 mg, 0.44 mmol) was dissolved in a solution of THF (10 ml). The mixture was stirred for 1 hour and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (hexane: EtOAc 7: 3 to 1: 1) to provide compound 1031(128 mg, 83% yield).

Step c:

1.0N aqueous lithium hydroxide (1.52 mL, 1.52 mmol) was added to a solution of compound 1031(100 mg, 0.22 mmol) in THF (6 mL) and MeOH (2 mL). The reaction mixture was stirred at 25 ℃ for 24 hours and then heated under reflux for 1 hour. The solution was made acidic by addition of 1.0N aqueous HCl and then extracted with EtOAc. The organic layer was washed with water (2 ×) and brine, then dried (MgSO)₄) Filtered and concentrated under reduced pressure. The residue was taken up in Et₂Trituration with O/hexane afforded compound 1003 as a white solid (80 mg, 83% yield). A mixture of compound 1003(38.0 mg, 0.085 mmol) and 0.02N aqueous NaOH (4.3 ml, 0.085 mmol) was sonicated in MeCN (3 ml). The resulting solution was lyophilized to give the corresponding sodium salt as a white solid (37 mg, 98% yield).

Example 5 (compound numbers 1019, 1020 and 1028):

step a:

a solution of DIAD (86. mu.L, 0.44 mmol) in THF (2.0 mL) was added to 3i (106 mg, 0.34 mmol), 5a (56.0 mg, 0.34 mmol) and PPh at 25 deg.C over 2 hours₃(114 mg, 0.44 mmol) was dissolved in a solution of THF (7 ml). The mixture was stirred for 1 hour and then concentrated under reduced pressure. The residue was purified by flash chromatography (Hexane: EtOAc 7: 3 to 1: 1) to give 5b (115) as a white solidMg, 73% yield).

Step b:

1.0N aqueous lithium hydroxide (1.0 mL, 1.0 mmol) was added to a solution of 5b (100 mg, 0.21 mmol) in MeOH (6 mL). The reaction mixture was stirred at 25 ℃ for 24 h, the solution was made acidic by addition of 1.0N aqueous HCl and then extracted with EtOAc. The organic layer was washed with water and brine and dried (MgSO)₄) Filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (hexane: EtOAc: AcOH 50: 1) to give first compound 1019 as a white solid (25 mg, 25% yield) and then compound 1020 as a white solid (48 mg, 50% yield). The corresponding sodium salt is obtained by treating with 0.02N NaOH aqueous solution.

Step c:

a 1.0M solution of dimethylamine in THF (5.0 ml, 5.0 mmol) and 1.0N aqueous lithium hydroxide (1.0 ml, 1.0 mmol) were added to a solution of 5b (50.0 mg, 0.11 mmol) in i-PrOH (3 ml). The reaction mixture was heated to reflux for 48 hours. To this mixture was added 1.0N aqueous HCl (2 ml) and extracted with EtOAc. The organic layer was washed with water and brine and dried (MgSO)₄) Filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (hexane: EtOAc: AcOH 50: 1) to provide compound 1028 as a white solid (18 mg, 35% yield). The corresponding sodium salt was treated with 0.02N NaOH.

Example 6 (compound number 1014):

step a:

to acid 6a (1.00 g, 5.55 mmol) was dissolved in CH₂Cl₂To a solution (50 ml) was added oxalyl chloride (0.73 ml, 8.3 mmol) and DMF (100 μ l). Mixing the reactionThe solution was stirred for 90 minutes, then EtOH (15 ml) was added and the resulting reaction mixture was stirred for an additional 1 hour. The reaction mixture was concentrated under reduced pressure, the resulting residue was diluted with EtOAc and washed successively with water and brine, then dried (MgSO)₄) Filtered and concentrated to give the ester 6b, which was used without further purification.

Step b:

to the ester 6b in CH₂Cl₂To the solution (50 ml) was added 1M BBr₃To a solution of dichloromethane (7.2 ml, 7.20 mmol). After 3 hours reaction at room temperature, the reaction mixture was cooled to 0 ℃ and EtOH (5 ml) was added. The reaction mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. The residue was diluted with EtOAc and successively with NaHCO₃Washed with saturated aqueous solution, water and brine, and then dried (MgSO)₄) Filtered and concentrated to dryness. The resulting residue was purified by flash chromatography (hexane: EtOAc 70: 30) to yield phenol 6c as a clear gum (802 mg, 74% of two-step overall yield).

Step c:

a solution of DIAD (87. mu.L, 0.44 mmol) in THF (2.0 mL) was added to 1h (100 mg, 0.33 mmol), Ph over 2 hours at room temperature₃P (104 mg, 0.44 mmol) and phenol 6c (65 mg, 0.34 mmol) were dissolved in a solution of THF (7.0 ml). The mixture was stirred for 4 hours and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (hexane: EtOAc 30: 70 to 50: 50) to give compound 6d as a white foam (46 mg, 29% yield).

Step d:

to a solution of ester 6d (44 mg, 0.09 mmol) in a mixture of THF (3 ml) and MeOH (1 ml) was added 1N aqueous lithium hydroxide (1.0 ml, 1.0 mmol). After 4 hours at room temperature, 1N HCl (2 ml) was added. The mixture was extracted with EtOAc. The organic layer was washed with water and brine and dried (MgSO₄) Filtering and concentrating to obtain compound 1 as white solid014(39 mg, 93% yield). The corresponding sodium salt was obtained by treating with 1 equivalent of aqueous sodium hydroxide and lyophilizing the resulting solution to give a fluffy white solid.

TABLE 1

Compound numbering	R2	R4	R5	R12	R13	R14	MS(ES1)m/z(MH)+
								1001	H	H	Me	Me	Me	COOH	447
1002	H	H	Me	Me	H	COOH	433
								1003	H	H	Me	Et	H	COOH	447
1004	H	H	Me	Me	OH	COOH	449
								1005	H	H	Me	Br	H	COOH	497/499
1006	H	H	Me	Me	H	CHMeCOOH	461
								1007	Cl	H	Me	Me	H	CH2CH2COOH	495/497
1008	H	H	Me	Me	H	CH2CH2COOH	461
								1009	Cl	H	Me	Me	H	CH＝CHCOOH	493/495
1010	H	H	Me	Me	H	CH＝CHCOOH	459
								1011	H	H	Me	Cl	H	CH2COOH	467/469
1012	H	H	Me	Br	H	CH2COOH	511/513
								1013	H	H	Me	Me	Me	CH2COOH	461
1014	H	H	Me	Me	H	CH2COOH	447
								1015	Cl	H	Me	Me	H	COOH	467/469
1016	Cl	Me	H	Me	H	COOH	467/469
								1017	Me	H	Me	Me	H	COOH	447
1018	H	Me	H	Me	H	COOH	433
								1019	OMe	H	Me	Me	H	COOH	463
1020	F	H	Me	Me	H	COOH	451
								1021	OEt	H	Me	Me	H	COOH	477

1022	H	H	Me	NO2	H	COOH	464
								1023	H	H	Me	Cl	H	COOH	453/455
1024	H	H	Me	Cl	NH2	COOH	434
								1025	H	H	Me	H	F	COOH	437
1026	H	H	Me	H	Cl	COOH	453/455
								1027	H	H	Me	CF3	H	COOH	487
1028	N(Me)2	H	Me	Me	H	COOH	476
								1029	H	H	H	Me	H	COOH	419
1030	H	H	Me	Me	H	COOMe	447
								1031	H	H	Me	Et	H	COOMe	461
1032	H	H	Me	Cl	H	COOMe	467/469
								1033	H	H	Me	CF3	H	COOMe	501
1034	OEt	H	Me	Me	H	COOMe	491
								1035	H	H	Me	H	F	COOMe	451

Reverse Transcriptase (RT) assay

Principle of measurement

Reverse transcriptase (1) is one of the many enzymes encoded by the human immunodeficiency virus (HIV-1), which is so named because it can transcribe DNA copies from an RNA template. This activity can be quantified in a cell-free enzymatic assay and assessed based on the observation of the ability of reverse transcriptase to transcribe radiolabeled DNA strand using 3H-dGTP substrate using a synthetic template, poly (r) (C) and biotinylated oligo d (G) primer. The assays described below utilize wild-type enzymes, which are the predominant form of the enzyme observed in HIV-1 infected patients, and may also utilize mutant RT enzymes (e.g., Y181C, prepared by site-directed mutagenesis in which the tyrosine residue of codon 181 is replaced by a cysteine residue) under similar assay conditions. The efficacy of a compound to inhibit a mutant enzyme can be determined by this assay.

Materials:

a) preparation of enzymes

The HIV-1 IIIB clone BH10 RT mutant was supplied as vector pKK233-2(Pharmacia) by Dr.C. -K.Shih (Boehringer Ingelheim pharmaceuticals Inc., U.S.A.). Briefly, HIV RT clone pKRT2, which contains only RT p66 gene regulated by the lactose operon/trc promoter, was obtained from dr.w Summers (university of yale) (2). Various specific amino acid substitutions were introduced into the wild type RT gene by site directed mutagenesis. The RT clone was subcloned into pKK233-2 bacterial expression vector. The clones provided include wild type, Vall06Ala, Tyr181Cys, Tyr188Leu, Gly190Ala and Pro236 Leu. Others were made by site-directed mutagenesis of the pKK233-2 RT clones themselves, including Lys103Asn, Lys103Asn/Tyr181Cys, Lys103Asn/Leu100lle, Lys103Asn/Pro225His, and Lys103Asn/Val108 lle.

b) Purification of enzymes

Purification of the recombinant reverse transcriptase was carried out by a combination of the aforementioned methods (3). Single colonies from fresh plates of transformed JM109 cells were precultured at 37 ℃. The pre-cultured seed was inoculated into 2 liters of growth medium. At OD₆₀₀RT gene expression was induced with IPTG (1 mM final) at 1.5 (5-6 hours at 37 ℃) and fermentation was continued for several more hours at 37 ℃. After centrifugation, the supernatant was removed and the cell pellet was collected and stored at-80 ℃ for purification. Cells were thawed overnight at 4 ℃ and suspended in lysis buffer (MES: 50mM, pH 6; EDTA: 1 mM; 10% v/v glycerol; 0.02% w/v vOBG; 0.02% w/v sodium azide). Lysozyme was added and the mixture was incubated in an ice bath for 40 minutes. After homogenization with Dounce in the presence of lysozyme and sonication, the cells were centrifuged for 30 minutes. The supernatant was collected (S1) and stored at 4 ℃. The pellet obtained after centrifugation was resuspended in extraction buffer (MES: 50mM, pH 6; KPO)₄: 50mM, pH 6; KCl: 100 mM; 10% v/v glycerol; 0.02% w/v OBG; 0.02% w/v sodium azide) and stirred at 4 ℃ for 30 minutes. This second mixture was centrifuged and the resulting supernatant was collected (S2). Repeating the above steps for more than 2 times, collecting supernatant S3 and S4, and extracting overnightAnd (S5). Polymin P (final concentration of 0.005%) was added to the combined supernatants to remove nucleic acids. The solution was stirred at 4 ℃ for 75 minutes and centrifuged for 1 hour. The supernatant (SS1) was precipitated with 20% w/v ammonium sulphate on ice and stirred at 4 ℃ for 1 hour. The mixture was then centrifuged and an additional 40% w/v ammonium sulfate (60% total) was added to precipitate the resulting supernatant (SS2), which was stirred for 1 hour and centrifuged again. The final precipitate (P1) was stored overnight at 4 ℃ before purification on the next day. All purification steps were performed at 4 ℃ unless otherwise specified. Resuspend pellet (P1) in MES-containing, 50mM, pH 6; KPO₄10mM, pH 6; KCl, 100 mM; 10% v/v glycerol; 0.02w/v OBG; and 0.02w/v sodium azide. This suspension was dialyzed overnight against the same buffer using a 12-14kD MWCO dialysis bag. The dialysate was centrifuged and the resulting supernatant filtered through a Millex-PF 0.8 micron filtration apparatus, the filtered sample was loaded onto a hydroxyapatite column (30 ml bed volume) and eluted with the same buffer, 220 ml KPO containing a linear gradient of 10-300 mM₄The bound enzyme is eluted with the same buffer. Fractions containing the p66/p51 heterodimer were collected (as determined by 8% SDS-PAGE and Western blotting) and subjected to further purification. The RT-containing fractions were diluted 2-fold with a solution containing Bis-Tris propane (50mM, pH7.0), OBG (0.02% w/v), glycerol (10% v/v) and sodium azide (0.02% w/v) and then loaded onto a Hi-Trap Hepparin Sepharose column (5 ml bed volume) and eluted with the same buffer. The bound RT was then eluted with 75 ml of the same buffer containing a linear gradient of 0-1M ammonium sulfate. Fractions containing RT were collected by SDS-PAGE and Western blot analysis. The protein concentration in this collection was determined by the Bradford method using BSA as a standard. The final enzyme preparation was incubated in a medium containing MES (50mM, pH6), KPO₄(300mM, pH6), KCl (175mM), glycerol (10% v/v) and sodium azide (0.02% w/v) were dialyzed, then aliquoted and frozen at-80 ℃.

The determination step comprises:

the radioactive enzyme assay was modified to a format suitable for 96-well microtiter plates and used strandsAvidin scintillation was performed adjacent to the beads. This assay will be briefly described below. HIV-1RT was frozen and diluted appropriately with Tris/HCl (50mM, pH7.8) containing NaCl (60mM), magnesium chloride hexahydrate (2mM), DTT (6mM), GSH (2mM) and Chaps (0.02% w/v) to give approximately 3nM of enzyme. To 30 microliters of this enzyme solution was added 10 microliters of inhibitor solution (50 μ M to 2.5nM inhibitor dissolved in the same assay buffer as described above, containing 15% v/vDMSO). This microtiter plate was preincubated for 15 minutes at room temperature before proceeding to the next step. In this pre-incubation step, the highest and lowest concentrations of inhibitor were 12.5. mu.M and 0.62nM, respectively, and the DMSO concentration was 3.75% v/v. Then 10. mu.l of substrate solution was added to initiate the enzyme reaction. The final reaction mixture contained Tris/HCl (50mM, pH7.8), NaCL (60mM), magnesium sulfate hexahydrate (2mM), DTT (6mM), GSH (2mM), Chaps (0.02% w/v), DMSO (3% v/v), Poly rC (179nM), biotin dG₁₅(18nM)、dGTP(288nM)、³H-dGTP (71nM) and enzyme (1-2 nM).

The maximum and minimum concentrations of inhibitor during this incubation step were 10. mu.M and 0.5nM, respectively. After addition of the substrate, the microtiter plates were covered with plastic seals and incubated for 1 hour at 37 ℃ in a dry incubator. The reaction was then stopped by adding 75. mu.l of EDTA (0.5 mol/l) containing 5 mg/ml of streptavidin adjacent beads.

The microtiter plates were shaken at medium speed for 2 minutes and incubated at room temperature for 1 hour. Then 75 microliters of 7M cesium chloride solution was added, the microtiter plate was shaken at a medium speed for 2 minutes and further incubated at room temperature for 1 hour. The microtiter plates were then covered with plastic and used to apply TopCount-NXT^TMThe microplate scintillation fluorescence counter (Packard) counts. Count 60 seconds for each well. The end of each row contains a blank well and a control well.

Percent inhibition was calculated as follows:

the compounds of the invention were tested for inhibition of RT Wild Type (WT) and mutant enzymes using the assays described above. The results of the measurements are given in Table 2 as IC₅₀The (nM) format is listed.

To confirm the inhibitory potency of the compounds against HIV replication, they were also tested in the following human T cell culture (Syncytia) assay.

ELISA assay for determining activity in cell culture

The ability of the compounds of the invention to inhibit HIV replication in cell culture was tested in a 96-well plate assay. Compounds and cell growth media were diluted with complete RPMI1640 (consisting of RPMI1640 + 10% fetal bovine serum, 10. mu.g/ml gentamicin and 10. mu.M beta-mercaptoethanol). The T lymphocyte cell line C8166 was infected multiple times with viruses encoding wild type and mutant reverse transcriptases at an infection rate of 0.001. The cells were then cultured for three days in the presence of a series of dilutions of the compound of the invention. Supernatants from eight identical wells were collected and tested using a commercially available HIV-1 p24 antigen detection kit (Bechman-Coulter)^®) The concentration of extracellular p24 was determined. The level of inhibition (percent inhibition) was calculated by the following formula:

the results of these measurements are reported in Table 2 as EC₅₀The (nM) format is listed.

References (incorporated by reference herein)

Benn, s, et al, Science 230: 949, 1985.

D' Aquila, r.t. and summmers, w.c.j.acq.imm.def.syn.2: 579, 1989.

3.a) Warren, T.C. et al, Protein Expression & Purification 3: 479, 1992; b) kohlstaedt, l.a. science 256 (5065): 1783, 1992.

TABLE 2

Inhibition of wild type and RT mutant strains by Compounds of formula I

Numbering	IC50(WT)(nM)	IC50K103N/Y181C(nM)	EC50(WT)(nM)	EC50K103N/Y181C(nM)
					1001	C	A	C	C
1002	C	A	C	C
					1003	C	A	C	C
1004	C	A	C	A
					1005	C	A	C	C
1006	C	A	NT	NT
					1007	C	B	C	B
1008	C	A	C	A
					1009	C	C	C	C
1010	C	C	C	C
					1011	C	A	NT	NT
1012	C	A	NT	NT
					1013	C	A	NT	NT
1014	C	A	C	A
					1015	C	B	C	C
1016	C	A	NT	NT
					1017	C	A	C	C
1018	C	A	C	C
					1019	C	A	C	C
1020	C	A	C	C
					1021	C	A	NT	NT
1022	B	A	B	A
					1023	B	A	C	A
1024	C	A	NT	NT
					1025	B	NT	NT	NT

1026	B	NT	NT	NT
					1027	B	A	NT	NT
1028	C	A	NT	NT
					1029	C	A	C	NT
1030	C	C	C	C
					1031	C	C	NT	NT
1032	C	C	NT	NT
					1033	C	A	NT	NT
1034	C	B	NT	NT
					1035	C	A	NT	NT

The symbols in the table illustrate:

IC₅₀and EC₅₀: a > 100 nM; b-100 nM-50 nM; c is less than 50 nM; and NT is not detected

Claims (23)

1. A compound of formula I:

wherein the content of the first and second substances,

R²is H, halogen, (C)_1-4) Alkyl, O (C)_1-4) Alkyl, HN (C)_1-4Alkyl) or N (C)_1-4Alkyl radical)₂；

R⁴Is H or CH₃；

R⁵Is H orCH₃Provided that R is⁴And R⁵Different;

R¹²is H, halogen, (C)_1-4) Alkyl, CF₃Or NO₂；

R¹³Is H, (C)_1-4) Alkyl, halogen, OH or NH₂Provided that R is¹²And R¹³Not H at the same time; and

R¹⁴is COOR^14aWherein R is^14aIs H or (C)_1-6) An alkyl group; or R¹⁴Is (C)_2-4) alkenyl-COOR^14aWherein R is^14aThe definition is the same as above; or R¹⁴Is (C)_1-4) alkyl-COOR^14aWherein R is^14aThe definition is the same as that of the above,

or a salt thereof.

2. A compound of claim 1, wherein R²Is H, halogen, (C)_1-4) Alkyl, O (C)_1-4) Alkyl or N (C)_1-4Alkyl radical)₂And R is⁴And R⁵Are not identical.

3.A compound of claim 2, wherein R²Is H, Cl, F, (C)_1-4) Alkyl, O (C)_1-4) Alkyl or N (C)_1-4Alkyl radical)₂。

4. A compound of claim 3, wherein R²Is H, Cl, F, CH₃OMe or OEt.

5. A compound of claim 4, wherein R²Is H.

6. A compound of claim 1, wherein R⁴Is H.

7. A compound of claim 1, wherein R⁵Is CH₃。

8. A compound of claim 1, wherein R¹²Is halogen, (C)_1-4) Alkyl, CF₃Or NO₂。

9. A compound of claim 8, wherein R¹²Is Br, Cl, CH₃Or CH₃CH₂。

10. The compound of claim 9, wherein R¹²Is CH₃Or CH₃CH₂。

11. A compound of claim 1, wherein R¹³Is H, CH₃Halogen, OH or NH₂。

12. The compound of claim 11, wherein R¹³Is H, CH₃Or OH.

13. The compound of claim 12, wherein R¹³Is H.

14. A compound of claim 1, wherein R¹⁴Is COOH, COOMe, (C)_2-4) alkenyl-COOH or (C)_1-4) alkyl-COOH.

15. The compound of claim 14, wherein R¹⁴Is COOH, CH ═ CH-COOH, CH₂COOH or CH₂CH₂COOH。

16. The compound of claim 15, wherein R¹⁴Is COOH.

17. A compound of formula I according to claim 1:

wherein the content of the first and second substances,

R²is H, Cl, F, CH₃OMe or OEt; r⁴Is H; r⁵Is CH₃；R¹²Is Br, Cl, CH₃Or CH₂CH₃；R¹³Is H, CH₃Or OH; and R¹⁴Is COOH, CH ═ CH-COOH, CH₂COOH or CH₂CH₂COOH；

Or a salt thereof.

18. The compound of claim 17, wherein R²Is H; r⁴Is H; r⁵Is CH₃；R¹²Is CH₃Or CH₃CH₂；R¹³Is H; and R is¹⁴Is COOH; or a salt thereof.

19. A pharmaceutical composition for the treatment or prophylaxis of HIV infection comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, according to claim 1, and a pharmaceutically acceptable carrier.

20. Use of the pharmaceutical composition of claim 19, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing HIV infection.

21. The use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of HIV infection.

22. The use of claim 21, wherein the medicament is administered in combination with an antiretroviral drug.

23. Use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of perinatal HIV-1 infection from mother to infant before delivery.