Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/18—Sulfonamides

Definitions

• This invention relates to the use of sulfonamide substituted diphenyl urea compounds to treat cystic fibrosis or non-cystic fibrosis bronchiestasis.
• CXCR2 is a well-characterized G-protein coupled receptor for a number of chemokines that share the Glu-Leu-Arg motif including interleukin-8 (IL-8, CXCL8) and growth regulated oncogene alpha, beta and gamma, (GRO ⁇ , ⁇ , ⁇ or CXCLl, 2,3 ) that are known to be involved in the recruitment of neutrophils to a site of injury [Reutershan, J. (2004) Drug News Perspect 19:615-623].
• CXCR2 is expressed primarily on neutrophils (PMN), but can be expressed on other leukocytes as well such as monocytes.
• Antagonism of CXCR2 has been shown to be effective in blocking the recruitment of PMN to the lung in response to stimuli such as LPS, cigarette smoke, or ozone exposure. It is expected that antagonism of CXCR2 would be of utility in preventing the recruitment of neutrophils to the lung in response to a wide spectrum of conditions related to disease, such as cystic fibrosis or non-cystic fibrosis bronchiectasis.
• Cystic fibrosis is a disease primarily characterized by recurrent cycles of infection and inflammation, particularly neutrophil predominant pulmonary cellular infiltration. It is the most common genetic disease in Caucasians. Cystic fibrosis is a disease involving epithelial cells throughout the body, causing mucus in the body to become thick, dry and sticky. Symptoms include blocked intestines at birth, salty sweat or skin, diarrhea, breathing problems, lung infection, persistent cough, wheezing, clubbing of the fingers, rectal prolapse and polyps in the nose or sinuses. Non-CF bronchiectasis
• bronchiectasis of the lung caused by conditions other than cystic fibrosis is characterized by cough, sputum production, recurrent infection and exacerbation.
• the lung inflammation and destruction in non-CF bronchiectasis is caused in-part by exuberant neutrophilic inflammation.
• a CXCR2 inhibitor would thus be expected to decrease neutrophilic damage to the lung, decrease inflammation, and thus, preserve lung function and diminish symptoms associated with the disease.
• cystic fibrosis In people with cystic fibrosis, buildup of thick mucus obstructs the airways, making breathing difficult and creating an environment for infection-causing bacteria to grow. To fight infection, neutrophils migrate to the lungs, releasing enzymes such as elastase which can damage the lungs. Neutrophils also release DNA and participate in killing of bacteria, which further thickens the mucus. The thicker mucus, in turn, causes more obstruction, which causes more infection and inflammation. The natural history of cystic fibrosis is such that there is an inexorable progression of disease symptoms resulting in continued loss of pulmonary function with increasing morbidity and mortality.
• Treatments for cystic fibrosis lung disease presently include inhaled hypertonic saline and DNAse to aid in airway clearance, as well inhaled antibiotics, and antiinflammatory agents such as inhaled corticosteroids or oral azithromycin.
• Other agents, such as high dose ibuprofen or oral steroids, which are effective anti-inflammatory agents have their use limited due to their gastrointestinal effects and adverse effects on bone density.
• ibuprofen or oral steroids which are effective anti-inflammatory agents
• CXCR2 agonists such as IL-8/CXCL8 and ENA-78/CXCL5 are highly expressed in the airways of patients with cystic fibrosis.
• IL-8 is considered to play a pivotal role in chronic airway inflammation through its actions as a neutrophil chemoattractant. The recruitment of activated neutrophils into the lung results in release of chromatin as well as proteases and oxidants. In addition, neutrophil killing of bacteria can release DNA.
• IL-8 is also elevated in the serum of patients with cystic fibrosis, where it may be involved in systemic processes such as osteoporosis or weight loss.
• CXCRl and CXCR2 expression is increased on peripheral blood neutrophils in patients with cystic fibrosis. Sputum concentrations of IL-8, MPO, and DNA appear to correlate with pulmonary function in subjects with cystic fibrosis. CXCR2 is also expressed on other cell types within the lung such as airway smooth muscle cells. CXCR2 has been shown to mediate smooth muscle cell contractile responses, suggesting a possible role for the receptor in regulating airway hyperreactivity. The dysregulated expression of IL-8 and CXCR2 in patients with cystic fibrosis suggests that inhibition of CXCR2 may provide a therapeutic benefit to patients with cystic fibrosis.
• the present invention relates to a method of treating cystic fibrosis in a human subject, previously diagnosed as having cystic fibrosis, in need thereof.
• the method involves administering to the subject having cystic fibrosis, N-[4-chloro-2-hydroxy-3- (piperazine-l-sulfonyl)phenyl]-N'-(2-chloro-3-fluorophenyl)urea, or a pharmaceutically acceptable salt thereof, in an amount effective to treat cystic fibrosis, or symptoms, such as reduced lung function, cough, exacerbations and mucus accumulation in the lung, associated with cystic fibrosis in that subject.
• the present invention also relates to a method of slowing the progression of cystic fibrosis in a human subject in need thereof.
• the method involves administering to the subject N-[4-chloro-2-hydroxy-3-(piperazine- 1 -sulfonyl)phenyl]-N'-(2-chloro-3- fluorophenyl)urea, or a pharmaceutically acceptable salt thereof, in an amount effective to slow the progression of cystic fibrosis, or the symptoms of cystic fibrosis in the subject.
• the present invention also relates to a method of alleviating the symptoms of cystic fibrosis in a human subject in need thereof.
• the method involves administering to the subject N-[4-chloro-2-hydroxy-3-(piperazine-l-sulfonyl)phenyl]-N'-(2-chloro-3- fluorophenyl)urea, or a pharmaceutically acceptable salt thereof, in an amount effective to alleviate the symptoms of cystic fibrosis in the subject.
• the present invention also relates to a method of treating the inflammatory response, such as increased lung neutrophilia or mucus hypersecretion, associated with cystic fibrosis in a patient who has been diagnosed with cystic fibrosis.
• This method involves administering to the subject N-[4-chloro-2-hydroxy-3-(piperazine-l- sulfonyl)phenyl]-N'-(2-chloro-3-fluorophenyl)urea, or a pharmaceutically acceptable salt thereof, in an amount effective to decrease the inflammation associated with cystic fibrosis in the subject.
• the present invention is directed to various methods of treating cystic fibrosis, or the symptoms associated with cystic fibrosis in a mammal, suitably a human in need thereof, by administering to said mammal or human, an effective amount of N-[4-chloro-2- hydroxy-3-(piperazine- 1 -sulfonyl)phenyl]-N'-(2-chloro-3-fluorophenyl)urea or a pharmaceutically acceptable salt thereof.
• cystic fibrosis encompasses, but is not limited to, reducing inflammation in the lung present in a subject, reducing mucus accumulation in the lung, reducing excessive mucus production, reducing pulmonary neutrophilia, reducing mucus production generally, decreasing mucus dysfunction, reducing mucus viscosity leading to improved mucociliary clearance, improving lung function, reducing risk of exacerbations, diminishing cough, reducing the frequency and/or severity of exacerbations which can include hospitalizations, thereby reducing the number of hospitalizations for a subject have cystic fibrosis.
• Another embodiment of the invention is directed to a method of slowing the progression of cystic fibrosis in a mammal, suitably a human in need thereof, which comprises administering to said mammal, or human, an effective amount of N-[4-chloro-2- hydroxy-3-(piperazine- 1 -sulfonyl)phenyl]-N'-(2-chloro-3-fluorophenyl)urea, or a pharmaceutically acceptable salt thereof.
• successful treatment of slowing the progression of cystic fibrosis encompasses, but is not limited to, reducing pulmonary neutrophilia, reducing mucus production, decreasing mucus dysfunction, reducing mucus viscosity leading to improved mucocilliary clearance, improving lung function, reducing the risk of exacerbations, diminishing cough., reducing frequency and/or severity of exacerbations, which can include hospitalizations, thereby reducing the number of hospitalizations for a subject have cystic fibrosis.
• This invention also provides for a method of alleviating or reducing the symptoms of cystic fibrosis which comprises administering to a mammal, suitably a human in need thereof, an effective amount of N-[4-chloro-2-hydroxy-3-(piperazine-l-sulfonyl)phenyl]- N'-(2-chloro-3-fluorophenyl)urea, or a pharmaceutically acceptable salt thereof.
• symptoms of cystic fibrosis include, but are not limited to, breathing problems, lung infection, persistent cough, wheezing, excessive mucus production and clubbing of the fingers. Therefore successful treatment with this selective CXCR2 antagonist is expected to improve lung function, reduce the number and severity of infection induced exacerbations (and associated hospitalizations) and improve patient's quality of life ultimately resulting in increased survival.
• Airway neutrophilia has been observed in infants with CF, and increases throughout life, is associated with decreased lung function, thickened mucus, reduced microbial clearance, and protease-mediated lung destruction. Therefore, another aspect of this invention is the treatment of pediatrics and infants diagnosed with cystic fibrosis.
• the compound, N-[4-chloro-2-hydroxy-3-(piperazine- 1 -sulfonyl)phenyl]-N'-(2- chloro-3-fluorophenyl)urea, and/or its pharmaceutically acceptable salts offer an alternative for the treatment of cystic fibrosis, representing a different class of compounds to currently available treatment options.
• the mainstay of initial therapy for cystic fibrosis includes inhaled hypertonic saline and DNAse to aid in airway clearance. Inhaled antibiotics and anti-inflammatory agents are also utilized.
• Patients with cystic fibrosis are currently treated with several classes of therapeutic agents which, in some patients, are either ineffective or have significant side effects.
• the present compound offers an alternative treatment option which may provide a better benefit/risk ratio to the patient.
• a subject diagnosed as having cystic fibrosis such as one who has been genetically identified as an asymptomatic individual, or a patient identified by a salty sweat test, or is a patient identified by CT as having bronchiectasis but who is asymptomatic may be treated with an effective amount of N-[4-chloro-2-hydroxy-3- (piperazine-l-sulfonyl)phenyl]-N'-(2-chloro-3-fluorophenyl)urea, or a pharmaceutically acceptable salt thereof
• Another aspect of the invention is the use of N-[4-chloro-2-hydroxy-3-(piperazine- l-sulfonyl)phenyl]-N'-(2-chloro-3-fluorophenyl)urea, or a pharmaceutically acceptable salt thereof in the treatment of non-cystic fibrosis bronchiestasis.
• the compound used in the method of the invention can be administered as a free base or as a pharmaceutically acceptable salt thereof.
• suitable pharmaceutically acceptable salts are well known to those skilled in the art, and include salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methane sulfonic acid, ethane sulfonic acid, toluenesulfonic acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandelic acid.
• Pharmaceutically acceptable salts may also be formed with pharmaceutically acceptable cations.
• Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations.
• One embodiment of the invention is the hydrochloric acid salt of N-[4-chloro-2- hydroxy-3-(piperazine-l-sulfonyl)phenyl]-N'-(2-chloro-3-fluorophenyl)urea.
• Another embodiment of the invention is the p-toluenesulfonic acid salt of N- [4- chloro-2-hydroxy-3-(piperazine-l-sulfonyl)phenyl]-N'-(2-chloro-3-fluorophenyl)urea.
• compound refers to N-[4-chloro-2-hydroxy-3- (piperazine- 1 -sulfonyl)phenyl] -N'-(2-chloro-3 -fluorophenyl)urea and any pharmaceutically acceptable salt thereof of said urea.
• This invention also relates to use of a pharmaceutical composition comprising an effective, amount of N-[4-chloro-2-hydroxy-3-(piperazine-l-sulfonyl)phenyl]-N'-(2-chloro- 3-fiuorophenyl)urea or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or diluent in any the methods described herein.
• the present compound and a pharmaceutical composition incorporating said compound, or salt thereof may conveniently be administered by any of the routes conventionally used for drug administration, for instance orally, parenterally, or via inhalation.
• the present compounds may also be administered in conventional dosages in combination with other known, second therapeutically active compounds which are also used for the treatment of cystic fibrosis, or symptoms associated with a patient being diagnosed with cystic fibrosis.
• the present compound may be administered in conventional dosage forms prepared by combining with standard pharmaceutical carriers according to conventional procedures. It will be appreciated that the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
• the carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• the pharmaceutical carrier employed may be, for example, either a solid or liquid.
• solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
• liquid carriers are syrup, peanut oil, olive oil, water and the like.
• the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
• a wide variety of pharmaceutical forms can be employed.
• the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
• the amount of solid carrier will vary widely but preferably will be from about 25mg to about Ig.
• the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquids or nonaqueous liquid suspensions.
• the present compound may be employed via suitable methods of administration, including oral, parenteral, intravenous, intramuscular, subcutaneous, intranasal and intraperitoneal administration. Appropriate dosage forms for such administration may be prepared by conventional techniques.
• the daily oral dosage regimen will preferably be from about 0.1 to about 3 mg/kg of total body weight given in divided doses if necessary
• the daily parenteral dosage regimen about 0.001 to about 3mg/kg of total body weight.
• the optimal quantity and spacing of individual dosages of a present compound will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques.
• the optimal course of treatment i.e., the number of doses of a present compound of or a pharmaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
• the compound used in the methods of the present invention may be tested in the following assays.
• [125j] jL_g (human recombinant, IM249) was obtained from Amersham Corp., Arlington Heights, IL, with specific activity 2000 Ci/mmol. All other chemicals were of analytical grade.
• the Chinese hamster ovary membranes were prepared according to Haour, et al., J 1 Biol. Chem.. 249 pp 2195-2205 (1974), incorporated herein to the extent required to perform the present assay, except that the homogenization buffer was changed to 40 mM Tris-HCL pH 7.5 containing 1 mM MgSO ⁇ 0.5 mM EDTA (ethylene-diaminetetra-acetic acid), 1 mM PMSF ( ⁇ -toluene-sulphonyl fluoride), 2.5 mg/L Leupeptin and 0.1 mg/ml Aprotinin.
• Tris-HCL pH 7.5 containing 1 mM MgSO ⁇ 0.5 mM EDTA (ethylene-diaminetetra-acetic acid), 1 mM PMSF ( ⁇ -toluene-sulphonyl fluoride), 2.5 mg/L Leupeptin and 0.1 mg/ml Aprotinin.
• Membrane protein concentration was determined using Bio-Rad Reagent using bovine serum albumin as a standard. All binding assays were conducted using Scintillation Proximity Assay (SPA assay) using wheatgerm agglutinin beads in a 96-well micro plate (optiplate 96, Packard) format. The membranes, CHO-CXCRl or CHO-CXCR2, were pre-incubated with the beads in binding buffer; 20 mM Bis Tris propane, pH 8 containing 25 mM NaCl, 1 mM MgSO 4 , 0.1 mM EDTA at 4 0 C for 30 min prior to assay.
• SPA assay Scintillation Proximity Assay
• the compound was diluted in 100% DMSO at 20 times the final concentration (final InM to 1000 nM and 5% DMSO).
• the assay was performed in 0.1 ml reaction buffer containing binding buffer, membranes pre-treated with wheatgerm agglutinin beads, various concentrations of compound, 5% DMSO, 0.04% CHAP, 0.0025% BSA and 0.225 nM [125 j] IL-8.
• the 96-well plates were incubated on a shaking platform for 1 hour. At the end of the incubation the plates were spun for 5 min at 2000 RPM, and counted in a Top Count counter.
• the recombinant CXCRl receptor is also referred to herein as the non-permissive receptor and the recombinant CXCR2 receptor is referred to as the permissive receptor.
• a compound demonstrating an IC50 value of ⁇ 10 uM is considered active in the present assay.
• a CXCR2 antagonist represents a different class of agents as compared with those presently used in cystic fibrosis and are expected to slow the disease progression of cystic fibrosis, at least in part by breaking the cycle of neutrophilic inflammation and infection.
• mice (20-25 g) or Lewis rats (250-275 g) from the same breeding groups were received from Jackson labs and used in these studies. Animals were housed for a minimum of five days under quarantine prior to the initiation of the studies.
• the compound of Example 1 e.g. the hydrochloride salt
• the compound of Example 1 was weighed-out and placed into a glass homogenizer with the appropriate amount of PEG-400 to yield a final concentration of 10%.
• the compound was homogenized in the PEG-400, and then brought to the final volume with distilled H 2 O.
• a stock solution of 10% volume/volume of PEG-400 was prepared for further compound dilution and to yield the final vehicle. All dosing solutions were vortexed immediately prior to administration.
• mice Male Lewis rats (325-350 g) (Charles River Labs, St. Constance, Canada), were dosed orally with either vehicle or the compound of Example 1, one hour prior to lipopolysaccharide (LPS) exposure. Rats were placed in groups of 5 into an aerosol exposure box (14 x 10 x 9 inches, Rubbermaid® 0100 - 2). After the compound of Example 1 was administered, animals were monitored for safe recovery before being returned to a cage. One hour post-dosing rats were exposed for 15 minutes to LPS (100 ug/ml) which was nebulized with a Hospitak nebulizer at a flow rate of 5 L/min into the exposure box.
• LPS lipopolysaccharide
• BAL Bronchoalveolar lavage
• Total cell counts were performed manually on a hemocytometer after diluting the resuspended cells 1 :2 with Tuerke solution.
• Differential cell slides were made with re-suspended cells using a Cytospin (5 min, 300 RPM) to form a monolayer of cells on the slide surface.
• the cells were stained with methylene blue and eosin (DiffQuik) and then differential counts were performed by light microscopy. A minimum of 200 cells per slide were counted.
• the compound of Example 1 demonstrated dose dependent inhibition of LPS- induced neutrophil recruitment in the lungs as measured in BAL fluid.
• LPS exposure of rats caused a characteristic neutrophil influx into the lungs that was assessed by enumerating cell numbers from bronchoalveolar lavage fluid.
• Oral administration of the compound of Example 1 (0.3-30 mg/kg in 10 % PEG-400) to rats caused a dose-related inhibition of BAL neutrophilia compared to vehicle-treated rats.
• the vehicle-treated group had a mean of 4.97 ⁇ 0.52 xl O 6 neutrophils and a significant inhibition in BAL neutrophilia was observed at doses of 3, 10 and 30 mg/kg of the compound of Example 1.
• IX FACS Lysing Solution (Becton Dickinson, San Jose, CA) was added with immediate vigorous vortexing, followed by additional vortex after the solution was added to the last sample. Samples were then incubated for 10 minutes at room temperature. The leukocytes were then pelleted at ⁇ 300Xg and washed with DPBS. Cells were then resuspended in 650 ul of 1% paraformaldehyde or 300 uL of Cell Fix (0.25 % paraformaldehyde, 0.025 M sodium azide, DPBS and FACS flow). The FACS Lyse solution does not completely lyse rat red blood cells.
• LDS-751 was added to each sample within 5 minutes of analysis to enable elimination of red blood cells. Either 5 ul of a 0.02% methanol stock (Molecular Probes) or 3 ul of a 1.67 mg/ml ethanol stock (Exciton) was used. Samples were analyzed using an LSR or CANTOS II flow cytometer (Becton-Dickinson) gating on the neutrophil population in the side scatter versus forward scatter plot. FLl (green FITC fluorescence, directly relating to CDl Ib content) of this population was then measured as mean channel fluorescence.
• mice (Balb/c) were dosed orally with 15 or 30 mg/kg of the compound of Example 1, volume of 10 ml/kg, or vehicle 1 hour prior to the first cigarette and immediately after the last daily cigarette on each of the 3 days of smoke exposure. Mice were placed into a small plexiglass chamber, 6 animals at a time. The chamber was fitted with an intake and outflow port. A cigarette (Reference 4Al, University of Kentucky Tobacco Institute) was attached to the intake of a peristaltic pump, and the smoke was driven into the intake port of the chamber containing the mice. An air-flow (250 ml/min) was also delivered into the chamber and mixed with the cigarette smoke.
• a cigarette Reference 4Al, University of Kentucky Tobacco Institute
• mice were exposed to the cigarette smoke for 4 minutes, the time required for the cigarette to burn down. Mice were exposed to 3 cigarettes per day for 3 days, with a minimum time of 2 hours between cigarettes. On day 4, mice were euthanized with Fatal Plus (i.p.) and bronchoalveolar lavaged with phosphate-buffered saline (5 x 0.7 ml). Cells were spun down, total cell counts were performed and slides were made on a cytospin and stained with Diff-Quick for differential cell counts. Cell counting was performed by counting 200 cells in cytospin samples by standard microscopy techniques. The percentage number of cells present was calculated and the total number of macrophages and neutrophils, in the BAL sample was then determined.
• Fatal Plus i.p.
• phosphate-buffered saline 5 x 0.7 ml
• Cigarette smoke exposure induced a significant recruitment of neutrophils in BAL fluid compared to untreated control mice.
• Oral treatment with the compound of Example 1 caused an inhibition of neutrophils by 56% at 15 and 30 mg/kg doses.
• Exposure of mice to 3 cigarettes per day for 3 days resulted in neutrophil recruitment into the lungs as measured by BAL approximately 18 hours after the last cigarette.
• There was no significant increase in any other cell types detected in BAL fluid after cigarette smoke exposure including macrophages, lymphocytes and eosinophils.
• mice were dosed orally with the compound of Example 1, one hour prior to ozone exposure. Mice were exposed to ozone (3 parts/million, ppm) or filtered air for 6 hrs, and BAL were performed at 24 post completion of the ozone exposure. Lewis rats were dosed orally with Example 1 1 hr prior to ozone exposure (3 ppm, 3 hrs), and BAL were performed at 4 hr post ozone exposure. In both cases, cytospins were obtained followed by differential cell staining using May-Grunwald and Giemsa solutions. Cell counting was performed by counting 300 cells, the percent of differential cells was calculated, and the total number of neutrophils, in the BAL was determined based on total cell counts.
• ozone caused an increase in BAL neutrophils, for example, from 0.07xl0 5 + 0.05 cells in air-exposed mice to 3.72xl0 5 +1.03 in ozone exposed mice.
• Treatment with the compound of Example 1 caused a dose dependent decrease in neutrophils (70 % inhibition at 30 mg/kg, p.o.; 36 % inhibition 10 mg/kg, and 6 % @ 3 mg/kg). Only the inhibition at 30 mg/kg was statistically significant using a one-way ANOVA with Bonferroni's correction, to p ⁇ 0.05.
• Nuclear magnetic resonance spectra were recorded at either 300 or 400 MHz using, respectively, a Bruker ARX 300 or Bruker AVANCE 400 spectrometer.
• CDCI3 is deuteriochloroform
• DMSO-d6 is hexadeuteriodimethylsulfoxide
• CD3OD is tetradeuteriomethanol. Chemical shifts are reported in parts per million ( ⁇ ) downfield from the internal standard tetramethylsilane.
• FTIR Fourier transform infrared
• FTIR spectra were recorded on a Nicolet 510 infrared spectrometer.
• FTIR spectra were recorded in transmission mode, and band positions are reported in inverse wave numbers (cm'l).
• Mass spectra were taken on either a SCIEX5 or Micromass instruments, using electrospray (ES) ionization techniques. Elemental analyses were obtained using a Perkin-Elmer 240C elemental analyzer. Melting points were taken on a Thomas-Hoover melting point apparatus and are uncorrected. All temperatures are reported in degrees Celsius.
• ODS refers to an octadecylsilyl derivatized silica gel chromatographic support. 5 ⁇ Apex-ODS indicates an octadecylsilyl derivatized silica gel chromatographic support having a nominal particle size of 5 ⁇ , made by Jones Chromatography, Littleton, Colorado.
• YMC ODS-AQ® is an ODS chromatographic support and is a registered trademark of YMC Co. Ltd., Kyoto, Japan.
• PRP- 1® is a polymeric (styrene-divinylbenzene) chromatographic support, and is a registered trademark of Hamilton Co., Reno, Nevada)
• Celite® is a filter aid composed of acid- washed diatomaceous silica, and is a registered trademark of Manville Corp., Denver, Colorado.
• the following Examples are intended to be illustrative only and not limiting in any way.
• 3,4-dichloroaniline 100 g was dissolved in TBME (660 mL) and cooled to 10-15 0 C.
• Sodium hydroxide 94 g of a 30% aqueous solution
• Trimethylacetyl chloride 84 mL was added at such a rate as to keep the internal temperature below 35 0 C.
• the addition was complete (10-15 min), the mixture was maintained at 30-35 0 C for about 30 min, and then cooled to 0-5 0 C over 30-40 minutes.
• reaction mixture was held at 0-5 0 C for 1 hour, and then filtered, rinsing first with 90:10 water/methanol (400 mL) and then water (600 niL.) Drying at 50-55 0 C under vacuum afforded product as off-white crystals. A yield of 127 g was obtained.
• micronized seeds of product (0.5 g) were charged in a minimal amount of acetonitrile (5 mL). The reaction mixture was then heated to 53-57 0 C over ⁇ 40 minutes, and held at that temperature for at least 4 hours. The reaction was cooled to 0-5 0 C, the product isolated by filtration, washed with acetonitrile (250 mL), and dried under vacuum at 55-60 0 C. A yield of 52.24g was obtained.
• the mixture was heated to 40-45 0 C and once a clear solution was observed, a clarifying filtration was performed, adding the solution to reactor 2 (containing the starting material solution) and maintaining the temperature in reactor 2 at 50-60 0 C.
• the mixture was heated to reflux, and held at 70-80 0 C until the reaction was complete. -3500 mL of solvent was removed by atmospheric distillation.
• the reactor was then charged with 2.5 L water followed by 4 L ACN, and the temperature adjusted to 70-80 0 C. After dissolution was observed, the resulting solution was cooled to 64-68 0 C. After 5- 10 minutes, milled product Form III seeds (5 g) were added in a minimal amount of acetonitrile, and held at 64-68 0 C for one hour.
• the mixture was cooled to 0-5 0 C over 2 hours and held at 0-5 0 C for ⁇ 30 minutes before isolating the product by filtration.
• the solid product was washed with 2.5L of acetonitrile, and dried under vacuum at 50-60 0 C. A yield of 480 g was obtained.

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