US 2004O1066O1A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0106601A1 Harris et al. (43) Pub. Date: Jun. 3, 2004

(54) METHOD OF INCREASING NEUTROPHIL (57) ABSTRACT PRODUCTION USING OPTICALLY-PURE (R)-2,3-BENZODIAZEPINES Compounds according to formula I: (76) Inventors: Herbert W. Harris, Merion, PA (US); Robert F. Kucharik, Glenmoore, PA

(US) I Correspondence Address: DRINKER BIDDLE & REATH ONE LOGAN SQUARE 18TH AND CHERRY STREETS PHILADELPHIA, PA 19103-6996 (US) (21) Appl. No.: 10/309,527 (22) Filed: Dec. 3, 2002 Publication Classification (51) Int. Cl...... A61K 31/5513 wherein R. R. R. R", R and n are as defined herein, are (52) U.S. Cl...... 514/221 administered to increase neutrophil levels in mammels. US 2004/0106601 A1 Jun. 3, 2004

METHOD OF INCREASING NEUTROPHIL widely used 1,4-benzodiazepine (BZ) anxiolytics Such as PRODUCTION USING OPTICALLYPURE diazepam (Valium(R) and chlordiazepepoxide (Librium(R). (R)-2,3-BENZODIAZEPINES The 1,4-benzodiazepines, in addition to having Sedative hypnotic activity, also possess muscle relaxant and anticon FIELD OF THE INVENTION Vulsant properties that, though therapeutically useful in 0001. The present invention relates to the use of 2,3- Some disease States, are nonetheless potentially untoward benzodiazepines in increasing neutrophil production in Side effects. Thus, the 1,4-benzodiazepines, though Safe when administered alone, may be dangerous in combination mammals. with other CNS drugs including alcohol. BACKGROUND OF THE INVENTION 0005 Tofisopam, in contrast, is a non-sedative anxiolytic 0002 2,3-Benzodiazepines that has no appreciable Sedative, muscle relaxant or anti convulsant properties. See Horvath et al., Progress in Neu 0003) Certain 2,3-benzodiazepines have been explored robiology, 60 (2000),309-342; the entire disclosure of which extensively for their potent CNS modulating activity. Com is incorporated herein by reference. In clinical Studies, pounds Such as tofisopam (Grandaxine), girisopam, and tofisopam improved rather than impaired psychomotor per norisopam (structures shown below with the atom number formance and showed no interaction with ethanol (Id.). ing System indicated for tofisopam) have demonstrated These observations comport with data that show that tofiso Substantial anxiolytic and antipsychotic activity. pam does not interact with central benzodiazepine (BZ)

receptors and binds only weakly to peripheral BZ receptors. Additional Studies have shown that tofisopam enhances mitogen-induced lymphocyte proliferation and IL-2 produc tion in vitro. (Id) 0006 Other 2,3-benzodiazepines, though structurally Similar to tofisopam, have been investigated and shown to have varying activity profiles. For example, GYKI-524.66 and GYKI-53655 (structures shown below) act as noncom petitive glutamate antagonists at the AMPA (C-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid) site, and have demonstrated neuroprotective, muscle relaxant and anticon Vulsant activity (Id.). Another group of 2,3-benzodiazepines that have been investigated are represented by the compound GYKI-52895, and show activity as selective dopamine uptake inhibitors with potential use in antidepressant and Tofisopam anti-Parkinsonism therapy (Id.).

HCO W CH N / O O eN { /S-( O eN CH

HN Nerisopam HN

GKY-53655 HCO \ CH N / O HCO eN { NH O eN

C Girisopam HN 0004 Tofisopam has been shown in humans to have an GKY-52895 activity profile that is significantly different from that of US 2004/0106601 A1 Jun. 3, 2004

needed, they are released from the bone marrow as immature -continued cells, called bands or Stab cells. CH 0012 Neutropenia is a blood disorder wherein the num ber of neutrophils in the blood is abnormally low as assessed N by an Absolute Neutrophil Count (ANC). An ANC is / acquired by performing a differential blood cell count in COG,O eN which percentages of cell types are recorded as well as the total number of cells. The differential blood cell count is done by Spreading a drop of blood on a microScope Slide. The Slide is Stained and examined under a microscope. One hundred white cells are counted and identified as either neutrophils, bands, lymphocytes, monocytes, eosinophils or HN basophils. Any a typical or immature cells also are counted. Cells are identified by the shape and appearance of the GKY-524.66 nucleus, the color of cytoplasm (the background of the cell), and the presence and color of granules. The percentage of each cell type is reported. Some instruments may be used to 0007 Tofisopam is a racemic mixture of (R)- and (S)- perform an automated differential. enantiomers. This is due to the asymmetric carbon, i.e., a 0013 A neutrophil shortage corresponds to an increased carbon with four different groups attached, at the 5-position risk of microbial infection. The blood of healthy human of the benzodiazepine ring. The molecular Structure and adults contains about 2500 to 6000 neutrophils per mm. In conformational properties of tofisopam have been deter children under the age of Six, the count may be lower. mined by NMR, CD and X-ray crystallography (Visy et al., Various sources have set the threshold for the diagnosis of Chirality 1:271-275 (1989)). The 2,3-diazebine ring exists neutropenia at different measured neutrophil levels ranging as two different conformers. The major conformers, (+)R from an ANC of about 2000 neutrophils per mm to about and (-)S have the 5-ethyl group in a quasi-equatorial posi 1500 neutrophils per mm. See The Merck Manual 17" Ed. tion, while in the minor conformers, (-)R and (+)S, the 1999, Section 11, Chapter 135, and Wallner, et al., U.S. Pat. 5-ethyl group is positioned quasi-axially. Thus, racemic No. 6,300,314, Oct. 9, 2001, the entire disclosures of which tofisopam can exist as four molecular Species, i.e., two are incorporated herein by reference. Severe neutropenia is enantiomers, each of which exists as two conformations. diagnosed when the ANC falls below 500 neutrophils per The Sign of the optical rotation is reversed upon inversion of mm. The symptoms, of increased risk of infection depend the diazepine ring from one conformer to the other. In crystal on the Severity of the neutropenia and on the duration of the form, tofisopam exists only as the major conformations, with disorder. dextrorotatory tofisopam being of the (R) absolute configu ration. (Toth et al., J. Heterocyclic Chem., 20:709–713 0014) Neutropenia treatable by methods of the present (1983); Fogassy et al., Bioorganic Heterocycles, Van der invention may be a chronic disorder. Neutropenia as a Plas, H. C., Otvös, L, Simongi, M., eds. Budapest Amster chronic disorder may be further classified as congenital, dam: Akademia; Kiado-Elsevier, 229:233 (1984)). cyclical and idiopathic neutropenia. Chronic congenital neu tropenia is inherited by a small number of individuals. The 0008 Differential binding of these two conformations of most Severe form of congenital neutropenia is Kostmann's tofisopam has been reported in binding Studies with human Syndrome and there are other, milder variations. Symptoms albumin (Simongi et al. Biochem. Pharm., 32(12), 1917 include frequent infections and feverS. 1920, 1983). The two conformers have also been reported as existing in equilibrium (Zsila et al., Journal of Liquid 0015 Cyclical neutropenia results from a regulatory Chromatography & Related Technologies, 22(5), 713-719, defect at the hematopoietic Stem cell level that causes 1999; and references therein). oscillations in production of neutrophils as well as other types of blood cells. Individuals with this disorder will have 0009. The optically pure (R)-enantiomer of tofisopam neutrophil counts of about 100 neutrophils per mm for three (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8- to six days out of every cycle. The neutrophil count ranges dimethoxy-5H-2,3-benzodiazepine) has been isolated and from Severe to moderate neutropenia levels through most of shown to possess the nonsedative anxiolytic activity of the the cycle. racemic mixture. See U.S. Pat. No. 6,080,736; the entire disclosure of which is incorporated herein by reference. 0016 Chronic idiopathic neutropenia refers to severe chronic neutropenia that does not clearly fall into either of 0010 Neutropenia the above classifications. Individuals Suffering from chronic idiopathic neutropenia typically acquire the disorder after 0.011 Neutrophils, also called polymorphonuclear leuko having normal neutrophil counts earlier in life. It is esti cytes, are the most numerous of the blood cells known as granulocytes. Neutrophils are the largest cell population mated that neutropenia may occur as a congenital or idio involved in acute inflammatory response. They are thus an pathic disorder in an estimated frequency of one per 200,000 important component of natural immunity, responding in the population. quickly to chemotactic Stimuli. Neutrophils destroy foreign 0017 Neutropenia may also occur secondary to another particles Such as bacteria by enveloping and digesting them, condition Such as cancer or Acquired Immunodeficiency a process called phagocytosis. Neutrophils increase in Syndrome. Neutropenia may also occur Secondary to an response to bacterial infection. When many neutrophils are event Such as a drug therapy. Thus, neutropenia may result US 2004/0106601 A1 Jun. 3, 2004 from physiological disorders that directly affect the immune an effective amount of at least one compound according to System. For example, diminished neutrophil production will formula I: result when leukemia, myeloma, lymphoma or a metastatic Solid tumor Such as, for example, breast or prostate cancer, infiltrate and replace bone marrow. Transient neutropenia is often associated with Viral infections. Chronic neutropenia is often associated with immunodefiency resulting from a viral infection, for example, Acquired Immunodeficiency Syn drome (AIDS) resulting from infection with Human Immu nodeficiency Virus (HIV). Autoimmune neutropenia may be asSociated with circulating antineutrophil antibodies. 0.018. A much more common cause is neutropenia as a Side effect of drug therapy, particularly cancer chemo therapy, radiation therapy for cancer and bone marrow transplantation associated with cancer therapy. Neutropenia Secondary to drug therapy can thus be Subdivided into two groups. The first involves immune-mediated neutropenia that may arise from drugs that act as haptens to Stimulate 0023 wherein: antibody formation. Acute hyperSensitivity reactions Such as 0024) R' is -(C-C7)hydrocarbyl or -(C- those caused by diphenylhydantoin and phenobarbital may C.)heteroalkyl, last a few dayS. However, chronic hyperSensitivity reactions may last for months or years. See The Merck Manual, 17" 0025) R is selected from the group consisting of Ed., Id. -H, and -(C-C)hydrocarbyl, wherein R and R' may combine to form a carbocyclic or heterocyclic 0019. The second area of drug-induced neutropenia 5- or 6-membered ring; involves the Severe neutropenia that predictably occurs after large doses of cytoreductive cancer drugs and which also 0026 R is independently selected from the group accompanies ionizing radiation therapy. These cytotoxic consisting of -O(C-C)alkyl, -OH, -O-acyl, therapies induce neutropenia because of the proliferative -SH, -S(C-C)alkyl, -NH, -NH(C-C)alkyl, nature of neutrophil precursor cells and the normal rapid -N((C-C)alkyl), -NH-acyl, -NO and halo turnover rate of circulating neutrophils. See The Merck gen, Manual, 17" Ed., Id. The risk of neutropenia secondary to cancer chemotherapy or radiotherapy depends on the type 0027 n is 1, 2 or 3; and Stage of the cancer and the type, the dosage and the 0028) R' and Rare independently selected from the Schedule of cancer treatment. Approximately 1.4 million group consisting of-O(C-C)alkyl, -OH, O-acyl, cancer patients in the US received chemotherapy in 2001. -SH, -S(C-C)alkyl, -NH, NH-acyl and halo About one half of chemotherapy patients develop neutrope gen, wherein R' and R may combine to form a 5, 6 nia. At present, less than 10% of chemotherapy patients or 7-membered heterocyclic ring, or a pharmaceuti receive prophylactic treatment to prevent neutropenia. cally-acceptable Salt of Such a compound; 0020. Therapy that exists currently for raising neutrophil 0029 wherein the administered compounds accord levels consists primarily of filgrastim (Nupogen E) and more ing to formula I comprise an R-enantiomer with recently, pegfilgrastim (Neulasta"), a longer acting deriva respect to the absolute conformation at the 5-position tive of filgrastim. Filgrastim is a recombinant version of a of the benzodiazepine ring, and is Substantially free human protein, G-CSF (granulocyte-colony Stimulating fac of the corresponding S-enantiomer of the same com tor), that Selectively stimulates the production of white pound. blood cells. G-CSF is currently the drug of choice for neutropenia. Since both of these drugs are recombinant 0030 Preferably, the administered compound of formula proteins they are not active orally and must be administered I, or a pharmaceutically-acceptable Salt of Such a compound, by injection. In addition, protein-based drugs are often comprises 85% or more by weight of the (R)-enantiomer. More preferably, administered compound of formula I, or a subject to rapid metabolism. The elimination half-life of pharmaceutically-acceptable Salt of Such a compound, com Nupogen(R) is 3.5 hours and of NeulastaTM is in the range of prises 90% or more by weight of the (R)-enantiomer. Even 15-80 hours. more preferably, the administered compound of formula I, or 0021 New agents are needed which are useful in the a pharmaceutically-acceptable Salt of Such a compound, treatment of neutropenia. In particular, agents are needed comprises 95% or more by weight of the (R)-enantiomer. that demonstrate biological activity when administered via Most preferably, the administered compound of formula I, or routes other than injection. Particularly, agents that may be a pharmaceutically-acceptable Salt of Such a compound, orally active are needed, as they may serve to enhance comprises 99% or more by weight of the (R)-enantiomer. patient compliance. 0031. According to another embodiment of the invention, there is provided a method of treating an individual afflicted SUMMARY OF THE INVENTION with neutropenia, comprising administering to the individual 0022. In one embodiment of the invention there is pro an effective amount of a compound of formula I; wherein the Vided a method of increasing the absolute neutrophil count administered compounds according to formula I comprise an in an individual, comprising administering to the individual R-enantiomer with respect to the absolute conformation at US 2004/0106601 A1 Jun. 3, 2004 the 5-position of the benzodiazepine ring, and is Substan 0055 According to a further preferred embodiment of the tially free of the corresponding S-enantiomer of the same administered compound: compound. 0056) R' is -CHCH; 0032. According to another embodiment of the invention, there is provided a method of preventing neutropenia in an 0057 R’ is -CH: individual who is at risk of developing neutropenia, Said method comprising administering to Said individual an 0.058 R, R' and Rare independently selected from effective amount of at least one compound according to the group consisting of -OH and -OCH; formula I; wherein the administered compounds according 0059) n is 2; and wherein R comprises substituents to formula I comprise an R-enantiomer with respect to the at the 3- and 4-positions of the phenyl ring, or a absolute conformation at the 5-position of the benzodiaz pharmaceutically-acceptable Salt of Such a com epine ring, and is Substantially free of the corresponding pound. S-enantiomer of the same compound. 0060 Preferably, compounds according to formula I, for 0033. In a preferred embodiment of the administered compound: administration, are Selected from the group consisting of: 0061 (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl 0034) R' is —(C-C)alkyl; 7,8-dimethoxy-5H-2,3-benzodiazepine; 0035) R is selected from the group consisting of 0062 (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl -H and -(C-C)alkyl; 7-hydroxy-8-methoxy-5H-2,3-benzodiazepine; 0036), R is independently selected from the group 0063 (R)-1-(3-hydroxy-4-methoxyphenyl)-4-methyl consisting of-O(C-C)alkyl, -O-acyland-OH; 5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine; 0037 n is 1, 2 or 3; and 0064 (R)-1-(3-methoxy-4-hydroxyphenyl)-4-methyl 0038) R' and R are independently selected from 5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine; -O(C-C)alkyl, -O-acyl and -OH, wherein, R" and R' may combine to form a 5, 6 or 7-membered heterocyclic 0065 (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl ring, or a pharmaceutically-acceptable Salt of Such a com 7-methoxy-8-hydroxy-5H-2,3-benzodiazepine; pound. 0066 (R)-1-(3-methoxy-4-hydroxyphenyl)-4-methyl 0.039 According to another preferred embodiment of the 5-ethyl-7-hydroxy-8-methoxy-5H-2,3-benzodiazepine; administered compound: and 0040) R' is -CHCH; 0067 (R)-1-(3-hydroxy-4-methoxyphenyl)-4-methyl 5-ethyl-7-hydroxy-8-methoxy-5H-2,3-benzodiazepine; 0041) R' is –C-C)alkyl; or a pharmaceutically-acceptable Salt thereof. 0042) R, RandR are independently selected from 0068 More preferably, the compound according to for the group consisting of -OH and -OCH; and mula I, for administration is (R)-1-(3,4-dimethoxyphenyl)- 4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine; or 0043 n is 1, 2 or 3; a pharmaceutically-acceptable Salt thereof. 0044 or a pharmaceutically-acceptable salt of Such a compound. 0069. The compound, (R)-1-(3,4-dimethoxyphenyl)-4- methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine, the 0.045 According to a further preferred embodiment of the R-enantiomer of tofisopam, is shown in the Structure dia administered compound: gram below. 0046) R' is —CHCH;

0047 R’ is -CH: HCO 0048) R, RandR are independently selected from the group consisting of -OH and -OCH; and 0049 n is 1, 2 or 3; or a pharmaceutically-accept able Salt of Such a compound. 0050. According to a further preferred embodiment of the administered compound: 0051) R' is -CHCH; 0.052 R is –CH: 0053) R, R and Rare independently selected from HC the group consisting of -OH and -OCH; and (R)-tofisopam 0054 n is 2; or a pharmaceutically-acceptable salt of Such a compound. US 2004/0106601 A1 Jun. 3, 2004

DEFINITIONS number of carbon atoms and one or two heteroatoms Selected from the group consisting of O, N, and S. Nitrogen 0070 The term “acyl” means a radical of the general and Sulfur atoms may be optionally oxidized to the N-oxide formula-C(=O)-R, wherein -R is hydrogen, hydrocar and Sulfoxide or Sulfone, respectively. In addition, a nitrogen byl, amino, alkylamino, dialkylamino hydroxy or alkoxy.' heteroatom may be optionally quaternized. The heteroat Examples include for example, acetyl (-CO=O)CH), pro om(s) may be placed at any position of the heteroalkyl pionyl (-CO=O)CHCH), benzoyl (-C(=O)CH), phe group, including between the rest of the heteroalkyl group nylacetyl (-C(=O)CHCH), carboethoxy and the fragment to which it is attached, as well as attached (-COCH2CH), and dimethylcarbamoyl to the most distal carbon atom in the heteroalkyl group. (-C(=O)N(CH)). When the R group in the acetyl radical Preferred are (C-C)heteroalkyl. More preferred are (C- is alkoxy, alkyl amino or dialkyl amino, the alkyl portion is C.)heteroalkyl. Examples include: -O-CH-CH preferably (C-C)alkyl, more preferably (C-C)alkyl. CH, -CH-CHCH-OH, -CH-CH-NH-CH, When the R is hydrocarbyl, it is preferably (C- -CH-C(=O)-CH, -CH-N=N-CH-CH, C.)hydrocarbyl. When R is hydrocarbyl, it is preferably CH-S-CH-CH, -CHCH-S(=O)-CH and alkyl, more preferably (C-C)alkyl. -CH-CH-NH-SO-CH. Up to two heteroatoms 0071. The term “alkyl”, by itself or as part of another may be consecutive, Such as, for example, -CH-NH Substituent means, unless otherwise Stated, a Straight, OCH, or -CH-CH2-S-S-CH. More preferred are branched or cyclic chain hydrocarbon radical, including di heteroalkyl groups containing one or two oxygen atoms. and multi-radicals, having the number of carbon atoms 0078 When two groups may “combine to form a car designated (i.e. C-C means one to six carbons). Alkyl bocyclic or heterocyclic 5- or 6-membered ring,” a carbocy groups include Straight chain, branched chain or cyclic clic ring is preferably Saturated. Preferred heterocyclic rings groups, with Straight being preferred. Examples include: are Saturated rings containing one or two heteroatoms methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, selected from N, O and S. Heterocyclic rings annulated to pentyl, neopentyl, hexyl, cyclohexyl and cyclopropylm the benzodiazepine Seven-membered ringin this way ethyl. (C-C)alkyl is preferred. Most preferred is (C- include, for example, furan, dihydrofuran, tetrahydrofuran, C.)alkyl, particularly ethyl, methyl and isopropyl. pyran, dihydropyran, tetrahydropyran, thiophene, dihy 0.072 The term “alkoxy” employed alone or in combi drothiophene, tetrahydrothiophene, pyrrole, dihydropyrrole, nation with other terms means, unless otherwise Stated, an pyrrolidine, pyridine, dihydropyridine, tetrahydropyridine alkyl group having the designated number of carbon atoms, and piperidine. as defined above, connected to the rest of the molecule via 0079 When two groups may “combine to form a 5-, 6- or an oxygen atom, Such as, for example, methoxy, ethoxy, 7-membered heterocyclic ring, preferred heterocyclic rings 1-propoxy, 2-propoxy (isopropoxy) and the higher are 5- or 6-membered rings containing one or two heteroa homologs and isomers. Preferred are (C-C)alkoxy. More toms selected from N, O and S. More preferred are hetero preferred is (C-C)alkoxy, particularly ethoxy and meth cyclic rings containing one heteroatom Selected from N, O Oxy. and S. Heterocyclic rings annulated to the benzodiazepine 0073. The term “amine” or “amino” refers to radicals of phenyl ring in this way include, for example, furan, dihy the general formula -NRR", wherein R and R' are indepen drofuran, dioxane, dioxolane, pyran, dihydropyran, tetrahy dently Selected from hydrogen or a hydrocarbyl radical, or dropyran, thiophene, dihydrothiophene, pyridine, dihydro wherein R and R' combined form a heterocycle. Examples of pyridine, tetrahydropyridine, piperidine, pyrrole, amino groups include: -NH2, methylamino, diethylamino, dihydropyrrole, imidazole, dihydroimidazole, thiazole, anilino, benzyl amino, piperidinyl, piperazinyl and indoli dihydrothiazole, Oxazole, and dihydrooxazole. nyl. Preferred hydrocarbyl radicals are (C-C)hydrocarbyl 0080. The term “substituted” means that an atom or radicals. Preferred are hydrocarbyl radicals that are alkyl group of atoms has replaced hydrogen as the Substituent radicals. More preferred are (C-C)alkyl. attached to another group. For aryl and heteroaryl groups, the term “substituted” refers to any level of substitution, 0.074 The term “aromatic” refers to a carbocycle or namely mono-, di-, tri-, tetra-, or penta-Substitution, where heterocycle having one or more polyunsaturated rings hav Such Substitution is permitted. The Substituents are indepen ing aromatic character (4n+2) delocalized at (pi) electrons). dently Selected, and Substitution may be at any chemically 0075. The term “aryl” employed alone or in combination accessible position. with other terms, means, unless otherwise Stated, a carbocy 0081. The phrase “optically active” refers to a property clic aromatic System containing one or more rings (typically whereby a material rotates the plane of plane-polarized light. one, two or three rings) wherein Such rings may be attached A compound that is optically active is nonSuperimposable on together in a pendent manner, Such as a biphenyl, or may be its mirror image. The property of nonSuperimposablity of an fused, Such as naphthalene. Examples include phenyl, anthracyl, and naphthyl. object on its mirror image is called chirality. 0082 The property of “chirality” in a molecule may arise 0.076 The term “hydrocarbyl” refers to any moiety com from any structural feature that makes the molecule nonsu prising only hydrogen and carbon atoms. This definition perimposable on its mirror image. The most common Struc includes for example alkyl, alkenyl, alkynyl, aryland benzyl tural feature producing chirality is an asymmetric carbon groups. Preferred are (C-C)hydrocarbyl. atom, i.e., a carbon atom having four nonequivalent groups 0077. The term “heteroalkyl” by itself or in combination attached thereto. with another term means, unless otherwise Stated, a stable 0083) The term “enantiomer” refers to each of the two Straight or branched chain radical consisting of the Stated nonsuperimposable isomers of a pure compound that is US 2004/0106601 A1 Jun. 3, 2004 optically active. Single enantiomers are designated accord 0091. The selection of an individual at increased risk for ing to the Cahn-Ingold-Prelog System, a set of priority rules developing neutropenia may take into account the presence that rank the four groups attached to an asymmetric carbon. of known risk factors. Such factors may include, for See March, Advanced Organic Chemistry, 4" Ed., (1992), p. example, cancer requiring chemotherapy or therapeutic ion 109. Once the priority ranking of the four groups is deter izing radiation; a disease that affects the immune System mined, the molecule is oriented So that the lowest ranking directly, Such as for example Acquired Immunodefiency group is pointed away from the viewer. Then, if the descend Syndrome (AIDS); or the presence of a virus such as Human ing rank order of the other groups proceeds clockwise, the Immunodeficiency Virus (HIV) known to cause AIDS. molecule is designated R and if the descending rank of the other groups proceeds counterclockwise, the molecule is DETAILED DESCRIPTION OF THE designated S. In the example below, the Cahn-Ingold-Prelog INVENTION ranking Sequence id A>B>C>D. The lowest ranking atom, D 0092 According to the present invention, (R)-2,3-benzo is oriented away from the viewer. diazepines of formula I, and pharmaceutically-acceptable Salts thereof, may be used to increase neutrophil levels in an A. A. individual as measured by an absolute neutrophil count. 0093. The use of compounds of formula I to raise neu C -k B B -k C trophil levels may be associated with one or more of Several therapeutic goals. Therapy to raise neutrophil levels may R configuration S configuration treat neutropenia that exists as a primary disease State. Alternatively, therapy according to method of the invention may treat neutropenia that is Secondary to another factor. 0084. The term “racemate” or the phrase “racemic mix Such factors include, for example, Viral infection, cancer or ture” refers to a 50-50 mixture of two enantiomers such that therapy with a drug that causes neutropenia. the mixture does not rotate plane-polarized light.). 0094. Therapy to raise neutrophil levels by methods of 0085. The term “substantially isolated”, or “substantially the present invention may also prevent neutropenia in free” of the other enantiomer or the term “resolved” or the instances wherein an individual is at risk of developing phrase “Substantially free” of the corresponding (S)-enanti neutropenia. Such instances include, for example, an indi omer, when used to refer to an optically active compound of vidual who anticipates beginning drug therapy using a drug formula I, means the (R)- and (S)-enantiomers of the com known or Suspected to cause neutropenia. pound have been separated such that the composition is 80% 0095 Numerous drugs have been shown to cause neu or more by weight a single enantiomer. tropenia as a Side effect. Such side effects have been 0.086 Thus, by “(R)-2,3-benzodiazepine substantially observed in drugs in a variety of drug classes including, for free of the (S)-enantiomer' is meant a 2,3-benzodiazepine example, thyroid inhibitors, antibiotics, neuropsychotropics, compound that comprises 80% or more by weight of its cardiovascular medications, analgesics, antimalarials, non (R)-enantiomer and likewise contains 20% or less of its Steroidal antiinflammatory agents, antihistamines and com (S)-enantiomer as a contaminant, by weight. binations thereof. See Lee, Wintrobe's Hematology, Lippin 0087. The phrase “effective amount” when used to cott, p. 1862-1869, and van der Klauw, M M et al., Arch. describe therapy to an individual refers to an amount of a Intern. Med., 1999, 159(4), p.369-374, the entire disclosures compound of formula I which results in increasing neutro of which are incorporated herein. phil production as measured by the absolute neutrophil 0096 Drugs that have been shown to cause neutropenia count of the individual’s blood. An effective amount of a include, for example: trimethoprim, chloramphenicol, peni compound of formula I for treatment of neutropenia is an cillins, cephalosporins, aminoglycosides, tetracyclines, amount which raises the absolute neutrophil count in an nitroimidazoles, nitrofurantoin, flucytosine, rifampin, iso individual afflicted with neutropenia. An effective amount of niazid, ethambutol, dapSone, Sulfonamide antibiotics, clo a compound of formula I for the prevention of neutropenia miprimine, thiacetazone, dipyrone, SulfaSalazine, meSala is an amount which maintains the absolute neutrophil count Zine, ciprofloxacin, chloroquin, mebendazole, terbendafine, of the individual above a level of about 500 neutrophils per pyrimethamine, levamisole, ristocetin, griseofulvin, phe mm in an individual during a time interval coinciding with nothiazines, benzodiazepines, amoxapine, meprobamate, an increased risk of neutropenia. Conditions which are barbiturates, clozapine, risperidone, imipramine, asSociated with an increased risk of neutropenia include, for desipramine, thiothixene, haloperidol, Valproic acid, hydan example, a present or forthcoming regimen of cancer che toins, Succinimides, trimethadione, carbamazepine, motherapy. procainamide, quinidine, propafenone, captopril, propra nolol, hydralazine, methyldopa, ibuprofen, indomethacin, 0088. The term “individual” or “subject,” includes Sulindac, tolmetin, aspirin, aminopyine, phenylbutaZone, human beings and non-human animals. With respect to the diflunisal, benoxaprofen, allopurinol, colchicine, propylth disclosed methods of increasing neutrophil production, these iouracil, thiouracil, methimazole, carbimazole, thiocyanate, terms refer, unless the context indicates otherwise, to: potassium perchlorate, cimetidine, ranatadine, tripelen 0089 (a) an organism that is afflicted with a disorder namine, methaphenilene, thenalidine, mianserin, bro characterized by neutropenia; or mopheneramine, quinine, hydroxychloroquin, quinacrine, 0090 (b) an organism that is at increased risk for diaZOxide, dihydropyridines, ticlopidine, Vesnarinone, developing neutropenia, due, for example, to forth aprindine, imipenem/cilastatin, Zidovudine, fludarabine, coming cancer chemotherapy. acyclovir, turbinafine, aminoglutethimide, famotidine, US 2004/0106601 A1 Jun. 3, 2004

beZafibrate, flutamide, tamoxafen, penicillamine, retinoic present invention have a composition that is 85% by weight acid, metoclopramide, phenindone, dinitrophenol, or greater of the (R)-enantiomer, and 15% by weight, or less, ethacrynic acid, rauwolfia, ethanol, chlorpropamide, tolb of the (S)-enantiomer. More preferably, compounds used in utamide, thiazides, Spironolactone, methazolamide, aceta methods of the present invention have a composition that is Zolamide, levodopa and combinations thereof. See, 90% by weight or greater of the (R)-enantiomer and 10% by Oyesanme et al., Psychosomatics, 40:5, September-October, weight, or less, of the (S)-enantiomer. More preferably, 1999, p.414-421; the entire disclosure of which is incorpo compounds used in methods of the present invention have a rated herein by reference. Neutropenia induced by any of the composition that is 95% by weight or greater of the (R)- aforementioned drugs may be treated or prevented according enantiomer and 5% by weight, or less, of the (S)-enantiomer. to the present invention. Most preferably, compounds used in methods of the present 0097. A more common source of drug-induced neutro invention have a composition that is 99% by weight or penia involves the Severe neutropenia that predictably greater of the (R)-enantiomer and 1% by weight, or less, of occurs after large doses of cytoreductive cancer drugs and the (S)-enantiomer. which also accompanies ionizing radiation therapy. The 0100 Racemic 2,3-benzodiazepines may be synthesized, predictability of neutropenia in an individual undergoing as shown in Scheme 1, from the corresponding 2-benzopy chemotherapy for cancer provides a basis for methods of the rilium salt H by reaction with hydrazine hydrate, wherein X present invention for provide prophylactic administration. See The Merck Manual, 17" Ed., 1999 Chapter 135, “Leu is a counterion Such as, for example perchlorate: kopenia', the entire disclosure of which is incorporated herein by reference. Scheme 1 0.098 Instances wherein neutropenia may be prevented R1 include administration to individuals receiving cancer che R R2 motherapy or to individuals in preparation for imminent N cancer chemotherapy. Methods of the invention also include administration to an individual in association with, or in 2O hwdraziney hwdratey preparation for other events that have been shown to R5 X He increase the risk of the individual Subsequently developing neutropenia. Such factors include, but are not limited to: N Therapeutic radiation therapy, drug therapies other than - H(R) cancer chemotherapy wherein the individual is known or 2 Suspected to have a Sensitivity to the therapy that increases the risk of developing neutropenia; drug therapies other than H cancer chemotherapy wherein the drug is associated with a high incidence of neutropenia, an immunodeficiency Such Acquired Immunodefiency Syndrome (AIDS); or a virus known to cause immunodefiency, Such as for example Human Immunodefiency Virus (HIV). 0099] The (R)-2,3-benzodiazepines of formula I useful in the present invention may be prepared by one of Several methods. These methods generally follow the synthetic Strategies and procedures used in the Synthesis of 2,3- benzodiazepines Such as tofisopam and tofisopam analogs. See U.S. Pat. Nos. 3,736,315 and 4,423,044 (tofisopam syntheses) and Horvath et al., Progress in Neurobiology 60(2000) p.309-342 and references cited therein (prepara tion of tofisopam and analogs thereof), the entire disclosures of which are incorporated herein by reference. In the Syn thesis methods that follow, the products of the chemical 0101 Accordingly, hydrazine hydrate (98%, approxi Syntheses are racemic (R)- and (S)-2,3-benzodiazepines. mately 3 equivalents based on the 2-benzopyrylium salt) is These racemic mixtures are Subsequently Separated using added dropwise to a stirred Solution of the 2-benzopyrylium known methods of resolution to produce the (R)-2,3-benzo Salt H in glacial acetic acid (approximately 1 mL/3 mmol of diazepines of formula I substantially free of the (S)-enanti 2-benzopyrylium salt). During this operation, the Solution is omers. By an “(R)-2,3-benzodiazepine” is meant a 2,3- maintained at an elevated temperature, preferably, 80-100 benzodiazepine that has an (R) absolute conformation by C. The Solution is then maintained a higher elevated tem virtue of a substitution at the 5-position of the benzodiaz perature, preferably 95-100° C., for about one hour. Then the epine ring to give a resolvable chiral carbon at the 5-posi reaction mixture is diluted with 2% aqueous Sodium hydrox tion. By an “(R)-2,3-benzodiazepine substantially free of the ide Solution (approximately 3 equivalents based on the (S)-enantiomer” or “an (R)-enantiomer of a compound of 2-benzopyrylium salt) and cooled. The product 2,3-benzo formula I Substantially free of the corresponding (S)-enan diazepine Separates as a Solid and is removed by filtration, tiomer' is meant a compound that comprises 80% or more washed with water and dried. The crude product may be by weight of the desired (R)-enantiomer and likewise con purified by taking it up in a polar aprotic Solvent Such as tains 20% or less of the (S)-enantiomer as a contaminant, by dimethylformamide (DMF) at an elevated temperature, pref weight. Preferably, compounds used in methods of the erably 100-130 C., and decolorizing the solution with US 2004/0106601 A1 Jun. 3, 2004

activated carbon. The carbon is removed by filtration and the Scheme 2, intermediate H is prepared from the correspond filtered solution is diluted with water. The purified product ing aryl ethanol derivative D via the isochroman interme precipitates out of the Solution and is collected by filtration. diate F. 0102) See Kórósi et al., U.S. Pat. No. 4,322,346, the 0104. Another variation for preparing 2,3-benzodiaz entire disclosure of which is incorporated herein by refer epines is illustrated in Scheme 3 and 4 (Examples 2 and 3). ence, disclosing three variations of the reaction protocol for The synthesis there proceeds from intermediate G without preparing a Substituted 2,3-benzodiazepine from the precur isolation of the intermediate benzopyrilium salt H. Sor benzopyrilium Salt. 0105 2-Benzopyrylium salts H may be synthesized from 0103 Retrosynthetically, the intermediate benzopyrilium intermediate 2-substituted phenyl ethanol derivatives D Salt, H, may be prepared from one of Several Starting through isochroman intermediate F, wherein X is a coun materials. According to one Such method, illustrated in terion Such as, for example, perchlorate:

Scheme 2 R1 O HO R1

5 5 5 2 R OE R°CH2MgX R PTSOH R R He(x - c1, B, I, He R4 R4 R4 A. B C

1. BH 2. HO

R1 CHO R1 R R2 - Sl is * R. R2

2x1 OH 4 R4 (R) R D N - (R) 2

F

CrO3 H2SO4 5° C.

R1 R2 R R R2 N O Ot R4 O perchloric acid R4 2 X

N N - H(R3), - H(R3), 21 21

G US 2004/0106601 A1 Jun. 3, 2004

0106 Accordingly, a substituted benzoic acid ester, A is 0112 Alternative methods for preparation of intermedi dissolved in a suitable solvent, preferably ether and cooled ate H Start with an aryl acetonide or indanone Starting to 0 C. Two equivalents of a Suitable Grignard reagent are material. See Kunnetsov, E. V., and Dorofeenko, G. N., Zh. added dropwise and the reaction is allowed to warm to room Org. Khim., 6, 578-581. and M. Vajda, Acta Chem. Acad. temperature and monitored for disappearance of Starting Sci. Hung, 40, p.295-307, 1964, respectively, the entire material. When the reaction is complete, it may be quenched disclosures of which are incorporated herein by reference. with a proton Source Such as acetic acid. Volatiles are removed in vacuo, and the product B is used for the next Step 0113 To synthesize a 2,3-benzodiazepine derivative of without purification. formula I having an amine Substituent, the Starting aromatic 0107 The O.C.-substituted benzyl alcohol B is taken up in amine components must be protected with a protecting a high boiling Solvent Such as toluene and a catalytic amount group or otherwise rendered unreactive in order for the of para-toluene sulfonic acid (p-TSOH). The mixture is amine to be rendered Stable to the reaction conditions warmed to reflux and may be monitored for disappearance employed in the reaction Schemes shown or referenced of Starting materials. When the reaction is complete, the above. A means of circumventing the need for a protecting Volatiles are removed in vacuo and the crude product C is group may be to use a starting material containing an purified by column chromatography. aromatic nitro group(S) in place of the desired aromatic amino group(s). The nitro group performs the same function 0108. The substituted styrene C is hydroxylated under as an amine protecting group in this Synthesis and it may, anti-Markovnikov conditions to give intermediate phenyl following the Synthesis Steps that are incompatible with an ethyl alcohol D. A solution of D, and of a suitably substituted amine Substituent, be then reduced to an amine. Reduction benzaldehyde E (1.2 eq) are added to anhydrous dioxane. of the aromatic nitro group can be done, for example, via The resulting Solution is then Saturated with gaseous HCl catalytic hydrogenation. Catalytic hydrogenation provides and warmed, preferably to reflux temperature for about one the capability to Selectively reduce the aromatic nitro group hour. The mixture is then cooled to room temperature, without reducing the olefin or other functionality in the poured into water, basified (preferably with aqueous Sodium intermediate. This synthetic strategy is disclosed in U.S. Pat. hydroxide) and extracted with an organic Solvent (preferably No. 4,614,740, wherein racemic 2,3-benzodiazepines were ethyl acetate). The extract is dried, filtered and concentrated prepared with amino groups at a position corresponding the under Vacuum. The resulting residue is purified, preferably R of formula I of the present invention. The entire disclo by crystallization, to yield F. sure of U.S. Pat. No. 4,614,740 is incorporated herein by 0109) To a stirred, cooled, (preferably to 0-5° C) solution reference. of F (2 g) in acetone (30 mL), is added dropwise a Solution 0114) Resolution of (R)-2,3-Benzodiazepines of Formula of chromium trioxide (2 g) in 35% sulfuric acid (20 mL). I The latter Solution is added at a rate Such that the reaction temperature remains below 5° C. After the addition is 0115 The synthetic procedures shown (or referenced) complete, the reaction mixture is allowed to rise to room above produce racemic mixtures of 2,3-benzodiazepines. In temperature and is stirred at room temperature for two hours. order to prepare (R)-2,3-benzodiazepines of formula I that The reaction mixture is then poured into water and extracted are useful in methods of the present invention, the racemic with an organic Solvent, preferably ethyl acetate. The mixture must be resolved. organic extract is washed with water and then with ice-cold 10% aqueous Sodium hydroxide. The aqueous alkaline frac 0116 Racemic 2,3-benzodiazepines may, for example, be tion is then acidified, preferably with dilute aqueous hydro converted to the (S)-dibenzoyltartaric acid salt, which is a chloric acid, and extracted with an organic Solvent, prefer diastereomeric mixture of SS and RS configurations. The ably, chloroform. The chloroform extract is dried, filtered pair of diastereomers (R.S) and (S,S) possess different and concentrated under Vacuum to give G. The crude residue properties, e.g., differential Solubilities, that allow for the use may further be purified by column chromatography. of conventional Separation methods. Fractional crystalliza tion of diastereomeric Salts from a Suitable Solvent is one 0110. The 2-O-acyl hydrocarbylbenzophenone G (5 g) is Such separation method. This resolution has been Success dissolved in glacial acetic acid (15 mL). To this mixture is fully applied to the resolution of racemic tofisopam. See added 60% perchloric acid (7.5 mL). The resulting mixture Hungarian Patent 178516 and also Toth et al., J. Heterocy is warmed to 100° C. (steam bath) for three minutes. The clic Chem., 20:09-713 (1983), the entire disclosures of mixture is allowed to cool to room temperature. Crystalli which are incorporated herein by reference. Zation of the crude product may begin Spontaneously at this point or may be induced by addition of ether or ethyl acetate. 0117. Alternatively, racemic-2,3-benzodiazepines may The product 2-benzopyrylium salt His removed by filtration be derivatized Via, for example, acylation of an aryl hydroxy and purified by recrystallization, preferably from ethanol or moiety, with a chiral acylating reagent, e.g., (S)-mandelic glacial acetic acid/ethyl acetate. acid. The resulting ester, has a Second chiral center, and thus exists as a diastereomeric pair Separable using conventional 0111. A similar Synthetic sequence for preparation of methods Such as crystallization or chromatography. Follow 2,3-benzodiazepines is disclosed in U.S. Pat. No. 3,736,315, ing the Separation, the chiral moiety with which the racemic the entire disclosure of which is incorporated herein by 2,3-benzodiazepine is derivatized, may be removed. reference. Synthetic Strategies for preparation of 2,3-benzo diazepines are also disclosed in Horvath et al., ProgreSS in 0118 Racemic 2,3-benzodiazepines may be separated Neurobiology 60(2000) p309-342 and references cited without diastereomer formation by differential absorption on therein; the entire disclosures of which are incorporated a chiral Stationary phase of a chromatography column, herein by reference. particularly a preparative HPLC column. Chiral HPLC US 2004/0106601 A1 Jun. 3, 2004 columns are commercially available with a variety of pack -continued ing materials to Suit a broad range of Separation applications. Exemplary Stationary phases Suitable for resolving the race mic 2,3-benzodiazepines include: 0119 (i) macrocyclic glycopeptides, Such as Silica bonded Vancomycin which contains 18 chiral centers Surrounding three pockets or cavities, 0120 (ii) chiral O-acid glycoprotein; 0121 (iii) human serum albumin; and 0122) (iv) cellobiohydrolase (CBH). 0123 Chiral C.-acid glycoprotein is a highly stable pro tein immobilized onto Spherical Silica particles that tolerates high concentrations of organic Solvents, high and low pH, and high temperatures. Human Serum albumin, though espe cially Suited for the resolution of weak and Strong acids, Zwitterionic and nonprotolytic compounds, has been used to resolve basic compounds. CBH is a very stable enzyme which has been immobilized onto Spherical Silica particles and is preferentially used for the Separation of enantiomers of basic drugs from many compound classes. 0.124. The resolution of tofisopam by chiral chromatog raphy using macrocyclic glycopeptide as a Stationary phase on a Chirobiotic VTM column (ASTEAC, Whippany, N.J.) is disclosed in U.S. Pat. No. 6,080,736. Fitos et al. (J. Chro matogr., 709 265 (1995)), discloses another method for resolving racemic tofisopam by chiral chromatography using a chiral O-acid glycoprotein as a stationary phase on a CHIRAL-AGPTM column (ChromTech, Cheshire, UK). S(+)-isomer The latter method separates the (R)- and (S)-enantiomers and also resolves the two conformers (discussed below) of each enantiomer. These chromatographic methods, may be 0.126 The present invention includes methods as used generally to Separate racemic 2,3-benzodiazepines into described herein that use any and all observable conforma individual (R)- and (S)-enantiomers. The Chirobiotic VTM tions of compounds of formula I (of the (R)- absolute column is available in a Semi-preparative size as employed configuration at carbon 5 of the benzodiazepine ring) which for the above separation 500 mmx10 mm). The stationary are biologically active in treatments to raise neutrophil phase of the Chirobiotic VTM column is commercially avail levels. able in bulk for packing of preparative chromatography 0127. Differential binding of the (+) and (-) conformers columns with larger Sample capacity. of 2,3-benzodiazepines generally, has been reported for tofisopam in binding Studies with human albumin (Simongi 0125 (R)- and (S)-enantiomers of 2,3-benzodiazepines et al. Biochem. Pharm., 32(12), 1917-1920, 1983). The (+) may also exist in two stable conformations that may be and (-) conformers of tofisopam have also been reported as assumed by the benzodiazepine ring, as generally depicted existing in an equilibrium (Zsila et al., Journal of Liquid below: Chromatography & Related Technologies, 22(5), 713-719, 1999; and references therein). 0128. It will be understood that compounds of formula I useful in the methods of the present invention may contain one or more chiral centers in addition to chiral center at the 5-position of the benzodiazepine ring of compounds of formula I. Such compounds may exist in, and may be isolated as pure enantiomeric or diastereomeric forms or as racemic mixtures. The present invention includes methods that use any possible enantiomers, diastereomers, racemates or mixtures thereof of formula I (dictated by a chiral center other than the 5-position of the benzodiazepine ring) which R(+)-isomer are biologically active in treatments to raise neutrophil levels. US 2004/0106601 A1 Jun. 3, 2004

0129. The compounds used in the methods of the present the present invention may be administered at different times invention may take the form of pharmaceutically-acceptable during treatment or prevention therapy. Salts. The term "salts', embraces Salts commonly used to form alkali metal salts and to form addition salts of free 0.135 The methods of the present invention may com acids or free bases. The term “pharmaceutically-acceptable prise administering (R)-2,3-benzodiazepines in the form of Salt' refers to Salts that possess toxicity profiles within a a pharmaceutical composition, in combination with a phar range So as to have utility in pharmaceutical applications. maceutically acceptable carrier. The active ingredient in Pharmaceutically unacceptable Salts may nonetheless poS such formulations may comprise from 0.1 to 99.99 weight SeSS properties Such as high crystallinity, which have utility percent. By “pharmaceutically acceptable carrier is meant in the practice of the present invention, Such as for example any carrier, diluent or excipient that is compatible with the utility in a Synthetic proceSS or in the resolution of enanti other ingredients of the formulation and to deleterious to the omers. Suitable acid addition Salts may be prepared from an recipient. inorganic acid or from an organic acid. Examples of Such 0.136 The compounds useful in methods of the invention inorganic acids are hydrochloric, hydrobromic, hydroiodic, may be administered for therapeutic effect by any route, for nitric, carbonic, Sulfuric and phosphoric acid. Appropriate example enteral (e.g., oral, rectal, intranasal, etc.) and organic acids may be selected from aliphatic, cycloaliphatic, parenteral administration. Parenteral administration aromatic, araliphatic, heterocyclic, carboxylic and Sulfonic includes, for example, intravenous, intramuscular, intraarte classes of organic acids, example of which are formic, rial, intraperitoneal, intravaginal, intravesical (e.g., into the acetic, propionic, Succinic, glycolic, gluconic, lactic, malic, bladder), intradermal, topical or Subcutaneous administra tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyru tion. Also contemplated within the Scope of the invention is Vic, aspartic, glutamic, benzoic, anthranilic, meSylic, Sali the instillation of drug in the body of the patient in a cyclic, Salicyclic, 4-hydroxybenzoic, phenylacetic, man controlled formulation, with Systemic or local release of the delic, embonic (pamoic), methaneSulfonic, ethaneSulfonic, drug to occur at a later time. For use to increase neutrophil benzeneSulfonic, pantothenic, 2-hydroxyethaneSulfonic, levels, the drug may be localized in a depot for controlled toluenesulfonic, Sulfanilic, cyclohexylaminoSulfonic, release to the circulation. Stearic, algenic, beta-hydroxybutyric, Salicyclic, galactaric and galacturonic acid. 0.137 The active agent is preferably administered with a pharmaceutically acceptable carrier Selected on the basis of 0.130) Suitable base addition salts of compounds of for the Selected route of administration and Standard pharma mula I useful in methods of the invention include for ceutical practice. The active agent may be formulated into example, metallic Salts made from calcium, magnesium, dosage forms according to Standard practices in the field of potassium, Sodium and Zinc or organic Salts made from pharmaceutical preparations. See Alphonso Gennaro, ed., N,N'-dibenzylethylenediamine, chloroprocaine, choline, Remington's Pharmaceutical Sciences, 18th Ed., (1990) diethanolamine, ethylenediamine, meglumine (N-methyl Mack Publishing Co., Easton, Pa. Suitable dosage forms glucamine) and procaine. All of these salts may be prepared may comprise, for example, tablets, capsules, Solutions, by conventional means from the corresponding compound parenteral Solutions, troches, Suppositories, or Suspensions. of formula I by reacting, for example, the appropriate acid or base with the compound of formula I. 0.138 For parenteral administration, the active agent may be mixed with a Suitable carrier or diluent Such as water, an 0131 The compounds useful in methods of the invention oil (particularly a vegetable oil), ethanol, Saline Solution, may be administered to individuals (mammals, including aqueous dextrose (glucose) and related Sugar Solutions, animals and humans) for treatment or prevention of neutro glycerol, or a glycol Such as propylene glycol or polyeth pen1a. ylene glycol. Solutions for parenteral administration prefer ably contain a pharmaceutically-acceptable water-Soluble 0132) For treating or preventing neutropenia, the specific Salt of the active agent. Stabilizing agents, antioxidizing dose of compound according to the invention to obtain agents and preservatives may also be added. Suitable anti therapeutic benefit will, of course, be determined by the oxidizing agents include Sulfite, ascorbic acid, citric acid and particular circumstances of the individual patient including, its salts, and sodium EDTA. Suitable preservatives include the size, weight, age and SeX of the patient. Also determi benzalkonium chloride, methyl- or propyl-paraben, and native will be the nature and Stage of the disease and the chlorbutanol. The composition for parenteral administration route of administration. For example, a daily dosage of from may take the form of an aqueous or nonaqueous Solution, about 100 to 1500 mg/kg/day may be utilized. Preferably, a dispersion, Suspension or emulsion. daily dosage of from about 100 to 1000 mg/kg/day may be utilized. More preferably, a daily dosage of from about 100 0.139 For oral administration, the active agent may be to 500 mg/kg/day may be utilized. Higher or lower doses are combined with one or more Solid inactive ingredients for the also contemplated. Neutrophil levels may be monitored in preparation of tablets, capsules, pills, powders, granules or the patient and the treatment regimen may be maintained other Suitable oral dosage forms. For example, the active until neutrophil levels reach a normal range. agent may be combined with at least one excipient Such as fillers, binders, humectants, disintegrating agents, Solution 0.133 For prophylactic administration, the compounds retarders, absorption accelerators, wetting agents absorbents useful in the practice of methods of the invention should be or lubricating agents. According to one tablet embodiment, administered far enough in advance of a known event that the active agent may be combined with carboxymethylcel increases the risk of neutropenia Such that the compound is lulose calcium, magnesium Stearate, mannitol and Starch, able to reach the Site of action in Sufficient concentration to and then formed into tablets by conventional tableting exert therapeutic effect. The pharmacokinetics of Specific methods. compounds may be determined by means known in the art and tissue levels of a compound in a particular individual 0140. The compositions of the present invention can also may be determined by conventional analyses. be formulated so as to provide slow or controlled-release of the active ingredient therein. In general, a controlled-release 0134. One or more compounds useful in the practice of preparation is a composition capable of releasing the active the present inventions may be administered Simultaneously, ingredient at the required rate to maintain constant pharma or different (R)-2,3-benzodiazepines useful in the practice of cological activity for a desirable period of time. Such dosage US 2004/0106601 A1 Jun. 3, 2004 forms can provide a Supply of a drug to the body during a 0144. The practice of the invention is illustrated by the predetermined period of time and thus maintain drug levels following non-limiting examples. in the therapeutic range for longer periods of time than other non-controlled formulations. EXAMPLES 0141 For example, U.S. Pat. No. 5,674,533 discloses controlled-release compositions in liquid dosage forms for Example 1 the administration of moguisteine, a potent peripheral anti Synthesis of (R)-1-(3,4-dimethoxyphenyl)-4-me tussive. U.S. Pat. No. 5,059,595 describes the controlled release of active agents by the use of a gastro-resistant tablet thyl-5-ethyl-7-hydroxy-8-methoxy-5H-2,3-benzodi for the therapy of organic mental disturbances. U.S. Pat. No. aZepine 5,591,767 discloses a liquid reservoir transdermal patch for 0145 A. Synthesis of Racemic 1-(3,4-dimethoxyphe the controlled administration of ketorolac, a non-Steroidal nyl)-4-methyl-5-ethyl-7-hydroxy-8-methoxy-5H-2,3-ben anti-inflammatory agent with potent analgesic properties. Zodiazepine: U.S. Pat. No. 5,120,548 discloses a controlled-release drug delivery device comprised of Swellable polymers. U.S. Pat. 0146 4.41 g (10 mmol) of 1-(3,4-dimethoxyphenyl)-3- No. 5,073,543 discloses controlled-release formulations methyl-4-ethyl-6,7-dimethoxyisobenzopyrilium chloride containing a trophic factor entrapped by a ganglioside hydrochloride is dissolved in methanol (35 mL) at a tem liposome vehicle. U.S. Pat. No. 5,639,476 discloses a stable perature of 40 C. After cooling to 20-25 C., hydrazine Solid controlled-release formulation having a coating hydrate (0.75 g, 15 mmol, dissolved in 5 mL methanol) is derived from an aqueous dispersion of a hydrophobic acrylic added. The reaction is monitored by HPLC and when polymer. The patents cited above are incorporated herein by complete, is evaporated to dryneSS. The residue is triturated reference. with cold water (3 mL), filtered and dried to yield the crude (RS)-1-(33,4-dimethoxyphenyl)-4-methyl-5-ethyl-7-hy 0142 Biodegradable microparticles can be used in the droxy-8-methoxy-5H-2,3-benzodiazepine which is subse controlled-release formulations of this invention. For quently triturated with hot ethyl acetate to yield the pure example, U.S. Pat. No. 5,354,566 discloses a controlled product. release powder that contains the active ingredient. U.S. Pat. No. 5,733,566 describes the use of polymeric microparticles 0147 B. Resolution of the Racemate to Produce (R)-1- that release antiparasitic compositions. These patents are (3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7-hydroxy-8- incorporated herein by reference. methoxy-5H-2,3-benzodiazepine: 0143. The controlled-release of the active ingredient can 0148 (RS)-1-(3,4-Dimethoxyphenyl)-4-methyl-5-ethyl be stimulated by various inducers, for example pH, tem 7-hydroxy-8-methoxy-5H-2,3-benzodiazepine (43 mg, dis perature, enzymes, Water, or other physiological conditions Solved in acetonitrile) is injected onto a Chirobiotic V or compounds. Various mechanisms of drug release exist. column (ASTEAC, Whippany, N.J.) Elution of the racemate For example, in one embodiment, the controlled-release with methyl-tert-butyl ether/acetonitrile 90/10 (v/v), at 40 component can Swell and form porous openings large mL/minute, is monitored at 310 nm, 2 mm path. enough to release the active ingredient after administration to a patient. The term “controlled-release component' in the 014.9 The R(+) enantiomer is the first compound to elute, context of the present invention is defined herein as a and is collected and dried. The R(-), S(+), S(-) enantiomers, compound or compounds, Such as polymers, polymer matri and Some residual R(+) enantiomer coelute and are collected ces, gels, permeable membranes, liposomes and/or micro in Subsequent fractions. Approximately 20% of the R(+) Spheres, that facilitate the controlled-release of the active isomer is converted to the R(-) isomer if left in the eluent for ingredient (e.g., (R)-tofisopam or a pharmaceutically-ac 24 hours. A stable 80/20 equilibrium (R(+) to R(-)) is ceptable Salt thereof) in the pharmaceutical composition. In observed between the conformers in the eluent Solution. another embodiment, the controlled-release component is Example 2 biodegradable, induced by exposure to the aqueous envi ronment, pH, temperature, or enzymes in the body. In Synthesis of racemic-1-(3-hydroxy-4-methoxyphe another embodiment, Sol-gels can be used, wherein the active ingredient is incorporated into a Sol-gel matrix that is nyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzo a Solid at room temperature. This matrix is implanted into a diazepine patient, preferably a mammal, having a body temperature 0150 Racemic-1-(3-hydroxy-4-methoxyphenyl)-4-me high enough to induce gel formation of the Sol-gel matrix, thyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine WS thereby releasing the active ingredient into the patient. Synthesized according to the route of Scheme 3.

Scheme 3 CH HO H3CO COOH H3CO COOEt H3CO EtOH EtMg CH H2SO4 H3CO H3CO H3CO to US 2004/0106601 A1 Jun. 3, 2004

-continued HO CHO HC

HCO CH HCO 1. BH 2. H2O2 bar H3CO

HC OH HC

BO CHO HCO CH HCO CH HCI -- --- O HCO HCO HCO

OBn OCH

H2SO4 CrO3

CH HC O HC O CH HCO HCO CH HCO CH W 1. NHNH2 H -e- N 2. HC Pd / O O H3CO eN H3CO H3CO

OH OH OBn OCH3 OCH OCH

0151. A. Esterification of 3,4-dimethoxybenzoic Acid to magnesium turnings in 10 mL of ether. After 5 mL of the Yield ethyl-3,4-dimethoxybenzoate(3943-77-9). iodoethane Solution had been added, a few grains of iodine were added and the mixture was heated to induce formation 0152. A solution of 200 g of 3,4-dimethoxybenzoic acid of the Grignard reagent. The remaining iodoethane Solution and 35 g of concentrated Sulfuric acid in 600 mL of absolute was then added. After the Grignard formation was complete, ethanol was heated at reflux overnight. The mixture was a solution of 5 g of ethyl 3,4-dimethoxybenzoate in ether concentrated and the residue poured into water. Methylene was added and the mixture was allowed to Stir at room chloride was added and the Solution washed Successively temperature overnight. The reaction was quenched by addi with water, dilute Sodium bicarbonate and water, then dried tion of Saturated ammonium chloride. The mixture was and concentrated. The residue was recrystallized from extracted with ether. The combined ether extracts were dried acetone/hexane. and concentrated to an oily residue. Yield: 5 g. 0153 B. Addition of Ethyl Magnesium Iodide to ethyl O155 C. Elimination of HO From 3-(3,4-dimethoxyphe 3,4-dimethoxybenzoate Acid to Yield 3-(3,4-dimethoxyphe nyl)pentan-3-ol to yield 4-((17)-1-ethylprop-1-enyl)-1,2- nyl)pentan-3-ol. dimethoxybenzene. 0154) A solution of 4.8 mL of iodoethane in 20 mL of 0156 A solution of 5 g of crude 3-(3,4-dimethoxyphe ether was added dropwise to a Suspension of 1.5 g of nyl)pentan-3-ol and 0.25g of p-tolenesulfonic acid in 80 mL US 2004/0106601 A1 Jun. 3, 2004

of benzene was heated at reflux for 1 hr with azeotropic 0166 H. Debenzylation of 3-(4,5-dimethoxy-2-4- removal of water. The mixture was then filtered through a methoxy-3-(phenylmethoxy)-phenyl pad of sodium bicarbonate and the filtrate concentrated. The carbonylphenyl)pentan-2-one to Yield 3-2-(3-hydroxy-4- residue was purified by distillation under reduced pressure. methoxyphenyl)carbonyl-4,5-dimethoxyphenylpentan-2- Yield: 2.9 g. OC. O157 D. Addition of HO to 4-((17)-1-ethylprop-1- 0167 A solution of 10 g of 3-(4,5-dimethoxy-2-4- enyl)-1,2-dimethoxybenzene to Yield 3-(3,4-dimethoxyphe methoxy-3-(phenylmethoxy)-phenyl nyl)pentan-2-ol. carbonylphenyl)pentan-2-one in methylene chloride con taining a Suspension of 0.9 g of 10% palladium on carbon 0158 To a solution of 26 g of 4-((17)-1-ethylprop-1- was hydrogenated at 80 psi for 1 hr. The mixture was filtered enyl)-1,2-dimethoxybenzene in tetrahydrofuran at 0°C. was through diatomaceous earth and the filtrate concentrated. added 189 mL of a 1.0M solution of borane-tetrahydrofuran Yield: 6.5 g. complex in tetrahydrofuran. The mixture was stirred for 3 hr 0168 I. Annulation of 3-2-(3-hydroxy-4-methoxyphe at 0°C., then 35.6 mL of 50% hydrogen peroxide was added, nyl)carbonyl-4,5-dimethoxyphenylpentan-2-one by Reac with simultaneous addition of SM Sodium hydroxide to tion with Hydrazine to Yield 1-(3-hydroxy-4-methoxyphe maintain the mixture at pH 8. The mixture was extracted nyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3- with ether. The combined ether extracts were dried and benzodiazepine. concentrated. 0169 A solution of 6.5g of 3-2-(3-hydroxy-4-methox 0159 E. Benzylation of 3-hydroxy-4-methoxybenzalde yphenyl)carbonyl-4,5-dimethoxyphenylpentan-2-one and hyde to Yield 4-methoxy-3-(phenylmethoxy)benzaldehyde 2.2 mL of hydrazine in 130 mL of ethanol was heated at (6346-05-0). reflux for 0.5 hr. After allowing the solution to cool to room temperature, it was Saturated with HCl gas. The mixture was 0160 A solution of 100 g of 3-hydroxy-4-methoxyben then concentrated to a volume of about 5 mL, basified with Zaldehyde and 135 g of benzyl bromide in 500 mL of concentrated ammonium hydroxide, and extracted with acetone containing a Suspension of 137 g of potassium methylene chloride. The combined methylene chloride carbonate was heated at reflux overnight. The mixture was extracts were dried and concentrated, and the residue recrys filtered, the filtrate concentrated and the residue recrystal tallized from ethyl acetate/hexane. Yield: 0.97g lized from toluene/hexane. 0170 The product 1-(3-hydroxy-4-methoxyphenyl)-4- 0.161. Yield: 65 g. methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine was analyzed by HPLC, elemental analysis, GC/MS, proton 0162 F. Reaction of 3-(3,4-dimethoxyphenyl)pentan-2- NMR and differential scanning calorimetry (DSC). The data ol with 4-methoxy-3-(phenylmethoxy)benzaldehyde to are as follows: Yield 4-(4-ethyl-6,7-dimethoxy-3-methylisochromanyl)-1- methoxy-2-(phenylmethoxy)benzene. 0171] Purity: 99.29% by HPLC (% area). Column: Betasil Phenyl 4.6x150 mm. Mobile Phase: 0163 A Solution of 14 g of 4-methoxy-3-(phenyl Acetonitrile:0.01M Phosphate Buffer (70:30). Flow methoxy)benzaldehyde and 15 g of 3-(3,4-dimethoxyphe Rate: 0.5 mL/min. Wavelength: 254 nm. nyl)pentan-2-ol in 0.3 L of dioxane was saturated with 0172 GC-MS; M/e=358; with the fragmentation pat hydrogen chloride gas. The mixture was heated at reflux for tern matching the proposed Structure. 3 hr, Saturated again with hydrogen chloride gas and allowed to Stir at room temperature overnight. It was then poured into 0173 DSC: Temperature program 100° C. to 300° C. water, basified with dilute sodium hydroxide and extracted at 5 C./min, indicated molar purity=99.75% and melt with methylene chloride. The combined methylene chloride ing point of 158.6 C. extracts were dried and concentrated. 0174 Elemental analysis (calculated/analysis): % 0164. G. Ring-Opening of 4-(4-ethyl-6,7-dimethoxy-3- C -68.09/68.08; % H-6.61/6.57; N- 7.53/7.35. Calcu methyliso-chromanyl)-1-methoxy-2-(phenylmethoxy)ben lated values include 0.02 equivalents of ethyl acetate and Zene to Yield 3-(4,5-dimethoxy-2-(4-methoxy-3-(phenyl 0.09 equivalents of residual water. methoxy)phenylcarbonylphenyl)pentan-2-one. 0175 NMR (DCCL) (performed on GE QE 300): 1.08 0165) To a solution of 30 g of crude 4-(4-ethyl-6,7- ppm (t, 3H); 1.99 (s.3H); 2.11 (m, 2H); 2.75 (m, 1H); 3.75 dimethoxy-3-methylisochromanyl)-1-methoxy-2-(phenyl (s, 3H); 3.93 (s, 3H); 3.97 (s, 3H); 6.46 (bs, 1H); 6.72 (s, methoxy)benzene in 450 mL of acetone at 5° C. was added 1H); 6.86 (m, 2H); 7.18 (d. 1H); 7.48 (s, 1H). a solution of 30 g of chromic oxide in 300 mL of 35% Example 3 Sulfuric acid. The mixture was Stirred at room temperature for 2 hr., neutralized by adding cold 10% sodium hydroxide Synthesis of 1-(3,4-dimethoxyphenyl)-4-methyl-5- and concentrated to remove acetone. Then, water was added ethyl-7 methoxy-8-hydroxy-5H-2,3-benzodiazepine and the mixture was extracted with methylene chloride. The combined methylene chloride extracts were dried and con 0176 Racemic 1-(3,4-dimethoxyphenyl)-4-methyl-5- centrated. The residue was purified by column chromatog ethyl-7-methoxy-8-hydroxy-5H-2,3-benzodiazepine was raphy on Silica gel. Yield: 10 g Synthesized according to the route of Scheme 4. US 2004/0106601 A1 Jun. 3, 2004

Scheme 4 HCO COOH HCO COOEt HCO COOEt EtOH BnBr H2SO4 HO HO BO Ms HC OH H3C HO H3CO CH H3CO CH H3CO CH 1. BH p-TSOH CH 2. HO BO BO BO

HCI

HCO CHO

HCO HC HC O

HCO CH HCO CH CrO3 He O H2SO4 O BO BO

OCH OCH OCH OCH 3 PdY6. 3 HC O

H3CO CH HCO 1. NHNH2 2. HCI O HO HO

OCH OCH3 OCH3

0177 A. Esterification of 3-methoxy-4-hydroxybenzoic Successively with water, dilute Sodium bicarbonate and Acid to Yield ethyl-3-methoxy-4-hydroxybenzoate. water, then dried and concentrated. Yield: 118 g 0.178 A Solution of 100 g of 3-methoxy-4-hydroxyben 0179 B. Benzylation of ethyl-3-methoxy-4-hydroxyben zoic acid and 17 g of concentrated sulfuric acid in 300 mL Zoate to Yield ethyl-3-methoxy-4-benzyloxybenzoate. of absolute ethanol was heated at reflux overnight. The 0180 A solution of 118 g of ethyl-3-methoxy-4-hydroxy mixture was concentrated and the residue poured into water. benzoate and 86 mL of benzyl bromide in 600 mL of acetone Methylene chloride was added and the solution washed containing a Suspension of 124 g of potassium carbonate US 2004/0106601 A1 Jun. 3, 2004

was heated at reflux overnight. The mixture was filtered, the with methylene chloride. The combined methylene chloride filtrate concentrated and the residue recrystallized from extracts were dried and concentrated. The residue was acetone. purified by column chromatography on Silica gel. Yield: 18 0181 C. Addition of Ethyl Magnesium Iodide to ethyl 9. 3-methoxy-4-benzyloxybenzoate to Yield 3-(3-methoxy-4- 0191) H. Debenzylation of 3-(4-benzyloxy-5-methoxy-2- benzyloxyphenyl)pentan-3-ol. {3,4-dimethoxyphenylcarbonylphenyl)pentan-2-one to Yield 3-2-(3,4-dimethoxyphenyl)carbonyl)-4-hydroxy-5- 0182 Iodoethane (112 mL) was added dropwise to a methoxyphenylpentan-2-one. Suspension of 35 g of magnesium turnings in 160 mL of ether. After the formation of ethyl magnesium iodide was 0192 A solution of 18 g of 3-(4-benzyloxy-5-methoxy complete, a Solution of 142 g of ethyl 3-methoxy-4-benzy 2-3,4-dimethoxyphenylcarbonylphenyl)pentan-2-one in loxybenzoate in ether was added and the mixture was methylene chloride containing a Suspension of 2 g of 10% allowed to Stir at room temperature for 3 days. The reaction palladium on carbon was hydrogenated at 80 psi for 1 hr. was quenched by addition of Saturated ammonium chloride. The mixture was filtered through diatomaceous earth and the The layers were Separated and the ether layer was dried and filtrate concentrated. Yield: 15 g. concentrated to an oily residue. Yield: 110 g. 0193 I. Annulation of 3-2-(3,4-dimethoxyphenyl)car 0183) D. Elimination of HO from 3-(3-methoxy-4-ben bonyl)-4-hydroxy-5-methoxyphenylpentan-2-one by Reac Zyloxyphenyl)pentan-3-ol to Yield 4-((17)-1-ethylprop-1- tion with Hydrazine to Yield 1-(3,4-dimethoxyphenyl)-4- enyl)-1-benzyloxy-2-methoxybenzene. methyl-5-ethyl-7-methoxy-8-hydroxy-5H-2,3- benzodiazepine. 0184. A solution of 110 g of crude 3-(3-methoxy-4- benzyloxyphenyl)pentan-3-ol and 7 g of p-tolenesulfonic 0194 Asolution of 14 g of 3-2-(3,4-dimethoxyphenyl acid in 2 L of benzene was heated at reflux for 4 hr with )carbonyl)-4-hydroxy-5-methoxyphenylpentan-2-one and azeotropic removal of water. The mixture was then filtered 4.7 mL of hydrazine in 280 mL of ethanol was heated at through a pad of Sodium bicarbonate and the filtrate con reflux for 0.5 hr. After allowing the solution to cool to room centrated. The residue was purified by column chromatog temperature, it was Saturated with HCl gas. The mixture was raphy on neutral alumina. then concentrated to a volume of about 5 mL, basified with concentrated ammonium hydroxide, and extracted with 0185 E. Addition of HO to 4-((1Z)-1-ethylprop-1-enyl)- methylene chloride. The combined methylene chloride 1-benzyloxy-2-methoxybenzene to Yield 3-(3-methoxy-4- extracts were dried and concentrated, and the residue recrys benzyloxyphenyl)pentan-2-ol. tallized from ethyl acetate/hexane. Yield: 1.5 g. 0186 To a solution of 96 g of 4-((17)-1-ethylprop-1- 0195 The product 1-(3,4-dimethoxyphenyl)-4-methyl-5- enyl)-1-benzyloxy-2-methoxybenzene in tetrahydrofuran at ethyl-7-methoxy-8-hydroxy-5H-2,3-benzodiazepine was 0° C. was added 510 mL of a 1.0M solution of borane analyzed by HPLC, elemental analysis, GC/MS, proton tetrahydrofuran complex in tetrahydrofuran. The mixture NMR and differential scanning calorimetry (DSC). The data was stirred for 3 hr at 0°C., then 204 mL of 25% hydrogen are as follows: peroxide was added. The mixture was adjusted to pH 8 by 0196) Purity: 98.36% by HPLC (% area). Column: addition of SM sodium hydroxide and extracted with ether. Betasil Phenyl 4.6x150 mm. Mobile Phase: The combined ether extracts were dried and concentrated. Acetonitrile:0.01M Phosphate Buffer (70:30). Flow Yield: 102 g. Rate: 0.5 mL/min. Wavelength: 254 nm. 0187 F. Reaction of 3-(3-methoxy-4-benzyloxyphenyl 0197) GC-MS; M/e=358; with the fragmentation pat )pentan-2-ol with 3,4-dimethoxybenzaldehyde to Yield 4-(4- tern matching the proposed Structure. ethyl-6-methoxy-7-benzyloxy-3-methyliso-chromanyl)-1,2- 0198 Differential scanning calorimetry (DSC): Tem dimethoxybenzene. perature program 100° C. to 300° C. at 5° C./min, 0188 A Solution of 46 g of 3,4-dimethoxybenzaldehyde indicated molar purity=99.14% and melting point of and 100 g of crude 3-(3-methoxy-4-benzyloxyphenyl)pen 146.2° C. tan-2-ol in 0.3 L of dioxane was saturated with hydrogen 0199 Elemental analysis (calculated/analysis): % chloride gas. The mixture was heated at reflux for 3 hr, then C-68.14/68.12; % H-6.63/6.63; N-7.43/7.20. The cal poured into water, basified with dilute sodium hydroxide and culated values include 0.1M of residual ethyl acetate. extracted with methylene chloride. The combined methylene chloride extracts were dried and concentrated. 0200) NMR (DCCL) (performed on GE QE 300): 1.08 ppm (t, 3H); 1.96 (s, 3H); 2.10 (m, 2H); 2.77 (m, 1H); 3.91 0189 G. Ring-Opening of 4-(4-ethyl-6-methoxy-7-ben (s, 3H); 3.93 (s, 3H); 3.98 (s, 3H); 5.73 (bs, 1H); 6.70 (s, Zyloxy-3-methylisochromanyl)-1,2-dimethoxybenzene to 1H); 6.80 (d. 1H); 6.95 (s, 1H); 7.00 (d. 1H); 7.58 (s, 1H). Yield 3-(4-benzyloxy-5-methoxy-2-3,4-dimethoxyphe nylcarbonyl phenyl)pentan-2-one. Example 4 0190. To a solution of 50 g of crude 4-(4-ethyl-6-meth Resolution of 1-(3-hydroxy-4-methoxyphenyl)-4- oxy-7-benzyloxy-3-methyliso-chromanyl)-1,2-dimethoxy methyl-5-ethyl-7,8-dimethoxy-5H-2.3-benzodiaz benzene in acetone at 5 C. was added a solution of 50 g of epine chromic oxide in 500 mL of 35% sulfuric acid. The mixture 0201 The enantiomers of racemic-1-(3-hydroxy-4-meth was Stirred at room temperature for 2 hr, neutralized by oxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-ben adding cold 10% sodium hydroxide and concentrated to Zodiazepine are resolved by chiral chromatography as fol remove acetone. Water was added and the mixture extracted lows. US 2004/0106601 A1 Jun. 3, 2004

0202 Racemic-1-(3-hydroxy-4-methoxyphenyl)-4-me thyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine is TABLE 2-continued loaded onto a semipreparative (500 mmx10 mm) Chirobi otic V column (ASTEC, Whippany, N.J.). Elution of the Change in absolute neutrophil count and enantiomeric mixture with methyl-tert-butyl ether/acetoni % change over the 16-day study. trile (90/10 V/V), at a flow rate of 40 mL/min, is monitored Mean Neutrophil Standard Mean at 310 nm. Fraction size is 10-20 mL and fractions are No. count (day 16) deviation 76 change Standard Subjected to analytical chromatography using the same Group animals (x 10 fulL) (x 10/u L) (day 16) deviation solvent composition on an analytical (150x4.6 mm) Chiro 1F 1O 0.5 O.21 1O 3.6 biotic V column. The fractions containing each isolated 2F 1O 2.4* 2.14 29* 19 enantiomer are processed by removing the elution Solvent in 3F 1O 6.O* 2.29 47: 9.8 4F 9 11.6* 7.99 63* 10.5 WCUO.

Example 5 0205 The normal range of absolute neutrophil counts is 0.4x10 cells/ul for male and 0.2-2.4x10 cells/ul for Increase in Neutrophil Production in 16-Day Study female rats. For each Sex, groups 2-4 were compared with in Rat. the control group (Group 1M or 1F). Group comparisons 0203 The present assay was performed as follows: were evaluated at the 5.0%, two-tailed probability level. Eighty test animals; Crl:CD(E) (SD)IGS BR rats; 40 female Data marked with * represents a Statistically significant (156-208 g) and 40 male (198-286 g); approximately 8 difference from the corresponding control group. Thus, this weeks old were divided into 4 groups each of male and Study demonstrates that neutrophil levels are significantly female animals as shown in Table 1, below. Animals were increased in a dose-dependant manner in the animals treated identified using an implanted microchip identification with R-tofisopam, which is a compound of formula I. device encoded with a unique number. Each animal was 0206 All references cited herein are incorporated by dosed daily via oral gavage with either a test Substance reference. The present invention may be embodied in other (R-tofisopam) or control, starting on Day 1 and through day Specific forms without departing from the Spirit or essential 15. The control article was 0.5% low viscosity carboxym attributes thereof and, accordingly, reference should be ethylcellulose (CMC) (Lot No. 74231) and 0.4% Tween 80(8) made to the appended claims, rather than to the foregoing (lot no. QJ1033) in reverse osmosis water. Specification, as indication the Scope of the invention. TABLE 1. What is claimed is: Dose group assignments for 16-day study in Rat 1. A method of increasing the absolute neutrophil count in Dose concentration an individual, comprising administering to Said individual Group No. animals Dose (mg/kg/day) (mg/mL) an effective amount of at least one compound of formula I: 1M 1O O O 2M 1O 1OO 1O 3M 1O 2OO 2O 4M 1O 400 40 1F 1O O O 2F 1O 1OO 1O 3F 1O 2OO 2O 4F 1O 400 40

0204 The animals were sacrificed on Day 16 (with the exception of three males and one female which died on test or was Sacrificed before the Scheduled termination, and which are not included in the mean neutrophil count data). Neutrophil counts in Table 2, below, are reported as a mean value of the count for the animals of each group Surviving and Sacrificed on Day 16. wherein: R" is -(C-C.)hydrocarbyl or -(C-C)heteroalkyl; TABLE 2 R is selected from the group consisting of -H, and Change in absolute neutrophil count and -(C-C)hydrocarbyl, % change over the 16-day study. wherein R' and R may combine to form a carbocyclic or Mean Neutrophil Standard Mean heterocyclic 5- or 6-membered ring; No. count (day 16) deviation 76 change Standard Group animals (x 10 fulL) (x 10/u L) (day 16) deviation R is independently selected from the group consisting of 1M 1O O.7 O.21 1O 2.O -O(C-C)alkyl, -OH, -O-acyl, -SH, -S(C- 2M 1O 5.7% 4.28 43* 15.7 C.)alkyl, -NH, -NH(C-C)alkyl, -N((C- 3M 1O 7.4% 4.42 45* 16.2 C.)alkyl), -NH-acyl, -NO and halogen; 4M 7 12.9% 7.69 57* 12.3 n is 1, 2 or 3; US 2004/0106601 A1 Jun. 3, 2004

R" and R are independently selected from the group (R)-1-(3-methoxy-4-hydroxyphenyl)-4-methyl-5-ethyl-7- consisting of -O(C-C)alkyl, -OH, O-acyl, -SH, hydroxy-8-methoxy-5H-2,3-benzodiazepine; and -S(C-C)alkyl, -NH2, NH-acyl and halogen; (R)-1-(3-hydroxy-4-methoxyphenyl)-4-methyl-5-ethyl-7- wherein, R" and R may combine to form a 5, 6 or hydroxy-8-methoxy-5H-2,3-benzodiazepine, Substan 7-membered heterocyclic ring, and tially free of the corresponding (S)-enantiomers; wherein, compounds according to formula I are (R)- or a pharmaceutically acceptable Salt thereof. enantiomerS Substantially free of the corresponding 6. The method of claim 5, wherein the compound is (S)-enantiomers, with respect to the absolute confor (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8- mation at the 5-position of the benzodiazepine ring, dimethoxy-5H-2,3-benzodiazepine, substantially free of the or a pharmaceutically-acceptable Salt of Such a com corresponding (S)-enantiomer; pound. or a pharmaceutically acceptable Salt thereof. 2. The method of claim 1, wherein: 7. A method of treating an individual afflicted with neutropenia, comprising administering to Said individual an R is selected from the group consisting of -H and effective amount of at least one compound of formula I:

-(C-C)alkyl, R is independently selected from the group consisting of -O(C-C)alkyl, -O-acyl and -OH, n is 1, 2 or 3; R" and R are independently selected from the group consisting of -O(C-C)alkyl, -O-acyl and -OH, wherein, R' and R may combine to form a 5, 6 or 7-membered heterocyclic ring; or a pharmaceutically-acceptable Salt of Such a com pound. 3. The method of claim 2, wherein: wherein: R" is —CHCH; R" is -(C-C)hydrocarbyl or -(C-C)heteroalkyl; R’ is -CH R" is selected from the group consisting of -H, and R. R" and Rare independently selected from the group -(C-C)hydrocarbyl, consisting of -OH and -O(C-C)alkyl; wherein R' and R may combine to form a carbocyclic or n is 1, 2 or 3; heterocyclic 5- or 6-membered ring; or a pharmaceutically-acceptable Salt of Such a com R is independently selected from the group consisting of pound. -O(C-C)alkyl, -OH, -O-acyl, -SH, -S(C- C.)alkyl, -NH, -NH(C-C)alkyl, -N((C- 4. The method of claim 3, wherein: C.)alkyl), -NH-acyl, -NO and halogen; R" is -CH2CH; n is 1, 2 or 3; R’ is -CH R" and R are independently selected from the group R. R' and Rare independently selected from the group consisting of -O(C-C)alkyl, -OH, O-acyl, -SH, consisting of -OH and -OCH; -S(C-C)alkyl, -NH2, NH-acyl and halogen; n is of 1, 2 or 3; wherein, R" and R may combine to form a 5, 6 or or a pharmaceutically-acceptable Salt of Such a com 7-membered heterocyclic ring; pound. wherein, compounds according to formula I are (R)- 5. The method of claim 4, wherein the compound is enantiomerS Substantially free of the corresponding Selected from the group consisting of: (S)-enantiomers, with respect to the absolute confor (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8- mation at the 5-position of the benzodiazepine ring, dimethoxy-5H-2,3-benzodiazepine; or a pharmaceutically-acceptable Salt of Such a com pound. (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7-hy 8. The method of claim 7, wherein the neutropenia treated droxy-8-methoxy-5H-2,3-benzodiazepine; is a Side effect of drug therapy. (R)-1-(3-hydroxy-4-methoxyphenyl)-4-methyl-5-ethyl-7, 9. The method of claim 8 wherein the drug therapy 8-dimethoxy-5H-2,3-benzodiazepine; comprises administration of at least one chemotherapeutic (R)-1-(3-methoxy-4-hydroxyphenyl)-4-methyl-5-ethyl-7, agent. 8-dimethoxy-5H-2,3-benzodiazepine; 10. The method of claim 8 wherein the drug therapy comprises administration of at least one therapy Selected (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7-meth from the group consisting of thyroid inhibitors, antibiotics, oxy-8-hydroxy-5H-2,3-benzodiazepine; neuropsychotropics, cardiovascular medications, analgesics, US 2004/0106601 A1 Jun. 3, 2004

antimalarials, nonsteroidal antiinflammatory agents, antihis wherein: tamines and combinations thereof. 11. The method of claim 10 wherein the drug therapy R" is -(C-C.)hydrocarbyl or -(C-C)heteroalkyl; comprises administration of at least one drug Selected from the group consisting of trimethoprim, chloramphenicol, R is selected from the group consisting of -H, and penicillins, cephalosporins, aminoglycosides, tetracyclines, -(C-C)hydrocarbyl, nitroimidazoles, nitrofurantoin, flucytosine, rifampin, iso niazid, ethambutol, dapSone, Sulfonamide antibiotics, clo wherein R' and R may combine to form a carbocyclic or miprimine, thiacetazone, dipyrone, SulfaSalazine, meSala heterocyclic 5- or 6-membered ring; Zine, ciprofloxacin, chloroquin, mebendazole, terbendafine, pyrimethamine, levamisole, ristocetin, griseofulvin, phe R is independently selected from the group consisting of nothiazines, benzodiazepines, amoxapine, meprobamate, -O(C-C)alkyl, -OH, -O-acyl, -SH, -S(C- barbiturates, clozapine, risperidone, imipramine, C.)alkyl, -NH, -NH(C-C)alkyl, -N((C- desipramine, thiothixene, haloperidol, Valproic acid, hydan C.)alkyl), -NH-acyl, -NO and halogen; toins, Succinimides, trimethadione, carbamazepine, procainamide, quinidine, propafenone, captopril, propra n is 1, 2 or 3; nolol, hydralazine, methyldopa, ibuprofen, indomethacin, R" and R are independently selected from the group Sulindac, tolmetin, aspirin, aminopyine, phenylbutaZone, diflunisal, benoxaprofen, allopurinol, colchicine, propylth consisting of -O(C-C)alkyl, -OH, O-acyl, -SH, iouracil, thiouracil, methimazole, carbimazole, thiocyanate, -S(C-C)alkyl, -NH2, NH-acyl and halogen; potassium perchlorate, cimetidine, ranatadine, tripelen wherein, R" and R may combine to form a 5, 6 or namine, methaphenilene, thenalidine, mianserin, bro mopheneramine, quinine, hydroxychloroquin, quinacrine, 7-membered heterocyclic ring, and diaZOxide, dihydropyridines, ticlopidine, Vesnarinone, wherein, compounds according to formula I are (R)- aprindine, imipenem/cilastatin, Zidovudine, fludarabine, enantiomerS Substantially free of the corresponding acyclovir, turbinafine, aminoglutethimide, famotidine, beZafibrate, flutamide, tamoxafen, penecillamine, retinoic (S)-enantiomers, with respect to the absolute confor acid, metoclopramide, phenindone, dinitrophenol, mation at the 5-position of the benzodiazepine ring, ethacrynic acid, rauwolfia, ethanol, chlorpropamide, tolb or a pharmaceutically-acceptable Salt of Such a com utamide, thiazides, Spironolactone, methazolamide, aceta pound. Zolamide, levodopa and combinations thereof. 12. The method of claim 7, wherein the neutropenia 14. The method of claim 13 wherein the risk of develop treated is a Side effect of ionizing radiation therapy. ing neutropenia is associated with a forthcoming drug 13. A method of preventing neutropenia in an individual therapy. who is at risk of developing neutropenia, Said method 15. The method of claim 14 wherein the drug therapy comprising administering to Said individual an effective comprises administration of at least one chemotherapeutic amount of at least one compound of formula I: agent. 16. The method of claim 13, wherein the risk of devel oping neutropenia is associated with a forthcoming ionizing radiation therapy. 17. The method of claim 13 wherein the risk of develop ing neutropenia is associated with immunodeficiency. 18. The method of claim 17 wherein said immunodefi ciency is caused by a cancer. 19. The method of claim 17 wherein said immunodefi ciency is caused by a virus. 20. The method of claim 19 wherein said virus is human immunodeficiency virus.