US 20100227853A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0227853 A1 Hoffman et al. (43) Pub. Date: Sep. 9, 2010

(54) INHIBITORS OF CYCLIC AMP Publication Classi?cation PHOSPHODIESTERASES (51) Int CL A61K 31/55 (2006.01) (75) Inventors: Charles S. Hoffman, Wenham, MA A61K 31/519 (2006-01) (Us); Frank Douglas My, A61P 35/02 (2006.01) WatertoWn, MA (U S),_ Arlene A61P 29/00 (2006.01) W man Petri Wa land MA (US) A611) 31/18 (200601) Y a Y ’ A61P 9/10 (2006.01) A61P 19/10 (2006.01) Correspondence Address: A611) 3 7/06 (2006-01) WOLF GREENFIELD & sACKs, P.C. A61K 31/53 77 (2006-01) 600 ATLANTIC AVENUE A61K 31/4015 (2006.01) BOSTON MA 02210 2206 US A61K 31/551 (200601) ’ ' ( ) A61K 31/4985 (2006.01) (52) US. Cl...... 514/217.06; 514/260.1; 514/255.05; (73) Assignee: Trustees of Boston College, 514/234.2; 514/424; 514/252.16; 514/267; Chestnut Hill, MA (US) 514/252.11; 514/218; 514/250 (57) ABSTRACT (21) App1.No.: 12/426,171 Recombinant ?ssion yeast cells and methods of using them are described, Which provide for identi?cation of chemical and biological inhibitors or activators of a target exogenous (22) Filed: Apr. 17, 2009 phosphodiesterase (PDE). The invention provides, in some aspects, compounds that inhibit cAMP PDE activity and Related US. Application Data compositions that include such compounds. The invention, in part, also includes methods of using cAMP PDE-inhibiting (60) Provisional application No. 61/124,657, ?led on Apr. compounds in the treatment of cAMP PDE-associated dis 18, 2008. eases and/ or disorders. Patent Application Publication Sep. 9, 2010 Sheet 1 0f 15 US 2010/0227853 A1

509 1/cgs2

900 girl-1 800 700 600 500 400 [5-GALACTOSIDASEACTIVITY 300 git2-7 200 100 j/> WILD TYPE I | 4 TIME AFTER CAMP REMOVAL (HOURS) Fig. 2 Patent Application Publication Sep. 9, 2010 Sheet 2 0f 15 US 2010/0227853 A1

GLUCOSE

ATP —>69 cAiVIP —> AMP

-Ura - PKA —I 5FOA ++ Fig. 3A

GLUCOSE E] ATP —> cAMP —> AMP I -u Fig.A 3B

GLUCOSE ACTIVATOR

ATP —> CAMP —> AMP I A -u *f Fig. 3c

GLUCOSE INHIBITOR >I< @ ATP —> cAMP —> AMP

I -Ura - PKA —I 5FOA ++ Fig. 3D Patent Application Publication Sep. 9, 2010 Sheet 3 0f 15 US 2010/0227853 A1

[ROLIPRAM] (MM) Fig. 4 Patent Application Publication Sep. 9, 2010 Sheet 4 0f 15 US 2010/0227853 A1

80

70 Z ||-|_J 60 2 a. 50-. m 4. 5 4o—

gE 30- +PDE4A+ROLIPRAM % 2O; -¢-PDE4B+ROLIPRAM <5 " +PDE2A+EHNA U __ 10-"

O | | | | | | | O 20 4O 6O 80 100 120 140 TIME (MINUTES) F|g.5A

EJDMSO EiZOpM mzoowvl (pmoI/mgpr.)CAMP

PDE4B ' PDE4A ' PDESA ' PDEZA ROLIPRAM ROLIPRAM ROLIPRAM EH NA Fig. 5B Patent Application Publication Sep. 9, 2010 Sheet 5 0f 15 US 2010/0227853 A1

2 5 O K

200K DEFINING I'DEAD" CELLS: WE USED VINCRISTINE AS POSITIVE CONTROL. ALMOST 100% OF "LIVE" POPULATION IS GONE.

FSC-A FSC-A Specimen_00I_vin_00I .fcs Specimen_O0I_vin_00I .fcs Event Count: 11967 Event Count: 11967 2 5 O K DEFINING "LIVE"CELLS: CELLS NOT TREATED WITH ANY 2I REAGENT. THE”LIVE" POPULATION IS CLEARLY VISIBLE. TWO O5 O SUBPOPULATIONS OF "DEAD"

a“ FSC-A FSC-A Fig. 6A Specimen_00I_ceIIs_00I .fcs Specimen_00I_ceIIs_0OI .fcs Event Count: 11445 Event Count: 11445 Patent Application Publication Sep. 9, 2010 Sheet 6 0f 15 US 2010/0227853 A1

IN VITRO APOPTOSIS ASSAY: s54, 40 MM

120-00— m FSC LYMPHOMA POP IN THIS ASSAY, s54 HOECHST LIVE TREATMENT LEADS TO

100.00- % {E CELLS

—I 80.00

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20.00—0.00 Q I |_u | a"<1- I |_u & Z a % 5 E a -20.00— {E 6 % Z 2 I44 3 5 E + Q. 0 (EACH ASSAY DONE IN TRIPLICATES) Fig. 6B Patent Application Publication Sep. 9, 2010 Sheet 7 0f 15 US 2010/0227853 A1

T AD“ | 727 N3 U | 2 | S2 4DI AL m0. 4321 E%EEEEW O50 i.m. EEEZD mR 700 0,.BSE28 101 E26221L QMCEWFZDm 401F200 m“65 mSEE Fig. 7A Patent Application Publication Sep. 9, 2010 Sheet 8 0f 15 US 2010/0227853 A1

LYMPH NODETUMOR LOAD 512 2.763

CELLSINTHELN EVENTS M1 (GFP-POSITIVE)OFTUMOR% MWU NTREATED 0 CONTROL.P 10° 101 102 103 104 UNTREATED1 s54TOX|C|TY CHOP_ TREATED FL1-H FigT7B Patent Application Publication Sep. 9, 2010 Sheet 9 0f 15 US 2010/0227853 A1

PROLIFERATION ANDVIABILITY OF PBMCs: DAY 3 MTT

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PROLIFERATION AND VlABlLlTY OF SlVmac239 INFECTED PBMCs: DAY 7 MTT 110 Patent Application Publication Sep. 9, 2010 Sheet 12 0f 15 US 2010/0227853 A1

SlVmac239 VIRAL PRODUCTION IN PBMCs I 3 4 2 2 11 6 4 9 8 I555 4062111 .. 1 1 8 9 0000520752 0000050505 169 SlVmac239 VIRAL 00000000000 ____.______PRODUCTION IN 62 AND 2iLA8 ROLIPRAIVI SIMILAR TO NO COMPOUND CONTROL IQZDOLEOOOz I223: #22:? IQZDOQEOOOz #2238 DAY 4 DAY 7 Fig. 9

Patent Application Publication Sep. 9, 2010 Sheet 14 0f 15 US 2010/0227853 A1

PDE7B AT VARIOUS DlLUTlONS vs COMPOUND#12 120

100- c a

s0

60

40- -<>- 1 ; 4k % CAMP HYDROLIZED +1; 8k % CAMP HYDROLIZED 209 +1;10k% CAMP HYDROLIZED —<>—1:20k% CAMP HYDROLIZED

O | | | | | | | | | | 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 Hg. 11

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I00

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—O—BRL 50481 —V—COMPOUND I2 O —I:I—COMPOUND28

O

INHIBITOR CONCENTRATION (IogM) Fig. 13 US 2010/0227853 A1 Sep. 9, 2010

INHIBITORS OF CYCLIC AMP expressed in the testes as Well as the brain. PDE11 is PHOSPHODIESTERASES expressed in skeletal muscle, prostate, kidney, liver, pituitary and salivary glands, and testes (BosWell-SmithV. et al., 2006, RELATED APPLICATIONS Brit JPharm 147:S252-57). [0001] This application claims the bene?t of US. provi [0006] The four PDE4 subfamilies are encoded by separate sional application 61/124,657, ?ledApr. 18, 2008. The entire genes (A, B, C, D) that generate many isoforms through the teachings of the referenced provisional application are incor use of alternative mRNA splicing and distinct promoters. porated by reference herein in its entirety. Isoforms generated by the four PDE4 subfamilies are each individually characterized by unique N-terminal regions. GOVERNMENT SUPPORT They can be divided into long forms, Which possess both the Upstream Conserved Region 1 (UCR1) and Upstream Con [0002] The invention Was supported, in Whole or in part, by served Region (UCR2) regulatory regions, While the short grant No. 1 R21 GM079662-01 from the National Institute of isoforms lack UCR1 and the super-short isoforms lack UCR1 General Medical Sciences of the National Institute of Health and also have a truncated UCR2. (NIH). The Government has certain rights in the invention. [0007] TWo PDE7 genes (PDE7A and PDE7B) have been identi?ed. PDE7A has three isoforms generated by alternate FIELD OF THE INVENTION splicing; PDE7A1 is restricted mainly to T cells and the brain, [0003] The present invention provides methods for treating PDE7A2 for Which mRNA is expressed in a number of cell in?ammatory diseases comprising either the administration types including muscle cells, and PDE7A3 found in activated of dual phosphodiesterase 4-phosphodiesterase 7 (PDE4/ T cells. The PDE7A1 and PDE7A2 isoforms have different PDE7) inhibitors, or the simultaneous or sequential co-ad sequences at the amino termini. PDE7A3 is similar to ministration of selective PDE4 inhibitors together With selec PDE7A1 in the amino terminus but has a different carboxy tive PDE7 inhibitors. The present invention further relates to terminal sequence than PDE7A1 and PDE7A2. PDE7B has pharmaceutical compositions containing these inhibitors, and approximately 70% homology to PDE7A in the enZymatic the use of these inhibitors in the treatment of in?ammatory core. diseases. [0008] PDEs are important drug targets. Many PDE-spe ci?c inhibitors have been developed and are currently being BACKGROUND OF THE INVENTION used or are being evaluated for use, such as KS-505a (PDE1); [0004] Phosphodiesterases (PDEs) play an important role EHNA (PDE2); Cilostamide, Enoxamone, Milrinone, Sigua in various biological processes by hydrolysing the key second Zodan (PDE3); Rolipram, Ro?umilast, Cilomilast (PDE4); messengers adenosine and guanosine 3',5'-cyclic monophos Sildena?l, Zaprinast (PDE5); Dipyridamole (PDE6); BRL phates (cAMP and cGMP respectively) into their correspond 50481 (PDE7), BAY 73-6691 (PDE9) (Boswell-Smith V. et ing 5'-monophosphate nucleotides. Therefore, inhibition of al., 2006, Brit JPharm 147:S252-57). PDE activity produces an increase of cAMP and cGMP intra [0009] PDE2 inhibitors Were developed for the treatment of cellular levels that activate speci?c protein phosphorylation sepsis, and Acute Respiratory Distress Syndrome (ARDS). pathWays involved in a variety of functional responses. At [0010] PDE3 inhibitors Were developed for the treatment of least 11 families of PDEs exist, some of Which (PDE 4, 7, 8) congestive heart failure, airWay diseases, and to treat fertility. are speci?c for cAMP, and others (PDE 5, 6, 9) for cGMP, PDE3 inhibitors have been shoWn to relax vascular and air While other family members have dual speci?city (PDE 1, 2, Way smooth muscle, inhibit platelet aggregation and induce 3, 10, 11). PDEs are expressed in a tissue and cell speci?c lipolysis. manner, and expression also changes depending on the cell [0011] PDE4 inhibitors Were developed for the treatment of state. For example, resting T lymphocytes express mainly in?ammatory airWays disease, asthma, chronic obstructive PDE3 and PDE4. HoWever, upon activation, T cells dramati pulmonary disease (COPD), allergic rhinitis, psoriasis, rheu cally upregulate PDE7 and appear to rely on this isoZyme for matoid arthritis, depression, schiZophrenia, AlZheimer’s Dis regulation of cAMP levels. ease, memory loss, cancer, dermatitis and multiple sclerosis. [0005] Three PDE1 genes have been identi?ed and are Inhibition of PDE4 has been associated With an anti-in?am expressed in heart, lung, and kidney tissue, as Well as in matory response associated With T cells as Well as monocytes, circulating blood cells and smooth muscle cells. PDE2 is macrophages, mast cells, basophils and neutrophils. The expressed in adrenal gland, heart, lung, liver, and platelets. majority of PDE4 selective inhibitors reported on to date The PDE3 family, expressed by the PDE3A and PDE3B serve to inhibit PDE4 isoforms from all four subfamilies With genes, are distributed in several tissues including the heart, either little or no PDE4 subfamily selectivity, While PDE4A lung, liver, platelets, adipose tissue, and in?ammatory cells. and PDE4B are the actual anti-in?ammatory targets. Over tWenty isoforms of PDE4 are expressed by four genes, [0012] PDE5 inhibitors Were developed for the treatment of and these are expressed in a Wide variety of tissues including erectile dysfunction and impotence, pulmonary hyperten heart, kidney, brain, liver, lung, the gastrointestinal track and sion, female sexual dysfunction, cardiovascular disease, pre circulating blood and in?ammatory cells. PDE5 (three iso mature ejaculation, stroke, leukaemia, and renal failure. forms) is expressed for example in the human corpus caver [0013] PDE7 inhibitors Were developed for the treatment of nosum (vascular) smooth muscle, lung, andplatelets. PDE6 is in?ammation. Increasing cAMP levels by selective PDE7 expressed by three genes in photoreceptors of the retina. inhibition appears to be a potentially promising approach to PDE7 proteins are expressed by tWo genes in skeletal muscle, speci?cally block T-cell mediated immune responses. heart, kidney, brain, pancreas, and T lymphocytes. PDE8 is [0014] There are side-effects associated With many PDE expressed by tWo genes in testes, eye, liver, skeletal muscle, inhibitors, Which limit their use. PDE1 inhibitors have dem heart, kidney, ovary, brain, and T lymphocytes. PDE9 is onstrated potent vasodilator activity. PDE3 inhibitors have expressed in kidney, liver, lung, and brain. PDE10 is demonstrated potent cardiac inotropic activity. Nausea, eme US 2010/0227853 A1 Sep. 9, 2010

sis and cardiac arrhythmias remain the major obstacles in the administration of a dual PDE4/7 inhibitor (PDE4A/7, development of PDE4 inhibitors, especially caused by inhi PDE4B/ 7, PDE4A/ 4B/ 7), can be used to increase therapeutic bition of PDE4D. PDE5 inhibitors affect PDE6 activity in the effectiveness, and/or reduce toxicity and/or side effects (such photoreceptors of the retina and can lead to visual distur as nausea) over presently-available approaches. The com bances consisting of altered color perception. There is an bined activity of PDE4 and PDE7 or dual PDE4/ 7 inhibitors unmet medical need to develop effective methods and iden tify effective PDE inhibitor compounds, including PDE may be especially useful in treating a Wide variety of immune inhibitors that speci?cally act on individual family members and in?ammatory disorders as an immunosuppressant and even on individual isoforms expressed from a single PDE therapy. PDE7 inhibitors act by inhibiting a very early stage gene, for treatment of immune and in?ammatory disorders. of the T cell activation cascade. PDE4 inhibition decreases the production of the pro-in?ammatory cytokines such as SUMMARY OF THE INVENTION Tumor Necrosis Factor alpha (TNF-ot) in monocytes and macrophages, as Well as affect granulocytes, such as neutro [0015] Described herein are PDE4 inhibitors (e.g., PDE4A phils. Dual PDE4/7 inhibitors or co-administration of selec inhibitors, PDE4B inhibitors), PDE7 inhibitors, combination tive PDE4 and PDE7 inhibitors are expected to be particularly inhibitors (e.g., PDE4A/4B, PDE4/7, such as PDE4A/7, useful in treating disorders that involve one or more in?am PDE4B/7, PDE4A/4B/7); methods in Which such inhibitors matory response alleviated, at lease in part, by PDE4 inhibi are used, including methods in Which an inhibitor is used to tion (e.g., via decreased mast cell, basophil and neutrophil treat a condition or disease (e.g., an in?ammatory disease, a degranulation and monocyte and macrophage production of neurological disease, memory loss, chronic lymphocytic leu pro-in?ammatory cytokines such as TNF-ot), and/ or are alle kemia, osteoporosis, HIV infection, cerebrovascular viated at least in part by PDE7 inhibition (e.g., through ischemia); and pharmaceutical compositions comprising at decreased T cell activation), e.g., disorders such as rheuma least one PDE4 inhibitor (e.g., PDE4A inhibitor, PDE4B toid arthritis, in?ammatory boWel disease (IBD), psoriasis, inhibitor), PDE7 inhibitor, PDE4/7 combination inhibitor) asthma, chronic obstructive pulmonary disease (COPD), and an appropriate carrier. The pharmaceutical composition lupus, visceral pain, osteoarthritis, osteoporosis, allergic can optionally additionally comprise at least one additional rhinitis, cancer, acquired immune de?ciency syndrome, drug. allergy, fertility diseases, and multiple sclerosis among oth [0016] PDE inhibitors Were identi?ed using methods ers. A PDE4-PDE7 inhibitor combination is also expected to described herein, such as high throughput drug screens on have a decreased potential for clinically signi?cant side genetically engineered ?ssion yeast strains that express drug effects compared to current immunosuppressants. targets (e.g., PDE4A and/or PDE4B, Which are anti-in?am matory targets). PDE inhibitors Were identi?ed based on their ability to stimulate groWth and compounds Were identi?ed BRIEF DESCRIPTION OF THE FIGURES because they Were effective in live cells. In addition, targets used in the assays are full-length proteins (as opposed to [0019] FIG. 1 shoWs groWth of ?ssion yeast strains carrying simply the catalytic domain) and the assay used included a mutations in the adenylate cyclase (git2) gene, the PDE built-in toxicity test, permeability test and stability test. The (cgs2) gene, or the gitl (a regulator of adenylate cyclase) gene inhibitors identi?ed display a very high degree of target speci on various groWth media. The arroWs point to tWo strains that ?city. Compounds identi?ed include inhibitors that act on tWo demonstrate that a reduction in PDE activity can restore of four PDE4 family enZymes and inhibitors that act on com 5FOA-resistant groWth to either a git2-7 or gitl-l mutant binations of PDE4 and PDE7 strains. One example of a com pound identi?ed is compound BC58, Which is an effective strain. Note that the git2 deletion strain (git2 A) remains PDE4A/ 4B inhibitor that exhibits limited/essentially no inhi 5FOA-sensitive even When carrying the cgs2-sl mutation. bition of PDE4D. Limited inhibition of PDE4D by a PDE [0020] FIG. 2 shoWs [3-galactosidase activity resulting inhibitor is desirable, in vieW of the fact that inhibition of from fbp l -lacZ expression as a function of time after removal PDE4D causes emesis and cardiac arrhythmias. Subtype of cAMP from the groWth medium. [3-galactosidase activity speci?city Was con?rmed by means of cAMP assays. Was measured at the time points indicated after cells Were [0017] As described herein and as shoWn in the tables, transferred from EMM medium containing 5 mM cAMP to Applicant has identi?ed compounds that are PDE4A inhibi EMM Without cAMP. tors; PDE4B inhibitors; PDE4A/4B inhibitors; PDE7 inhibi [0021] FIG. 3 shoWs schematic diagrams of cAMP-regu tors; and combination PDE4/ 7 inhibitors (PDE4A/7, PDE4B/ lated groWth phenotypes in ?ssion yeast strains expressing 7, PDE4A/4B/7). Inhibitors described herein can be used individually (e.g., a PDE4A inhibitor; a PDE4B inhibitor; a the fbpl-ura4 reporter. FIG. 3A is a diagram shoWing that PDE7 inhibitor; a combination inhibitor, such as PDE4A/7, glucose signaling leads to adenylylcyclase activation and a PDE4B/ 7, PDE4A/4B/ 7 or in combination With one or more cAMP signal, Which activates PKA to repress fbpl -ura4 tran other PDE inhibitor(s) (e.g., PDE4A inhibitor With a PDE4B scription. These cells cannot groW in medium lacking uracil inhibitor and/or a PDE7 inhibitor) or in combination With (-Ura), but do groW in medium containing 5FOA. FIG. 3B is another therapeutic agent/ drug that is also a PDE inhibitor or a diagram shoWing that cells carrying mutations in genes another therapeutic agent/ drug that is not a PDE inhibitor. required for glucose signaling have reduced adenylylcyclase [0018] Co-administration of PDE inhibitors, Which may be activity to loWer cAMP levels. This results in loW PKA activ selective for the PDE family, a speci?c PDE subfamily, or a ity and a failure to repress fbpl -ura4 transcription. These cells speci?c isoform of a PDE-subfamily member, such as a selec groW in medium lacking uracil (-Ura), but do not groW in tive PDE4 inhibitor With a selective PDE7 inhibitor, or medium containing 5FOA. FIG. 3C is a diagram shoWing a US 2010/0227853 A1 Sep. 9, 2010

screen for PDE activators carried out by taking a strain such as by MTT assay. The treatment of cells on Day 1 and Day 3, the one in panel A and screening for compounds that enhance leads to a 30-fold and 100-fold reduction in viral load on Days groWth in medium lacking uracil. The compounds identi?ed 4 and 7, respectively. include ones that stimulate PDE activity to loWer cAMP levels. FIG. 3D is a diagram showing a screen for PDE inhibi [0028] FIG. 10 shoWs the SFOA groWth response by tors carried out by taking a strain such as the one in FIG. 3B PDE7A- (left panel) and PDE7B-expressing (right panel) and screening for compounds that enhance groWth in SFOA ?ssion yeast strains. PDE inhibition alloWs groWth in SFOA medium. The compounds identi?ed include ones that inhibit medium as measured by optical density after 48 hours incu PDE activity to raise cAMP levels. bation at 30° C. Compound BC12 and six structural analogs [0022] FIG. 4 is a graph shoWing that deletion of pap1+ Were tested for groWth stimulation. For both PDE7A and enhances rolipram-mediated fbpl-lacZ repression. [3-galac PDE7B, BC12 is the most potent inhibitor, promoting groWth tosidase activity from tWo independent exponential phase at loW micromolar concentrations. cultures Was determined in pap 1 ’' (light gray bars) and paplA [0029] FIG. 11 is the in vitro enZyme assay data to demon (dark gray bars) gpa2' mutant strains groWn in EMM com strate that BC12 stimulates PDE7B activity in vitro. The plete medium containing various concentrations of rolipram PDE7B catalytic domain Was expressed in and puri?ed from as indicated, While receiving identical volumes of DMSO E. coli and tested for activity in an in vitro enZyme assay, (vehicle). Values are plotted as a percent of the vehicle-treated measuring the hydrolysis of cAMP to AMP. Four dilutions of cultures that did not receive rolipram. The ratio of fold-inhi the enZyme preparation Were tested at various compound bition in the pap 1 A strain versus the pap1+ strain is shoWn for concentrations Qi axis). The percent of cAMP hydrolyZed is each concentration of rolipram. plotted on the Y axis. The addition of compound BC12 increases cAMP hydrolysis. There is no evidence that PDE7B [0023] FIGS. 5A and 5B shoW graphs demonstrating that is activated in vivo by BC12, hoWever this result suggests that PDE inhibitors alter cAMP levels in yeast strains. FIG. 5A BC12 does not act by occupying the cAMP-binding site of the shoWs results When cAMP levels Were measured in exponen enzyme. tial phase cells immediately prior to 200 [1M drug addition [0030] FIG. 12 shoWs the effect ofBC12, BC28, BC54, and (rolipram for strains CHP1085 (PDE4A) and CHP1114 BC58 relative to rolipram (PDE4 inhibitor) and BRL50481 (PDE4B), and EHNA for strain LWP371 (PDE2A)), and 10, (PDE7 inhibitor) on IL2 secretion by concavalin A treated 30, 60, and 120 minutes after drug addition. Values represent Jurkat cells. the average and SD of tWo or three independent experiments. [0031] FIG. 13 is the in vitro enZyme assays of PDE7B FIG. 5B shoWs results When cAMP levels Were measured 60 activity in extracts made from rat pulmonary endothelial minutes after addition of either vehicle (DMSO), 20 [1M drug, cells. BC12 and BC28 inhibit With KI values that are approxi or 200 [1M drug as indicated. The strains used are as in FIG. 5A, together With strain CHP1141 (PDE8A). Values repre mately 100-fold loWer than that of the Glaxo SmithKline com sent the average and SD of tWo or three independent experi pound BRL50481. BC12 inhibits PDE7B, unlike the activa ments. tion seen With the E. col 1' -expressed enZyme. KI for BC 1 2 and [0024] FIGS. 6A and 6B shoW the in vitro assay of BC54 BC28 on PDE7 from rat pulmonary endothelial cells is action. Lymphoma cells are treated With the drug for 24 hours, approximately 200 nM, While the KI on PDE4 is about 25-30 then stained With Hoechst dye. The cells form a clearly micromolar. de?ned population on the fsc/ssc dot plot. Vincristine treat [0032] Also included are Tables 1 through 19. ment Was used as a positive control. Compound BC54 induces apoptosis in cell culture of tumor cells (almost 100% DETAILED DESCRIPTION OF THE INVENTION of “live” population is gone). BC54 is referred to in the ?gure as S54. [0033] Described herein are methods for treating a Wide [0025] FIGS. 7A and 7B shoW results ofan in vivo assay of variety of immune and in?ammatory disorders using PDE4 BC54 action. The results shoW that BC54 reduces the tumor inhibitor(s), PDE7 inhibitor(s); a combination of PDE4 load in lymph nodes of treated mice Who have received a inhibitors and PDE7 inhibitors, or dual PDE4A/4B, or dual transplanted tumor that groWs rapidly over the course of a PDE4/7 inhibitors (e.g., PDE4A/7, PDE4B/7, PDE4A/4B/7 Week. No signi?cant effect of BC54 Was shoWn on spleen inhibitors), Which may be selective for the PDE family, a tumor burden. speci?c PDE subfamily, or a speci?c isoform of a PDE subfamily member. Also described are compounds and com [0026] FIGS. 8A, 8B and 8C shoW the effect ofBC54 on the positions that include at least one PDE4 inhibitor (e.g., a proliferation and viability of PBMC cells. BC54 Was not PDE4A inhibitor, a PDE4B inhibitor); at least one PDE7 found to be very toxic to the cells. inhibitor, at least one combination inhibitor (e.g., PDE4A/ 4B [0027] FIG. 9 shoWs the assessment of antiviral activity of inhibitor, PDE4/7 inhibitor, such as PDE4A/7 inhibitor, BC54. PBMC cells Were isolated from uninfected rhesus PDE4B/ 7 inhibitor, PDE4A/4B/7 inhibitor) or a combination macaques and mito gen stimulated (PHA). The cells Were then of tWo or more such inhibitors. Such compositions may also incubated With SIVmac239 for 2 hours, folloWing Which they include a pharmaceutically acceptable carrier. When admin Were Washed to remove any free virus. BC54 Was then added istered to an individual, the compounds inhibit PDE4 and/or to the infected cells. Fresh compound Was added after 3 days. PDE7 activity in vivo and are useful for treating immune and The antiviral activity Was measured by SIV p27 ELISA on in?ammatory disorders. The selective PDE4 or PDE7 inhibi day 3 and 7, While the cell viability Was determined on day 7 tor compounds described herein, used alone or in combina US 2010/0227853 A1 Sep. 9, 2010

tion, and dual PDE4/7 inhibitors may be used. Combinations (e.g., combinations of tWo or more PDE4 inhibitors (e.g., -continued PDE4A inhibitor and PDE4B inhibitor); combinations of one or more PDE4 inhibitor With a PDE7 inhibitor) may be more effective than either a selective PDE4 inhibitor or a selective PDE7 inhibitor administered alone in the treatment of dis / N \ ease, through additive or synergistic activity resulting from O the combined inhibition of PDE4 and PDE7. Expression of O PDE7A, for example, increases When PDE4 is inhibited. [0034] Described herein are compounds that exhibit loW \ \N N \N—< : : toxicity against biological organisms in vitro. In some embodiments the compounds exhibit the ability to permeate biological organisms in vitro, e.g., to cross a biological mem brane. In some embodiments the compounds exhibit high bio-stability in biological organisms in vitro, e.g., are not rapidly degraded or are active for an extended period. [0035] There are numerous compounds described herein. They are grouped into Groups NI-NIII, as shoWn beloW. In certain embodiments the compounds are selected from com pounds of formula (NI) (Group NI). [0036] In one set of embodiments, the compound may have a structure as in formula NI,

(N1) O O R7 / N \ O O N N NR8, \ \ N N \ N

[0037] wherein: [0038] R7 and R8 can be the same or different and are optionally substituted carbonyl groups; and [0039] R9 is alkyl. [0041] In one embodiment, the compound has the folloW [0040] In some embodiments, R7 is C:OO)R“, Wherein ing structure, also referred to herein as “BC54”: R“ is optionally substituted alkyl; R8 is (C:O)Rb, Wherein Rb is optionally substituted alkyl or optionally substituted arylalkyl; and R9 is cyclohexyl. For example, the compound may have the structure,

0 0

O 0

[0042] Results described herein shoW that BC54 is a good PDE4 and PDE7 combination inhibitor, but does not act on all PDEs (no effect on PDE8A or PDE3A). It shoWs good activ ity in TNFO. and CLL assays. It is effective in mouse DLBCL model and in SIV cell culture. It affects PDE4B, but is Weaker than on other 4 s and 7 s.