NZ753660B2

NZ753660B2 - Compositions and methods comprising a polyamine

Info

Publication number: NZ753660B2
Application number: NZ753660A
Authority: NZ
Inventors: Travis Haussener; Sujeevini Jeyapalina; Ryan Looper; Hariprasada R Kanna Reddy; Paul R Sebahar; Dustin Williams
Original assignee: Curza Global Llc; University Of Utah Research Foundation
Priority date: 2013-05-22
Filing date: 2014-05-21
Publication date: 2021-06-29

Abstract

Compounds, compositions, and methods comprising a polyamine compound are described, which may be used to kill, disperse, treat, or reduce biofilms, or to inhibit or substantially prevent biofilm formation. In certain aspects, the present invention relates to compounds, compositions, and methods comprising polyamine compounds that have antimicrobial or dispersing activity against a variety of bacterial strains capable of forming biofilms. prising polyamine compounds that have antimicrobial or dispersing activity against a variety of bacterial strains capable of forming biofilms.

Description

COMPOSITIONS AND METHODS COMPRISING A POLYAMINE CROSS-REFERENCES TO RELATED ATIONS This application claims the beneﬁt of US. Provisional Application Nos. 6l/826,453 (ﬁled May 22, 2013), 61/826,761 (ﬁled May 23, 2013), 61/834,149 (ﬁled June 12, 2013); 61/836,555 (ﬁled June 18, 2013), 61/887,267 (ﬁled October 4, 2013), 61/902,135 (ﬁled November 8, 2013), and 61/938,111 (ﬁled February 10, 2014), as well as US. Non- Provisional Application Nos. 14/076,143 and 14/076,149 (both ﬁled November 8, 2013).

These applications are incorporated by reference in their entirety for all es.

FIELD OF THE INVENTION

[0002] The present invention is directed to polyamine compounds, compositions, and methods, which preferably have antimicrobial or sing activity t a variety of bacterial strains capable of forming bioﬁlms. Various aspects and embodiments relate lly to polyamine compounds and to methods of preparing or using such compounds.

BACKGROUND OF THE INVENTION

[0003] Antimicrobial nds, such as ional otics, have the ability to kill or to retard the growth of bacteria, fungi, and other microorganisms. Some antimicrobial compounds also are effective against viruses. Antimicrobial compounds are used in a wide variety of clinical settings, industrial applications, food production facilities and environmental applications all across the globe in an effort to reduce the risk of, for example, bacterial colonization and development of disease in people.

Traditional antibiotics are primarily derivatives or tic mimics of natural compounds ed by bacteria, plants, or fungi. These compounds typically have very speciﬁc methods of action against a cell wall/membrane component of bacteria, or an /protein in a metabolic y. Examples of traditional antibiotics on the market e penicillin, oxacillin, vancomycin, gentamicin, rifampicin and amoxicillin, among others.

Because bacteria have the ability to develop resistance genes to these antibiotics as a result of c mutations or acquired defense mechanisms that target the speciﬁc activity of the antibiotics, bacteria typically have the ability to develop resistance to traditional antibiotics. Increasingly more ent bacterial resistance has made traditional antibiotics to become less and less effective in a variety of applications.

Bacterial resistance to antibiotics represents one of the most underappreciated threats to modern society. See Zhang et al., Antibiotic resistance as a global threat: Evidence from China, Kuwait and the United States, Global Health 2, 6 (2006). Currently, more than 90% of clinical isolates of Staphylococcus aureus display resistance to penicillin. See Balaban et al., Control ofBiofilm Infections by Signal lation, Ch. 1, 1-11 (Springer, 2008). Recent s have even indicated that bacteria in natural ecosystems metabolize antibiotics as an energy source. See Leslie, Germs Take a Bite Out ofAntibiotics, Science 320, 33 (2008). The trend of bacterial resistance continues to increase as ted by almost daily scientiﬁc publications and world news reports of antibiotic resistant superbugs such as carbapenem-resistant Enterobacteriacea, vancomycin-resistant Enterococci, multidrug- resistant Pseudomonas aeruginosa and methicillin—resistant Staphylococcus aureus (MRSA).

See e. g., FoxNews.com. Europe in the Grip ofDrug-Resistant ugs (2011); k, M., TIME (2010); Arias et al., The rise of the Enterococcus.‘ beyond ycin resistance, Nat Rev Microbiol 10, 266-278 (2012); Jain, R. et al., Veterans aﬂairs initiative to prevent methicillin-resistant Staphylococcus aureus infections, N Engl J Med 364, 1419-1430 (2011); Nordmann et al., The real threat ofKlebsiella pneumoniae carbapenemase—producing bacteria, Lancet Infect Dis 9, 228-236 (2009); Aloush et al., Multidrug-resistant monas nosa.‘ riskfactors and al impact, crob Agents Chem 50, 43- 48 . In on to adversely ing civilian patients, antibiotic-resistant bacteria affect injured military personnel. Multiple reports from Operation Iraqi m/Operation Enduring Freedom have indicated that multidrug-resistant bacteria and antibiotic resistance constitute one of the most disconcerting aspects of military theater treatment. See e.g., Calhoun et al., Multidrug—resistant Organisms in Military Woundsfrom Iraq and Afghanistan, Clinical Orthopaedics and Related Research 466, 1356-1362 (2008); Murray et al., Bacteriology of War Wounds at the Time ofInjury, Military Medicine 171, 826—829 ; Huj er et al., Analysis ofAntibiotic Resistance Genes in rug—Resistant Acinetobacter sp. Isolatesfrom Military and Civilian Patients Treated at the Walter Reed Army Medical Center, Antmicrobial Agents and herapy 50, 4114-4123 (2006).

Multiple factors contribute to bacterial cells’ ability to resist the effects of antibiotics. See e. g., Morita et al., Antibiotic Inducibility ofthe MexXYMultidrug Efflux System ofPseudomonas aeruginosa: Involvement ofthe Antibiotic-Inducible PA54 71 Gene Product, Journal of Bacteriology 188, 1847-1855 ; Tran et al., Heat-Shock Protein ClpL/HSPI00 Increases Penicillin Tolerance in Streptococcus pneumoniae, es in Oto-rhino-laryngology 72, 126-128 (2011); Livorsi et al., nce Factors ofGram- Negative ia in Sepsis With a Focus on Neisseria meningitidis, Contributions to Microbiology 17, 31-47 ; , et al., Speciﬁc Ion Eﬂects on the Growth Rates of Staphylococus aureus ana1 Pseudomonas aeruginosa, Physical Biology 2, 1-7 (2005).

Amongst these factors is the ability of bacteria to develop a bioﬁlm. See e. g., Costerton et al., How bacteria stick, Sci Am 238, 86-95 (1978); Lawrence et al., Optical sectioning of microbial bioﬁlms, J Bacteriol 173, 6558-6567 (1991); ZoBell, The Eﬂect ofSolia1 Surfaces upon Bacterial Activity, Journal of iology 46, 39-56 (1943). Bioﬁlms have unique characteristics that allow them to withstand, or defend themselves against a variety of perturbations including exposure to antibiotics.

Biofilms are surface-attached communities of ia, often crobial, that produce a slimy, extracellular polysaccharide substance (EPS) that encapsulates them. The EPS provides protection, Leid et al., The Exopolysacharide Alginate Protects Pseudomonas aeruginosa Bioﬁlm Bacteriafrom IFN-y—Mediated Macrophage Killing, The Journal of logy 175, 7512-7518 (2005), as well as a e of nutrients, water and trace elements to sustain life. Costerton et al., The Bacterial alyx in Nature and Disease, Annual Review of Microbiology 35, 299-324 (1981). Biofilms are the predominant phenotype of bacteria in natural ecosystems. Gram-negative bacteria, Gram-positive bacteria, and mycobacteria, in addition to other unicellular organisms, can produce bioﬁlms.

Within the biofilm community, bacteria may have several methods of defending themselves against the biocidal effects of antibiotics. First, they have strength in numbers.

Biofilms may n millions or trillions of cells in a very small volume. Second, bacteria in a biofilm have the ability to rapidly transfer c material, such as plasmids, that cally code for the tion of molecules that protect them against antibiotics. Lujan etal., Disrupting Antibiotic Resistance Propagation by Inhibiting the Conjugative DNA Relaxase, PNAS 104, 12282—12287 (2007); Lederberg et al., Gene ination in Escherichia coli. Nature 158, 529-564 (1946). Rates of plasmid transfer in biofilms have been shown to be much higher than amongst planktonic bacteria, which are free-ﬂoating in an environment. Hausner et al., High Rates ofConjugation in Bacterial Bioﬁlms as Determined by Quantitative In Situ Analysis, Applied and Environmental iology 65, 3710-3713 (1999). Third, as a m community matures, it creates an oxygen gradient such that an oxygen-rich environment exists on the outer edges of a biofilm, whereas an oxygen-deprived, or anaerobic, area exists in the t portions of a bioﬁlm. Walters et al., Contributions ofAntibiotic Penetration, Oxygen Limitation, and Low Metabolic Activity to Tolerance ofPseua’omonas aeruginosa bioﬁlms t0 ﬂoxacin ana1 Tobramycin, Antimicrobial Agents and Chemotherapy 47, 317-323 ; Borriello et al., Oxygen Limitation Contributes to Antibiotic Tolerance ofPseudomonas aeruginosa in Bioﬁlms, Antimicrobial Agents and Chemotherapy 48, 2659-2664 . This may result in d metabolic activity in those cells that dwell in the interior of the biofilm. Importantly, traditional antibiotics are typically effective t bacterial cells that are y dividing, i.e., in a logarithmic phase of growth. Mandell, Interaction ofIntraleukocytic Bacteria and Antibiotics, The Journal of Clinical Investigation 52, 1673—1673 (1973); Gilbert et al., Inﬂuence ofGrowth Rate on Susceptibility to Antimicrobial : s, Cell Cycle, Dormancy, ana1 Stringent Response, Antimicrobial Agents and Chemotherapy 34, 1865-1868 (1990). Fourth, in a mature biofilm, water channels form throughout the community.

Stoodley et al., Liquidﬂow in bioﬁlm systems, App Env iol 60, 716 (1994).

These water channels have the ability to dlfﬁlSC, remove or prevent toxic byproducts as well as antibiotics from interacting with cells in the biofilm. For novel antimicrobial agents to be effective over the long term, addressing each of these four characteristics may se the potential for s in a variety of applications including healthcare, industrial, environmental, agricultural and sanitation industries. Furthermore, biofilms tend to secrete proteoglycan materials that create an extracellular matrix, which has the ability to potentially bind and hinder the activity of antibiotics.

Alternative approaches to killing bacteria include the use of antimicrobial agents that have fast-acting and nonspecific mode of activity against the cell membrane of bacteria.

These alternate compounds include detergents, squalamine, quaternary ammonium compounds, and naturally occurring antimicrobial peptides, among others. By ing and depolarizing the cell membrane in a cific fashion at a faster rate, agents that attack the cell membrane globally can kill bacteria before they have time to upregulate their defense mechanisms. In addition, modes of action of these alternate antimicrobials are not limited to a speciﬁc protein or enzyme within a metabolic pathway.

However, as important as it is to kill bacteria and prevent their ability to cause infections in humans or animals, or contaminate unwanted ses in industrial, agricultural or environmental applications, when bacteria are attached to a surface, it sometimes may be more beneﬁcial to not only kill bacteria, but also to cause them to “fall off’ of a surface as well, 6. g. disperse or dislodge ia in a bioﬁlm ity. In certain aspects, the present invention provides compounds, compositions, and s that have shown the y to disperse or dislodge bacterial cells in a bioﬁlm, such that the cells are no longer able to reattach and form new bioﬁlm communities, and, notably, the same compounds, compositions, and s kill substantially all bacteria cells in a bioﬁlm.

By sing a bioﬁlm and killing the cells within it, at least two beneﬁts are ed. This may be particularly important when considering the fact that although bacteria in a bioﬁlm, which may be attached to a surface, can be killed by an antimicrobial agent, the dead cells and extracellular matrix residues may provide an attachment point for viable bacteria to re-adhere and form a bioﬁlm once again with greater afﬁnity. If bioﬁlms are sed and killed, viable bacteria that are introduced to a surface will have reduced ability to preferentially adhere to that area. This can be particularly important in industrial ations wherein the formation of bioﬁlms on a surface can be problematic, as well as medical ations wherein bacteria may adhere to the surface of a medical device. It has been surprisingly discovered that itions of the present ion have signiﬁcant potential to eradicate bacteria within a bioﬁlm as well as cause the bioﬁlm to disperse or dislodge, resulting in a variety of potential applications across multiple settings.

Thus, there is a need for novel compounds, compositions, and methods that have potent antimicrobial and anti-bioﬁlm activity against a variety of bacterial strains, ally at high bacterial concentrations and against antibiotic-resistant bacteria.

BRIEF SUMMARY OF THE INVENTION It is an object of the present invention to provide novel compounds, itions, and methods having antimicrobial activity and dispersing activity against a wide variety of bacterial strains capable of forming bioﬁlms. In some preferred aspects, the invention provides compounds, compositions, and methods that are effective t antibiotic—resistant bacterial bioﬁlms.

It has been discovered that compounds, itions, and methods of the present invention rapidly disperse bioﬁlms and kill microorganisms such as bacteria, so that the microorganisms do not have an opportunity to upregulate their defense mechanisms. Thus, there may be a reduced risk of bacteria developing resistance to the compounds, compositions, and methods of the present invention. Furthermore, such compounds, compositions, and methods may not be limited to eradicating ia that are in log-phase growth. The ability of compounds, compositions, and methods of the present ion to se biofilms while demonstrating antimicrobial activity may address many of the characteristics that make biofilm communities difficult to treat using traditional antibiotics.

More specifically, by dispersing and killing bacteria in a biofilm, water channels and the bacterial community as a whole may be broken apart, allowing for broader distribution of antimicrobial agent(s) to a greater number, or even ntially all, of the cells within a biofilm.

Aspects of this disclosure feature methods of g, dispersing, dislodging, treating, and reducing biofilms as well as preventing or inhibiting m formation. In some embodiments, the method comprises exposing a biofilm to an effective amount of a ition of the t invention to y kill, disperse, dislodge, treat, , prevent, or t bacterial biofilms. [0016a] According to an embodiment, there is provided a non-therapeutic method for dispersing or killing a biofilm, the method comprising a step of contacting the biofilm with an anti-biofilm composition, thereby dispersing or killing the biofilm; wherein the anti-biofilm composition comprises a biocidal polyamine compound selected from the group consisting of Ra Ra A1 A5 A1 A5 A3 Ra , Ra A3 Ra , and a salt thereof; wherein: A1, A2, A3, and A5 are each an independently selected CR5; each Ra is an independently selected group of Formula V: R1a R1b N R2c R4 Rm R2d R3 ; each R1a and R1b is a member independently selected from hydrogen and alkyl; each R2a, R2b, R2c and R2d is a member independently selected from the group consisting of hydrogen, alkyl, and fluoroalkyl; 16584576_1 (GHMatters) P41348NZ01 each R3 is a member independently selected from the group consisting of -Z1-R4, -Z1-Y1-R4, -Y2-R4, and -Z1-Y1-Y2-Y3-R4; each Y1, Y2, and Y3 is an ndently selected group of Formula IA: Rm R2d each Z1 and Z2 is an independently selected -N(R4)-; each Rm is -CH2-; each m is an integer independently selected from 1 to 2; each R4 is a member independently selected from the group consisting of hydrogen, alkyl, fluoroalkyl, alkenyl, alkynyl, aryl, lkyl, heteroaryl, kyl, cycloalkylalkyl, and heteroarylalkyl; or, alternatively, for an -N(R4)2 group, one of the two R4 in the group is a member selected from the group consisting of -(CO)OR6a, -(CO)N(R6a)(R6b), and -C(NR6a)N(R6b)(R6c); or, alternatively, for an -N(R4)2 group, the two R4 groups join to form a heterocyclic ring; each R5 is a member independently selected from the group consisting of hydrogen, alkyl, hydroxyl, alkoxy, aminoalkoxy, alkylamino, alkylaminoalkoxy, alkenyl, alkynyl, aryl, aryloxy, arylamino, cycloalkyl, cycloalkoxy, lkylalkoxy, cycloalkylamino, cycloalkylalkylamino, heterocyclyl, heterocycyloxy, heterocycylamino, halo, haloalkyl, fluoroalkyloxy, heteroaryl, heteroaryloxy, heteroarylamino, arylalkyl, arylalkyloxy, arylalkylamino, heteroarylalkyl, heteroarylalkyloxy, heteroarylalkylamino, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; and each R6a, R6b, and R6c is a member independently selected from hydrogen and alkyl; wherein if R4 is -(CO)OR6a, R6a is alkyl; wherein the polyamine compound comprises at least six primary or secondary amino groups. [0016b] In another ment, the present invention provides an anti-biofilm composition comprising a biocidal ine compound used in methods according to the invention.

In an embodiment, the present invention provides a polyamine compound.

[0018] In a further ment, the t invention provides a composition for treatment of ms, the composition comprising, consisting of, or consisting essentially of a polyamine compound as set forth in any of the embodiments, aspects, or combination of aspects herein. 16584576_1 ters) P41348NZ01 In a further embodiment, the present invention provides a method of treating a biofilm comprising, consisting of, or consisting ially of the step of administering a polyamine compound, or a composition sing the polyamine compound, as set forth in any of the embodiments, aspects, or combination of aspects herein.

In a further embodiment, the present ion provides a method of making a ine compound, or a composition comprising, consisting essentially of, or consisting of the polyamine compound, as set forth in any of the embodiments, aspects, or combination of aspects herein.

These and other objects, aspects, and embodiments will become more apparent when read with the following ed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Various embodiments and aspects of the present invention are shown and described in nce to the numbered drawings. 16584576_1 (GHMatters) P41348NZ01 shows a simpliﬁed schematic representation of one embodiment of a class of polyamine compounds. through show exemplary ng materials or reactants that may be used to prepare certain c embodiments ofpolyamine compounds as set forth herein. shows ary polyamine chains that may be used to prepare certain speciﬁc embodiments of polyamine compounds as set forth herein. through show exemplary polyamine compounds according to aspects of the present invention. through show ary polyamine compounds according to aspects of the present invention. through show exemplary polyamine compounds ing to aspects of the present invention. shows an exemplary synthesis strategy to produce ine compounds according to certain s of the present invention.

[0030] shows another exemplary synthesis strategy to produce polyamine compounds according to certain aspects of the t invention. shows still r exemplary synthesis strategy to produce polyamine compounds according to certain aspects of the present invention. shows yet another exemplary synthesis strategy to produce polyamine compounds according to certain aspects of the present ion. shows the minimum inhibitory concentration (“MIC”) of ines and novel polyamine compounds according to certain aspects of the present invention.

A is a table showing certain polyamine compounds according to n aspects of the present invention;

[0035] B is a table g the effect bioﬁlm eradication concentration (“EBEC”) of certain polyamine compounds represented in and EBECs of certain polyamines. shows an example of microbial contamination in the oil and gas industry.

A shows a representative bacteria bioﬁlm.

B shows a representative bacteria bioﬁlm, such as the bioﬁlm shown in A, treated according to certain standards in the oil and gas industry.

C shows a representative bacteria bioﬁlm, such as the bioﬁlm shown in A, treated with a polyamine compound of the t invention. shows the results of treating bioﬁlms of Alcam'vorax borkumensz's grown on the e of galvanized steel with a polyamine compound of the present invention. shows a photograph demonstrating the ability ofVanicream-based cream to elute compounds CZ-25, CZ-52, and polymyxin B polytherapy formula and to eradicate bacteria as indicated by zones of clearing around the cream. The second row of plates is from a duplicate test of the same cream. shows ng electron microscopy (SEM) images of Ti coupons with MRSA bioﬁlms exposed to CZ-25 (left), CZ-52 (middle), or water only (right). The compounds CZ-25 and CZ-52 trated the y to disperse the majority of biofllms such that a monolayer of cells remained on the surface of the Ti. Those samples treated with water only had bioﬁlm ities that remained in all areas of the coupons. shows photographs ofMRSA biofilms being dispersed by the compound CZ-25. (Left) Bioﬁlms of MRSA grown on the surface of Ti coupons in a CDC biofllm r exposed to water for 2 hours. (Right) Bioﬁlms ofMRSA grown on the surface of Ti coupons exposed to the compound CZ—25 in water for 2 hours. Note the strings of biofilm dispersing from the surface of the metal. shows SEM images of biofllms nivarax barkumensz's grown on the surface of galvanized steel in a ﬂow system, then exposed to the compound CZ-25 (also known as PBC—25). shows raphs SEM images of bioﬁlms ofAlcanivorax borkumensz's grown on the surface of galvanized steel in a flow , then exposed to the compound CZ- 7 (also known as PBC-7). Glutaraldehyde, the standard of ent in oil and gas facilities, was compared as a control. shows a schematic of the stir tank bioﬁlm reactor. shows a photograph of a glass slide removed after 6 days in the stir tank bioreactor. shows a hypothetical mode of binding of spermidine to an exopolysaccharide. shows several polyamine-containing l products with crobial activity. shows a method for making several embodiments of the polyamine compound. shows a method for making yet another embodiment of the ine compound. shows a method for making still another embodiment of the polyamine compound. shows a method for making several other embodiments of the polyamine compound. shows a pH proﬁle of selected polyamine nds’ hydrochloride salts in deionized water.

I5 [0055] FIGS. 30A to 301 show the effects of the polyamine compounds CZ-86 and CZ—l 10 on various bacterial cell cultures. B shows a control without added polyamine compounds.

It will be appreciated that the drawings are illustrative and not limiting of the scope of the invention, which is deﬁned by the appended claims. The embodiments shown accomplish various aspects and objects of the ion; however, it will be understood that other aspects, features or modiﬁcations may be within the scope of the appended claims. It is appreciated that it is not possible to clearly show each element and aspect of the ion in a single ﬁgure, and as such, multiple ﬁgures are presented to separately illustrate various details of the invention in greater y. Similarly, not every ment need accomplish all advantages of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The invention and accompanying drawings will now be discussed in reference to the numerals provided therein so as to enable one skilled in the art to practice the t invention. The skilled artisan will understand, however, that the inventions bed below can be practiced without employing these speciﬁc details, or that they can be used for purposes other than those described herein. Indeed, they can be modiﬁed and can be used in conjunction with products and ques known to those of skill in the art in light of the present disclosure. The drawings and ptions are intended to be exemplary of various aspects of the invention and are not intended to narrow the scope of the ed claims.

Furthermore, it will be appreciated that the drawings may show aspects of the invention in isolation and the elements in one ﬁgure may be used in ction with elements shown in other ﬁgures.

It will be appreciated that reference throughout this speciﬁcation to aspects, features, advantages, or similar language does not imply that all of the aspects and advantages that may be ed with the present invention should be or are in any single embodiment of the invention. Rather, language ing to the aspects and advantages is understood to mean that a speciﬁc aspect, feature, advantage, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, sion of the aspects and advantages, and similar language, throughout this speciﬁcation may, but does not necessarily, refer to the same ment.

The described aspects, features, advantages, and characteristics of the invention may be combined in any suitable manner in one or more ﬁarther embodiments. rmore, one skilled in the relevant art will recognize that the invention may be practiced t one or more of the speciﬁc aspects or advantages of a particular embodiment. In other ces, additional aspects, features, and advantages may be recognized and claimed in certain embodiments that may not be present in all embodiments of the invention.

DEFINITIONS Unless otherwise , all technical and scientiﬁc terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or lent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be ng. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, including U.S. Appl. Nos. 61/482,522; 61/482523; 61/591,601; 61/616,944; 61/826,453; 61/826,761; 61/836,555; 61/834,l49; 13/379,191; 14/076,143; and 14/076,149 as well as Int’l Pat. Publ. Nos. , 2012/151555, and 2013/148230. In case of conﬂict, the present speciﬁcation, including these deﬁnitions, will control.

The terms “a,” “an,” and “the” as used herein not only includes aspects with one member, but also includes aspects with more than one member. For example, an embodiment including “a polyamine compound and an excipient” should be understood to present certain aspects with at least a second polyamine compound, at least a second excipient, or both.

The term ” as used herein to modify a cal value indicates a deﬁned range around that value. If “X” were the value, “about X” would generally indicate a value from 0.95X to 1.05X. Any reference to “about X” speciﬁcally indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.” When the quantity “X” only includes whole—integer values (e.g., “X carbons”), “about X” tes from (X-l) to (X+l). In this case, “about X” as used herein speciﬁcally indicates at least the values X, X-1, and X+1.

When “about” is applied to the ing of a numerical range, it s to both ends of the range. Thus, “from about 5 to 20%” is equivalent to “from about 5% to about %.” When “about” is applied to the ﬁrst value of a set of values, it s to all values in that set. Thus, “about 7, 9, or 11%” is equivalent to “about 7%, about 9%, or about 11%.”

[0064] The term “acyl” as used herein includes an alkanoyl, aroyl, heterocycloyl, or heteroaroyl group as deﬁned . Examples of acyl groups include, but are not limited to, acetyl, benzoyl, and nicotinoyl.

The term “alkanoyl” as used herein includes an alkyl-C(O)- group wherein the alkyl group is as deﬁned herein. Examples of alkanoyl groups include, but are not limited to, acetyl and propanoyl.

The term “agent” as used herein includes a compound or mixture of compounds that, when added to a composition, tend to produce a particular effect on the composition’s properties. For example, a composition comprising a ning agent is likely to be more viscous than an otherwise identical ative composition that lacks the thickening agent.

[0067] The term yl” as used herein es a straight or branched chain hydrocarbon containing at least one carbon-carbon double bond. The chain may contain an indicated number of carbon atoms. For example, “Cl-Cu alkenyl” indicates that the group may have from 1 to 12 (inclusive) carbon atoms and at least one carbon-carbon double bond.

When the indicated number of carbon atoms is 1, then the Ci alkenyl is double bonded to a carbon (i.e., a carbon equivalent to an oxo . In certain aspects, the chain es 1 to 12, about 2 to 15, about 2 to 12, about 2 to 8, or about 2 to 6 carbon atoms. Examples of an l group may include, but are not limited to, ethenyl (z'.e., , allyl, propenyl, butenyl, crotyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, dodecenyl, cyclopentenyl, cyclohexenyl, 2-isopentenyl, allenyl, butadienyl, pentadienyl, 3-(l,4- pentadienyl), and hexadienyl.

[0068] An alkenyl group can be unsubstituted or optionally tuted. When optionally substituted, one or more hydrogen atoms of the alkenyl group (e.g., from 1 to 4, from 1 to 2, or I) may be replaced with a moiety independently selected from ﬂuoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio, with the proviso that no hydrogen atom substituent on the -carbon double bond is ed by a hydroxy, amino, or thio group.

In some aspects, the alkenyl group is unsubstituted or not optionally substituted.

The term “alkyl” as used herein includes an aliphatic hydrocarbon chain that may be straight chain or branched. The chain may contain an indicated number of carbon atoms: For example, C1-C12 indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. If not otherwise indicated, an alkyl group contains from 1 to about 20 carbon atoms. In some aspects, alkyl groups have 1 to about 12 carbon atoms in the chain. In some s, alkyl groups (“lower alkyl”) have 1 to about 6 carbon atoms in the chain. Examples may e, but are not limited to, methyl, ethyl, propyl, isopropyl (iPr), l-butyl, 2—butyl, isobutyl (iBu), tert-butyl, pentyl, 2-methylbutyl, l,l-dimethylpropyl, hexyl, heptyl, octyl, nonyl, decyl, docecyl, cyclopentyl, or cyclohexyl.

[0070] An alkyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the alkyl group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from ﬂuoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, the alkyl group is unsubstituted or not ally tuted.

[0071] The term “alkoxy” as used herein includes a straight or ed chain saturated or unsaturated hydrocarbon ning at least one oxygen atom in an ether group (e.g., EtO-).

The chain may contain an indicated number of carbon atoms. For example, “Cl-Cu alkoxy” indicates that the group may have from 1 to 12 sive) carbon atoms and at least one oxygen atom. Examples of a C1-C12 alkoxy group include, but are not limited to, methoxy, ethoxy, isopropoxy, , n-pentoxy, isopentoxy, neopentoxy, and hexoxy.

An alkoxy group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the alkoxy group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently ed from ﬂuoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio, with the o that no hydrogen atom alpha to the ether oxygen is replaced by a hydroxy, amino, or thio group. In some aspects, the alkoxy group is unsubstituted or not optionally substituted.

[0073] The term “alkynyl” as used herein es a straight, branched, or cyclic hydrocarbon containing at least one carbon—carbon triple bond. Examples may include, but are not limited to, ethynyl, propargyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, or decynyl.

An alkynyl group can be unsubstituted or optionally substituted. When optionally tuted, one or more hydrogen atoms of the alkynyl group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from ﬂuoro, hydroxy, , amino, alkylamino, acylamino, thio, and alkylthio, with the proviso that no sp-hybridized hydrogen atom substituent is replaced by a hydroxy, amino, or thio group. In some aspects, the alkynyl group is unsubstituted or not optionally substituted. 2O [0075] The term “aroyl” as used herein includes an aryl-CO- group wherein aryl is as deﬁned herein. Examples include, but are not limited to, benzoyl, naphth—1—oyl and naphth- 2-oyl.

The term “aryl” as used herein includes cyclic aromatic carbon ring systems ning from 6 to 18 carbons. Examples of an aryl group include, but are not d to, phenyl, naphthyl, anthracenyl, tetracenyl, biphenyl and phenanthrenyl.

An aryl group can be unsubstituted or ally substituted. When optionally substituted, one or more hydrogen atoms of the aryl group (e.g., from 1 to 5, from 1 to 2, or 1) may be replaced with a moiety independently selected from alkyl, cyano, acyl, halo, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, the alkoxy group is tituted or not optionally substituted.

The term “arylalkyl” or “aralkyl” as used herein includes an alkyl group as deﬁned herein where at least one hydrogen substituent has been ed with an aryl group as deﬁned herein. Examples include, but are not limited to, benzyl, ylethyl, 4- methylbenzyl, and 1,1 ,-dimethylphenylmethyl.

A arylalkyl or aralkyl group can be unsubstituted or optionally substituted as per its component groups. For example, but without limitation, the aryl group of an arylalkyl group can be substituted, such as in 4-methylbenzyl. . In some aspects, the group is unsubstituted or not optionally substituted, especially if including a deﬁned substituent, such as a hydroxyalkyl or alkylaminoalkoxy group.

[0080] The term “cycloalkyl” as used herein includes a cyclic hydrocarbon group that may contain an indicated number of carbon atoms: For example, C3-C12 indicates that the group may have from 3 to 12 (inclusive) carbon atoms in it. If not otherwise indicated, a cycloalkyl group includes about 3 to about 20 carbon atoms. In some aspects, cyclo alkyl groups have 3 to about 12 carbon atoms in the group. In some s, cycloalkyl groups have 3 to about 7 carbon atoms in the group. es may include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4,4—dimethylcyclohexyl, and cycloheptyl.

A cycloalkyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more en atoms of the cycloalkyl group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from ﬁuoro, hydroxy, alkoxy, 2O amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, a substituted cycloalkyl group can orate an exo- or endocyclic alkene (e.g., cyclohexenyl). In some aspects, a lkyl group is unsubstituted or not optionally tuted.

The terms “disorder,” “disease,” and “condition” are used herein interchangeably for a condition in a subject. A disorder is a disturbance or derangement that affects the normal function of the body of a t. A disease is a pathological condition of an organ, a body part, or a system resulting from various causes, such as infection, genetic defect, or environmental stress that is characterized by an identiﬁable group of symptoms. A disorder or disease can refer to a bioﬁlm—related disorder or disorder caused by a planktonic ial phenotype that is characterized by a disease-related growth of bacteria.

[0083] The term “effective amount” or “effective dose” as used herein includes an amount ent to achieve the desired result and accordingly will depend on the ingredient and its desired . Nonetheless, once the desired effect is identiﬁed, determining the effective amount is within the skill of a person skilled in the art.

As used herein, “fluoroalkyl” includes an alkyl group wherein the alkyl group includes one or more ﬂuoro- substituents. es include, but are not d to, triﬂuoromethyl.

As used herein, al” substitution includes two or more substituents that are directly attached to the same atom. An example is 3,3-dimethyl substitution on a cyclohexyl or spirocyclohexyl ring.

As used , “halo” or “halogen” includes ﬂuoro, chloro, bromo, or iodo.

[0087] The term “heteroaryl” includes mono and bicyclic aromatic groups of about 4 to about 14 ring atoms (e. g., 4 to 10 or 5 to 10 atoms) containing at least one heteroatom.

Heteroatom as used in the term heteroaryl refers to oxygen, sulfur and nitrogen. A nitrogen atom of a heteroaryl is optionally oxidized to the corresponding N—oxide. es include, but are not limited to, pyrazinyl, l, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,4- thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, imidazo[l,2-a]pyridine, imidazo[2,l-b]thiazolyl, benzofurazanyl, l, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, l,2,4-triazinyl, and benzothiazolyl.

[0088] A heteroaryl group can be unsubstituted or optionally tuted. When ally tuted, one or more hydrogen atoms of the heteroaryl group (e.g., from 1 to 5, from 1 to 2, or 1) may be replaced with a moiety independently selected from alkyl, cyano, acyl, halo, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, the heteroaryl group is unsubstituted or not optionally substituted.

[0089] The term “heteroaroyl” as used herein includes a heteroaryl-C(O)- group wherein heteroaryl is as deﬁned herein. Heteroaroyl groups include, but are not limited to, thiophenoyl, noyl, pyrrolylcarbonyl, and pyridinoyl.

The term ocycloyl” as used herein includes a heterocyclyl-C(O)- group wherein heterocyclyl is as deﬁned herein. Examples include, but are not limited to, N—methyl prolinoyl and tetrahydrofuranoyl.

As used herein, “heterocyclyl” includes a non-aromatic saturated monocyclic or multicyclic ring system of about 3 to about 10 ring atoms (e.g., 5 to about 10 ring atoms, or 3 to about 6 ring atoms), in which one or more of the atoms in the ring system is an element or elements other than carbon, e. g., nitrogen, oxygen or sulfur. A heterocyclyl group optionally comprises at least one bridized atom (e.g. , a ring incorporating an carbonyl, endocyclic oleﬁn, or exocyclic oleﬁn). In some embodiments, a nitrogen or sulfur atom of the heterocyclyl is optionally oxidized to the ponding N-oxide, S-oxide or S,S-dioxide.

Examples of monocycylic heterocyclyl rings e, but are not limited to, piperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, l,3-dioxolanyl, 1,4- dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, and tetrahydrothiopyranyl.

A heterocycyl group can be unsubstituted or optionally tuted. When ally substituted, one or more hydrogen atoms of the group (e.g., from 1 to 4, from 1 to 2, or 1) may be ed with a moiety independently selected from fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, a substituted heterocycyl group can orate an exo— or endocyclic alkene (e.g., cyclohex—2—en—l—yl). In some aspects, the heterocycyl group is unsubstituted or not optionally substituted.

The term “hydrophobic moiety” or “hydrophobic group” as used herein includes a moiety or a functional group that repels water. Examples may include, but are not limited to, a non—polar alkyl moiety, such as an unsubstituted alkyl group having more than ﬁve carbons; a phenyl group; and an anthracenyl group.

As used herein, the terms “hydrophilic moiety" or “hydrophilic group” includes a moiety or a functional group that has a strong afﬁnity to water. Examples may include, but are not limited to, a charged moiety, such as a cationic moiety or an anionic moiety, or a polar uncharged , such as an alkoxy group or an amine group.

[0095] As used herein, the term “hydroxyalkyl” includes an alkyl group where at least one hydrogen substituent has been replaced with an alcohol (-OH) group. In certain aspects, the hydroxyalkyl group has one alcohol group. In certain s, the hydroxyalkyl group has one or two alcohol groups, each on a different carbon atom. In certain aspects, the hydroxyalkyl group has 1, 2, 3, 4, 5, or 6 alcohol . Examples may include, but are not limited to, hydroxymethyl, oxyethyl, and l—hydroxyethyl.

When any two substituent groups or any two instances of the same substituent group are endently ed” from a list of alternatives, the groups may be the same or different. For example, if Ra and Rb are independently selected from alkyl, ﬂuoro, amino, and hydroxyalkyl, then a molecule with two Ra groups and two Rb groups could have all groups be an alkyl group (e.g., four different alkyl groups). Alternatively, the ﬁrst Ra could be alkyl, the second Ra could be ﬂuoro, the ﬁrst Rb could be hydroxyalkyl, and the second Rb could be amino (or any other substituents taken from the group). Alternatively, both Ra and the ﬁrst Rb could be ﬁuoro, while the second Rb could be alkyl (i.e., some pairs of substituent groups may be the same, while other pairs may be different).

As used herein, “polyamine” includes a nd that has at least two amine groups, which may be the same or different. The amine group may be a primary amine, a secondary amine, a tertiary amine, or quaternary ammonium salt. Examples may include, but are not d to, aminopropane, 1,4-diaminobutane, hexamethylenediamine, dodecan- 1,12-diamine, spermine, spermidine, rmine, and norspermidine.

As used herein, “or” should in general be construed non—exclusively. For example, an embodiment of “a composition comprising A or B” would typically present an aspect with a composition comprising both A and B, and an embodiment of “a method to disperse or kill bioﬁlms” could disperse, kill, or a combination of both. “Or” should, however, be construed to exclude those aspects presented that cannot be ed without contradiction (e.g., a composition pH that is between 9 and 10 or between 7 and 8).

As used herein, “spirocycloalkyl” includes a cycloalkyl in which geminal substituents on a carbon atom are replaced to join in forming a bstituted ring. For example, but without limitation, for a —C(R1)(R2)- group that was part of a longer carbon chain, if R1 and R2 joined to form a cyclopropyl ring incorporating the carbon to which R1 and R2 were bonded, this would be a spirocycloalkyl group (i.e., spirocyclopropyl).

As used herein, “spiroheterocyclyl” includes a heterocycloalkyl in which l substituents on a carbon atom are replaced to join in forming a l,l-substituted ring. For example, but t limitation, for a —C(R1)(R2)- group that was part of a longer carbon chain, if R1 and R2 joined to form a idine ring incorporating the carbon to which R1 and R2 were bonded, this would be a spiroheterocyclyl group.

As used herein, the term “treat,” “treating,” or “treatment” es administering or applying a composition (e.g., a composition described herein) in an amount, manner (e.g., schedule of administration), and mode (e.g., route of administration) that is effective to improve a er or a m thereof, or to prevent, to retard, or to slow the progression of a disorder or a symptom thereof. Such improvements can include, but are not limited to, alleviation or ration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or , delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the rrence of disease, and ion, whether partial or total and whether detectable or undetectable.

This can be evidenced by, e.g., an ement in a parameter associated with a biofilm or with a biofilm-related disorder or an indication or symptom thereof, a biofilm- related industrial, agricultural, environmental, etc. condition, e.g., to a statistically significant degree or to a degree detectable to one skilled in the art. An effective amount, manner, or mode can vary depending on the surface, application, or subject and may be tailored to the surface, application, or subject. By ating a biofilm or preventing or slowing ssion of a biofilm or of a biofilm-related disorder or an indication or symptom thereof, or a biofilm-related industrial, ltural, environmental, etc. condition, a treatment can prevent or slow oration or corrosion resulting from a m or from a biofilm-related disorder or an indication or symptom thereof on an affected surface or in an affected or diagnosed subject.

“Treating” and “treatment” as used herein also include prophylactic ent. In certain embodiments, treatment methods comprise administering to a subject a therapeutically effective amount of a composition of the invention. The stering step may consist of a single administration or may comprise a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the ion, the age of the patient, the concentration of active agent in the composition, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a ular treatment or prophylaxis regime. Changes in dosage may result and become apparent by rd diagnostic assays known in the art. In some aspects, chronic administration may be required. For example, the compositions are administered to the subject in an amount, and for a duration, sufficient to treat the patient.

[0104] As used herein, a reference to a composition of formula A, B, C, or a salt thereof may indicate A, a salt ofA, B, a salt ofB, C, or a salt of C.

INE COMPOUNDS Aliphatic polyamines, for example, spermine, spermidine, norspermine, norspermidine, hexamethylenediamine, 1,12-diaminododecane, 1,3-diaminopropane, etc., have been shown to have some activity against a variety of bacterial strains when used alone.

In particular, it has been shown that norspermine and norspermidine may be effective in dispersing various biofilm s. However, it does not appear that these polyamines have sufficient bactericidal activity.

It has been surprisingly discovered that by attaching one or more polyamine chains to a lipophilic or hydrophobic , examples of which may include cyclic or aromatic IO backbone molecules, results in novel compounds that are e of dispersing biofilms and that also have substantial antimicrobial activity against a variety of strains of bacteria (e. g., by killing the bacteria).

Referring now to there is shown a simpliﬁed schematic of an exemplary polyamine compound of the present invention, generally indicated at 10. The polyamine compound 10 may be generally characterized as including a lipophilic or hydrophobic moiety , and one or more cationic es 30. In some ments, the polyamine compound may include one or more cationic residues 30 comprising primary amines 40 and one or more secondary amines 50. In some embodiments, the polyamine compound 10 may include one or more cationic residues 30 having a tertiary or quaternary amine (not shown).

[0108] As depicted in ary polyamine compounds 10 of the present ion may be amphipathic, e.g. the cationic residues 30 may be generally disposed along one face ofthe molecule, while the lipophilic or hydrophobic moiety 20 is generally ed along the opposite face of the molecule.

In certain embodiments, the present invention provides polyamine compounds and itions and methods comprising such compounds. In certain embodiments, the polyamine nds comprise one or more polyamine side chains (e.g., l, 2, 3, 4, 5, 6, 7, 8, ... n polyamine moieties/side chains) that may be the same or different. In certain embodiments, the amino groups of the one or more polyamine side chains may be ionizable.

In certain embodiments, the amino groups may be positively charged. In some embodiments, the polyamine side chains are branched. In some embodiments, the ine chains are linear.

FIGs. 2A-2H show exemplary backbone molecules, which may be used to prepare certain novel polyamine nds. It will be appreciated by those skilled in the art that alternative backbone molecules may be used to e polyamine compounds ing to certain aspects of the present invention. Without being bound by theory, it is believed that the lipophilicity or hydrophobicity of a backbone molecule of novel polyamine compounds may contribute to the antimicrobial ty or bioﬁlm dispersing activity of these novel compounds. shows exemplary ine chains, which may be used to prepare certain novel polyamine compounds according to certain s of the present ion. It will be appreciated by those skilled in the art that polyamine chains are intended to only be representative. Without being bound by theory, it is believed that increasing the number of primary or secondary amines of polyamine compounds of the present invention may increase the antimicrobial activity or bioﬁlm dispersing activity of these novel compounds.

FIGs. 4A-4P show exemplary polyamine compounds comprising a benzene backbones. FIGs. 5A and 5B show ary polyamine compounds comprising an anthracene ne. FIGs. 6A—6E show exemplary polyamine nds comprising a Vitamin D backbone. through show ary chemical synthesis strategies for preparing a variety of polyamine nds according to certain aspects of the present invention.

[0114] In certain embodiments, the one or more polyamine moieties may have at least three amino groups separated by three atoms either in a straight chain or cyclic molecule. In some particular embodiments, the one or more polyamine moiety or moieties may comprise norspermidine (also known as N—(3-aminopropyl)propane-l,3-diamine), norspermine (N'-[3- (3 -aminopropylamino)propyl]propane-l,3—diamine), or a combination thereof.

[0115] Exemplary compositions of the t invention may include a compound comprising a single ine chain or multiple polyamine chains attached to a base backbone compound to improve their antimicrobial activities or improve certain other features or aspects not present when polyamines are used alone such that the compositions may be designed for use in a speciﬁc application. Furthermore, it has been surprisingly ered that by increasing the number ofprimary and secondary amines within a single molecule an increase in the antimicrobial activity of a compound may result. Furthermore, exemplary novel polyamines may be used in combination (6.g. formulated as a composition) with y approved, commercial antimicrobial products (6.g. chlorhexidine gluconate) to improve the dispersing or killing ability of such compositions.

In some aspects, the invention provides a compound or composition that comprises, consists essentially of, or consists of a polyamine compound or composition used in any of the embodiments or aspects of the methods described herein.

In some aspects, the invention provides a composition for treating (e.g., dispersing or killing) bioﬁlrns, the composition comprising a polyamine compound selected from: p11J§A4 l2 l3 Ra Ra Ra Y WW gYAAB: 1&A45A: UYAiAa l2 l4 A2 A's l7 l2 i l l7 A's l7 \AB‘ \A3’ \A6’ \A3 L1 AB’ \AB' , , , ,andasalt thereof; wherein: each Ra is a member independently ed from and ; A1, A2, A3, A4, A5 , A6, A7, A8, and A9 are each an A11 member independently selected from N, CR3, and CR5; or, alternatively, a pair of adjacent AI1 members join to form an independently selected aryl, cycloalkyl, heterocyclyl, or cycloaryl ring that is fused with an AI1 ring at the pair’s AI1 ring positions; wherein at least one A11 member and at most ﬁve AI1 members are an independently selected CR3; each R”, Rlb, R”, and R1d is a member ndently selected from hydrogen, ﬂuoro, alkyl, and ﬂuoroalkyl; or, alternatively, an R121 and an R1b join to form an oxo group; each Rza, R213, R2°, de, R23, and R2f is a member independently selected from hydrogen, alkyl, lkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; alternatively, a pair of R2 members from the same Ra group independently ed from the group R2&1 and RZb, R20 and R“, and R23 and szjoin to form a member independently selected from spirocycloalkyl, eterocycyl, and oxo; or, alternatively, an R2a and an ch from the same Ra group join to form a ring independently selected from cycloalkyl and heterocycyl; each Rm is a member independently selected from —CR23R2b-, -CR2°R2d-, -C(R2a)=(R2b)-, -CC-, and -C(Rza)(RZb)-L2-C(R2°)(R2d)-; each m is an integer independently selected from 1 to 20; each L1 and L2 is a member independently selected from a bond, -O-, -C(O)O-, -NR4-, -NR4C(O)-, and —C(O)NR4-; each R3 is a member independently selected from , -Zl-Y1-R4,—Z1-Yl-Y2-R4, and -Z1-Y1-Y2-Y3-R4; each R4 is a member independently selected from hydrogen, alkyl, ﬂuoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, and arylalkyl, or, alternatively, for a - N(R4)2 group, one of the two R4 in the group is a member selected from -(CO)OR63-, (CO)N(R6a)(R6b), and 7C(NR6a)N(R6b)(R6°); or, alternatively, for an —N(R4)2 group, the two R4 groups join to form a heterocyclic ring; each R5 is a member independently selected from hydrogen, alkyl, hydroxyl, alkoxy, aminoalkoxy, mino, alkylaminoalkoxy, alkenyl, alkynyl, aryl, aryloxy, arylamino, cycloalkyl, cycloalkoxy, cycloalkylalkoxy, cycloalkylamino, lkylalkylamino, heterocyclyl, heterocycyloxy, heterocycylamino, halo, haloalkyl, ﬂuoroalkyloxy, heteroaryl, heteroaryloxy, heteroarylamino, arylalkyl, arylalkyloxy, arylalkylamino, heteroarylalkyl, arylalkyloxy, heteroarylalkylamino, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; each Y1, Y2, and Y3 is an independently selected group of Formula IA: each Z1 and Z2 is a member independently selected from NR4 and O; and each R6a, R6b, and R60 is a member independently selected from hydrogen, alkyl, fluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, cycloalkyl, and heteroarylalkyl, or, alternatively, two R6H members R681 and R61) or R681 and R60 join to form a heterocycyl ring; n the polyamine nd comprises at least two primary or ary amino .

In certain aspects, each R4 is a member independently selected from hydrogen, alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl, or, alternatively, for a -N(R4)2 group, one of the two R4 in the group is a member selected from -(CO)OR6a-, (CO)N(R63)(R6b), and —C(NR63)N(R6b)(R6°); and each R5 is a member ndently selected from hydrogen, alkyl, hydroxyl, alkoxy, alkylamino, alkenyl, alkynyl, aryl, aryloxy, arylamino, cycloalkyl, cycloalkoxy, cycloalkylamino, heterocyclyl, heterocycyloxy, heterocycylamino, halo, haloalkyl, fluoroalkyloxy, heteroaryl, heteroaryloxy, heteroarylamino, kyl, arylalkyloxy, arylalkylamino, heteroarylalkyl, heteroarylalkyloxy, heteroarylalkylamino, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl.

In another preferred aspect, the compounds or compositions of the embodiments and aspects described herein has the proviso that the polyamine compound is not of Formula IB: \N4 /R4 F? F'z4 IB wherein the —N(R4)2 groups are tertiary amines.

In some s, the compounds or compositions of the embodiments and aspects described herein has the proviso that the polyamine compound is not: R\N/\/\N4 NMN’R4 F'z4 F'w IB-2 wherein the —R4 groups are hydrogen or methyl.

In a preferred aspect, the ion presents the compounds or compositions described herein with the proviso that the polyamine compound is not of formula IB: R\WNMN4/\/\N/R4 4 or a salt thereof; IB n the —N(R4)2 groups are tertiary amines; and with the proviso that the polyamine compound is not of formula IC: RZ-HN-(CH2)p-NH-(CH2)q-NHCH2-Y-CHZNH-(CH2)S-NH-(CH2)t-NH-RZ (IC) or a salt thereof; wherein Y is selected from anthracenyl, naphthyl, and phenyl; wherein each RZ is independently selected from hydrogen and alkyl; and wherein p, q, t, and s are each an integer ndently ed from 3 and 4.

In some aspects, the composition comprises, consists essentially of, or ts of a polyamine nd of formula Ra Ra Ra Ra RaU 0Ra Ra Ra U L1 Ra L1 R5 Ll Ra L1 R5 R5 Ra, Ra R5 or a salt thereof; wherein: each Ra is ndently a group of Formula I: each R121 and R1b is a member independently selected from hydrogen, ﬂuoro, alkyl, and ﬂuoroalkyl; each Rza, R21), R2°, and R2d is a member independently selected from hydrogen, alkyl, fluoroalkyl, aryl, and arylalkyl; each Rm is an independently selected—CRzaRZb-g each m is an integer independently selected from 1 to 2; each L1 is a member independently selected from a bond and each R3 is an independently selected -Zl-Y1-R4; each R4 is a member independently selected from hydrogen, alkyl, ﬂuoroalkyl, alkenyl, and alkynyl; or, alternatively, for an -N(R4)2 group, one of the two R4 in the group is a member selected from -(CO)OR6a-, -(CO)N(R6a)(R6b), and -CCNR6a)N(R6b)(R6°); each R5 is a member independently ed from hydrogen, alkyl, hydroxyl, alkoxy, halo, lkyl, lkyloxy,and hydroxyalkyl; each Y1 is an independently selected group of Formula IA: each Z1 and Z2 is an independently selected NR4; and each R6a, R61), and R60 is a member independently selected from hydrogen and alkyl; wherein if R4 is -C(O)OR6a, R6&1 is alkyl; and wherein the polyamine compound comprises at least two primary or secondary amino groups.

In some aspects, each m is 1. In some aspects, at least one m is 1. In some aspects, each m is 2. In some aspects, at least one m is 2.

[0124] In some aspects, each Rza, RZb, R2°, and R2d is a member independently selected from hydrogen, alkyl, and lkyl. In some aspects, each Rza, RZb, ch, and R2d is a member independently selected from en, alkyl, ﬂuoroalkyl, and arylalkyl.

In some aspects, each R121 and R1b is a member ndently selected from hydrogen and alkyl.

[0126] In some aspects, the polyamine compound comprises at least four primary or secondary amino groups.

In some aspects, the polyamine compound is of formula or a salt thereof.

In some s, Ra is -CH2[NH(CH2)3]2NH2. In some aspects, R3 is an independently selected group of Formula VII: H’ 7ng VII.

[0129] In some aspects, each m is l.

In some aspects, R5 is hydrogen.

In some aspects, each R4 is a member independently selected from en and alkyl.

In some aspects, the polyamine compound is selected from H2N[(H2C)3HN]2H2C CHleH(CH2)3]2NH2 Ph H2N[(H2(3)3|‘|N]2|‘|2C CHleH(CH2)3]2NH2 and a salt thereof.

In some aspects, L1 is a bond.

In some aspects, Rm is -CH2-.

[0135] In some aspects, the polyamine nd is selected from H2N[(H20)3HN]2H2C CHleH(CH2)3]2NH2 H2N[(H2(3)3|'|N]2l’|2C CHleH(CH2)3]2NH2 l‘l2l\ll(l‘lzc)3l‘lN12H2C CHleH(CH2)3]2NH2 CHleH(CH2)3]2NH2, H2N[(H20)3HN]2H20MMCH2[NH(CH2)3]2NH2 CH2)3]2NH2 l‘l2l\ll(l‘l20)3l‘lN12H2CMCHleH(CH2)3]2NH2 CHleH(CH2)3]2NH2 OiPr H2N[(HZC)3HN]2HZC OD CHleH(CH2)3]2NH2 H2N[(H2C3)3HN]2H2C CHleH(CH2)a]2NH2, and a salt thereof.

In some aspects, each Ra is independently a group of Formula II: R13Nlb R.2 N R20 m R3 each of the An members is independently selected from CR3 and CR5; or, alternatively, a pair of adjacent AI1 members join to form a cycloalkyl, aryl, heterocyclyl, or cycloaryl ring; wherein at least one A11 member and at most three A11 members are independently selected CR3, each R”, Rlb, R”, and R1d is a member independently selected from hydrogen, ﬂuoro, alkyl, and lkyl; for each Ra member, at most two R”1 of the Ra member are selected from - C(R2a)=(R2b)-, -cc-, and -C(Rza)(R2b)-L2-C(R2c)(R2d)-; and each m is an integer independently selected from 1 to 16.

In some aspects, the polyamine compound is selected from: Ra R3 ﬁl J§A5 ﬁ1J§A4 A2 l4 l2 A ﬁS’A\\AB9iv \A3. \A3 L1 AG' and a salt thereof; wherein: at least one AIl member and at most three A11 members are independently selected CRa, each Rza, R21), R”, R”, R23, and R2f is a member independently selected from hydrogen, alkyl, ﬂuoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, and arylalkyl; alternatively, a pair of R211 members from the Ra group independently selected from the group R2a and R21), R20 and R”, and R2e and R2f join to form a ring independently selected from spirocycloalkyl and spiroheterocycyl; or, alternatively, the R221 and the R20 from the R3 group join to form a ring independently selected from cycloalkyl and heterocycyl; each m is an integer independently selected from 1 to 12; each R3 is a member independently selected from -Z1-Y1-R4 and -Zl-Y1-Y2-R4; and each Z1 and Z2 is an ndently selected NR4.

[0138] In some aspects, A1, A2, A3, A4, A5, A6, A7, A8, and A9 are each an AI1 member independently selected from CRa, and CR5; or, alternatively, a pair of adjacent AIl members join to form a lkyl, aryl, heterocyclyl, or heterocycloaryl ring; each Rza, R2b, R2°, RM, R26, and R2f is a member independently ed from hydrogen, alkyl, lkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl; each m is an integer independently selected from 1 to 10; each L1 and L2 is a member independently selected from a bond, -O-, and -NR4-; each R3 is a member independently ed from -Z1-R4, -Z1-Y1-R4, and -Z1-Y1-Y2-R4; and each Z1 and Z2 is an independently selected NR4.

[0139] In some aspects, the invention provides the polyamine nd selected from: Ra R8 ﬁ1/KT5 P11J§A4 5’A§Ae9 ' ' A\ 3—.A4 ‘l 2 A2 ,1 —.A7 A \A3 L1 A6 and a salt f; , , each R"1 is independently a group of a V: N R20 R“: ( Rm%R2d m R3 each of the An members is independently selected from CRa and CR5; or, alternatively, a pair of adjacent AIl members join to form a cycloalkyl, aryl, heterocyclyl, or heterocycloaryl ring; wherein at least one A11 member and at most three A11 members are independently selected CRa, each Rla, Rlb, R”, and R1d is a member independently selected from hydrogen, ﬂuoro, alkyl, and ﬂuoroalkyl; for each Ra member, at most two R”1 of the Ra member are selected from — C(Rza)=(R2b)-, —cc—, and )(R2b)-L2-C(R2°)(R2d)-; and each R3 is a member independently selected from -Zl-Y1-R4 and -Zl-Y1-Y2-R4.

[0140] In some aspects, the polyamine compound comprises at least four primary or secondary amino groups. In some aspects, the polyamine compound comprises at least six primary or secondary amino groups.

In some s, the invention provides the polyamine compound selected from Ra Ra 1km 1kg) Elam/k 13,1 Ra, Ra A Ra, and a salt thereof; wherein each A11 member is an independently selected CR5.

In some aspects, the invention es the polyamine compound selected from Ra Ra R5 and a salt thereof; and wherein R3 is an independently selected group of Formula VII: HI ( Rm)'\ m R3 VI].

In some aspects, the invention provides the ine compound selected from Ra R3 R5 and a salt thereof; and wherein R5 is hydroxyl, alkoxy, cycloalkoxy, or arylalkyloxy.

In some aspects, Rm is —CH2-. In some aspects, Ra is —CH2[NH(CH2)n]pNH2; each n is an integer independently selected from 3 to 12 ; and each p is an integer independently selected from 1 to 3. In some aspects, m is 1. In some aspects, each m is 1. In some aspects, at least one In is 1. In some aspects, each m is 2. In some aspects, at least one In is 2. In some aspects, R5 is selected from en, phenyl, and oxy.

In some aspects, the polyamine compound is selected from Ra Ra Ra and a salt thereof.

In some s, the polyamine compound is selected from: "'2N[(H20)2J'”\|]2l’|2C CHleH(CH2)3]2NH2 Ph "'2N[(H20)3|‘”\|]2|‘|2C CHleH(CH2)3]2NH2 H2N[(H2C)3HN]2H20 CH2)3]2NH2 CH2[NH(CH2)3]2NH2 and a salt thereof.

In some aspects, the polyamine compound is selected from: H2N[("'2C)3HleHzC CHleH(CH2)3]2NH2 I'I2N[(H20)3HN]2H2C CH2[NH(CH2)3]2NH2 H2N[(H2C)3HN]2H2C CH2)3]2NH2 CH2[NH(CH2)3]2NH2 and a salt thereof.

[0148] In some aspects, the polyamine compound is selected from Ra Ra Ra Ra ,TkA‘t H’K9 ‘TJW s/KAB ArkA4 Ra R31\iL1J\ 'A7 Raj]\A3’ 1 '11] A]5A: A3 A6! L1 AG’ Raj]\A:’JA\ L1 A6a Ra Ra R8J\A3J\LJ\A6TJ§A4 pf/gﬁRa8and a salt thereof; wherein each AIl member is an independently selected CR5.

In some s, the polyamine compound is selected from Ra Ra Ra RakA:/JA:L1J\ ﬂ1/J\A45'A\\A89 J11 /J\A4 &A8 “1‘7 ’J\ 1 ' A7 A6 Ra A3 L1 A6 and a salt f; , , wherein each AI1 member is an independently selected CR5.

In some aspects, the polyamine compound is selected from Ra Ra Ra Ra Ra : Ra Ra : Ra L1 R3 L1 R5 L1 Ra L1 R5 URS URa Ra R5 and a salt thereof.

In some aspects, Ra is an independently selected group of Formula VII: V11.

In some aspects, R5 is en. In some aspects, L1 is selected from a bond and O.

In some aspects, Rm is —CH2-. In some aspects, m is 1. In some s, each m is 1. In some aspects, at least one m is 1. In some aspects, each m is 2. In some aspects, at least one mis2.

[0153] In some aspects, Ra is —CH2[NH(CH2)n]pNH2; each n is an integer independently selected from 3 to 12; and each p is an integer independently selected from 1 to 3.

In some aspects, the ine compound is selected from H2N[(H20)3HNl2HzC CHleH(CH2)3]2NH2 H2N[(H20)3HN]2H2C CHleH(CH2)3]2NH2 H2N[(H2C)3HN]2H2C CHleH(CH2)312NH2 CH2[NH(CH2)3]2NH2 H2N[(H2C)3HN]2H2C CH2[NH(CH2))3]2NH2 CH2[N H((CH2)3]2NH2 "'2C)3HleHzC OO CHleH(CH2)3]2NH2 CHleH(CH2)3]2NH2 OiPr H2N[(H20)3HN]2H2C O CHleH(CH2)3]2NH2 H2N[(H20)3HN]2H2C (CH2)3]2NH2, and a salt f.

In some aspects, R5 is hydroxyl, alkoxy, lkoxy, heterocycyloxy, aryloxy, arylalkyloxy, heteroaryloxy, or heteroarylalkyloxy. In some aspects, R5 is hydroxyl, alkoxy, cycloalkoxy, heterocycyloxy, arylalkyloxy, heteroaryloxy, or heteroarylalkyloxy. In some aspects, R5 is hydroxyl, alkoxy, cycloalkoxy, or arylalkyloxy. In some aspects, R5 is hydroxyl, alkoxy, or cycloalkoxy. Preferably, R5 is alkoxy.

In some aspects, Ra is H(CH2)n]pNHR4; each n is an integer independently selected from 3 to 12; and each p is an integer independently selected from 1 to 3.

Preferably, R4 is alkyl, cycloalkyl, or arylalkyl; more preferably, R4 is alkyl.

In some aspects, Ra is -CH2[NH(CH2)n]pNHR4; each n is an integer independently selected from 3 to 12; and each p is an integer ndently selected from 1 to 3.

Preferably, n is 3. More preferably, R4 is not hydrogen.

[0158] In some aspects, the polyarnine compound is: or a salt thereof. Preferably, R4 is isobutyl.

In n aspects, the invention provides a polyamine composition for treatment of biofilms, the composition comprising, consisting essentially of, or consisting of a polyamine compound of formula or a salt thereof; wherein: each Ra is an independently selected group of Formula I: A1, A2, A3, A4, A5, A6, A7, A8, and A9 are each an A11 member independently selected from N, CR3, and CR5; n at least one A11 member and at most ﬁve AIl s are independently selected CRa; each R121 and R1b is a member independently ed from hydrogen, ﬂuoro, alkyl, and ﬂuoroalkyl; each Rza, R21), RR, and R2d is a member independently selected from hydrogen, alkyl, alkyl, aryl, and arylalkyl; each Rm is a member independently selected from —CR2aR2b- and -C(R2a)(R2b)-L2- C(R2°)(R2“)-; each m is an integer independently selected from 1 to 20; each L1 is a member independently selected from a bond and -O-; each L2 is a member independently selected from a bond, -O-, and -NR4-; each R3 is a member independently selected from -Z1-R4, -Zl-Y1-R4, and -Z1-Y1-Y2- each R4 is a member independently selected from hydrogen, alkyl, ﬂuoroalkyl, alkenyl, l, aryl, and arylalkyl; or, alternatively, for a 2 group, one of the two R4 in the group is a member selected from R6a-, -(CO)N(R6a)(R6b), and -C(NR63)N(R6b)(R6C); each R5 is a member independently selected from hydrogen, alkyl, hydroxyl, alkoxy, alkylamino, aryl, aryloxy, heterocyclyl, halo, ﬂuoroalkyl, ﬂuoroalkyloxy, heteroaryl, arylalkyl, arylalkyloxy, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; each Y1 and Y2 is an independently selected group of Formula IA: 1 0 IA each Z1 and 22 is a member independently selected from NR4 and 0; each R6a, Réb, and R60 is a member independently selected from en and alkyl; wherein if R4 is —C(O)OR6a, R6a is alkyl; and wherein the biocidal polyamine compound comprises at least two primary or secondary amino groups.

In some aspects, the composition comprises a polyamine compound of formula 1%,“ 5’A§A89 €12 J\ q I A\A3/ 2A7 L1 A6 or a salt thereof.

In some aspects, each A1, A2, A3, A4, A5, A6, A7, A8, and A9 are each an A11 member ndently selected from N, CRa, and CR5 ; wherein at least one AI1 member and at most ﬁve AI1 members are independently ed CRa.

In some aspects, each R1&1 and R1b is a member independently selected from hydrogen, ﬂuoro, alkyl, and ﬂuoroalkyl.

In some aspects, each Rza, RZb, R20, and R2d is a member independently selected from hydrogen, alkyl, ﬂuoroalkyl, aryl, and arylalkyl.

[0164] In some aspects, each Rm is a member independently ed from —CR2aR2b- and _C(R2a)(R2b)_L2_C(R20)(R2d)_ .

In some aspects, each R6a, R61”, and R60 is a member independently selected from hydrogen and alkyl; wherein if R4 is —C(O)OR6a, R621 is alkyl.

In some aspects, each L1 is a member ndently ed from a bond and -O-; and each L2 is a member independently selected from a bond, -O-, and -NR4-.

[0167] In some aspects, each R5 is a member independently selected from hydrogen, alkyl, hydroxyl, alkoxy, alkylamino, aryl, aryloxy, cyclyl, halo, alkyl, fluoroalkyloxy, heteroaryl, arylalkyl, arylalkyloxy, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl.

In some s, each R6a, R61”, and R60 is a member independently selected from hydrogen and alkyl; wherein if R4 is R6a, R6a is alkyl.

[0169] In some aspects, the invention provides a polyamine compound selected from: (a) a compound of Formula IC: Rq R3 Rm R1 ( N\ RQNWLT‘KNMLNH /—NH HN q H I n H R1 R” N/\l/CDRHR)p R1 (b) a compound of Formula ID: Rm’“ R1 R3\ Ea NR3R§N~(27LQN'IR4 4 1 hair“: I R R R R \/j— W) P /\’/K R1 RP R5 (R6)Z or a salt thereof; 1 5 wherein: each R1, Rm, Rn, RP, and Rq is independently a substituent independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, kyl, and heteroarylalkyl; each R2 is independently a substituent independently selected from hydrogen, alkyl, alkoxy, alkylamino, alkenyl, alkynyl, aryl, aryloxy, arylamino, halo, haloalkyl, aryl, heteroaryloxy, heteroarylamino, arylalkyl, alkylaryloxy, alkylarylamino, heteroarylalkyl, heteroalkylaryloxy, heteroalkylarylamino, Rm Rm R1 R1 R: N?“ R: WT» N N n if R1 R1 and R1 R1 Rn each 2 is is independently an integer independently selected from 1 to each m, n, p, and q is independently an integer independently ed from 1 to 20; each R3 is independently a substituent independently selected from hydrogen, each R4 is independently a substituent independently ed from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, R1 R1 and R1 R1 R" , ; and each R5 is independently a substituent independently selected from alkyl, alkoxy, alkylamino, alkenyl, alkynyl, aryl, aryloxy, arylamino, halo, kyl, heteroaryl, heteroaryloxy, heteroarylamino, arylalkyl, alkylaryloxy, alkylarylamino, heteroarylalkyl, heteroalkylaryloxy, hetReroalkylarylamino, Rm Rm R1 R1 3 (R6)Z R1 m a R1 09% RN N N n f R4 R1 Rnn R3NY R1 R1 R1 R1 R” I/J and , , Rq R3 RmmFlz3 R1 R\WWW“!3 NR3R§N4QtNR4’ 7:]— Hop R1 R4 R1 R” R1 RD 4 R6)z ;and each R6 is independently a substituent independently selected from hydrogen, alkyl, , alkylamino, alkenyl, alkynyl, aryl, y, arylamino, halo, haloalkyl, heteroaryl, heteroaryloxy, heteroarylamino, arylalkyl, alkylaryloxy, alkylarylamino, arylalkyl, heteroalkylaryloxy, and heteroalkylarylamino.

In some aspects, the polyamine compound is selected from: (a) a compound of Formula IE: (b) a compound of Formula IF: wherein: each R1, Rm, R“, R”, and Rq is independently a substituent independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; each R2 is independently a substituent independently selected from en, alkyl, , alkylamino, alkenyl, alkynyl, aryl, aryloxy, arylarnino, heteroaryl, heteroaryloxy, heteroarylamino, arylalkyl, alkylaryloxy, alkylarylarnino, heteroarylalkyl, heteroalkylaryloxy, alkylarylamino, R1 M22, R1 09:» N N n ,6 R1 R1 and R1 R1 Rn each m, n, p, and q is independently an integer independently selected from 1 to 20; each R3 is independently a substituent ndently selected from hydrogen, Rm Rm R1 R1 R1 my R1 991% N N “a“ R1 R1 ,and R4 R1 Rn each R4 is independently a tuent independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, aryl, arylalkyl, heteroarylalkyl, Rrn Rm R1 R1 Riwa R1 mi: N N “z“ R1 R1 and R1 R1 R” , ; and each R5 is independently a substituent independently selected from alkyl, alkoxy, alkylamino, alkenyl, alkynyl, aryl, aryloxy, arylamino, heteroaryl, heteroaryloxy, arylamino, arylalkyl, alkylaryloxy, alkylarylarnino, heteroarylalkyl, heteroalkylaryloxy, heteroalkylarylamino, In some s, the polyamine compound is CH2[NH(CH2)3]2NH2 In still some aspects, R2 is hydrogen.

In yet some aspects, R5 is aryl or aryloxy.

In still yet some aspects, R5 is In some aspects, the polyamine compound is 2NA<\/\NH HN/\/)~NH2 2 2 or a salt thereof.

In some aspects, the polyamine compound is umwm (CH2)3]2NH2 or a salt thereof.

In some aspects, the polyamine compound is H NNN NwlNH or a salt thereof.

In some aspects, the polyamine compound is HNNN N/\/>NH of) or a salt thereof.

In some aspects, the invention provides a polyamine compound of Formula LP or a salt f: E Elwfw“Nww-MMWNRW) F «J H 2', wherein: M is selected from: (a) a C1-C13 alkyl, alkenyl or alkynyl group, and (b) —(L—NH)Z—L—, where L is a C3-C13 alkyl, alkenyl or alkynyl group, and where z is an integer from zero to 6; x is an integer from 1 to 6; and R1 and R2 each is independently selected from hydrogen and C1—C3 alkyl group.

[0180] In some aspects, the polyamine nd of Formula I-P may comprise a hydrophobic phenyl group and up to six hydrophilic polyamine . Each of the hydrophilic polyamine chains may be the same, or some of the hydrophilic polyamine chains may be same, or none of the hydrophilic polyamine may be the same. Additionally, each hydrophilic polyamine chain may se a neutral amine, a cationic ammonium salt, or both.

In some aspects, the polyamine compound may comprise a compound of Formula II-P or a salt thereof, wherein n is an integer from 1 to 13, x is an integer from 1 to 6, and R1 and R2 each may independently be hydrogen or a C1-C3 alkyl group. .» \ ff {”9 ”'22" "'" H {V ‘X \W?’ II-P In some aspects, the polyamine compound may comprise a compound of Formula III-P or a salt f, n n is an integer from 1 to 13, and the hydrophobic phenyl group is substituted with two hydrophilic polyamine chains at the ortho-, meta-, or para-position.

,A r’ I; rcmnmwziﬁ Kg; H I III-P In yet some aspects, the polyamine compound may comprise a compound of Formula IV-P or a salt thereof, wherein each m and n is an independently selected integer from 1 to 13.

H N “Mi, CH“RpmN”W?.»»~”’"\‘:§ “N } Nmiitﬁﬁér _______NH H a H \gf’f’j l 5 IV-P In some aspects, m is 12 (i.e., nd IV-l or a salt thereof). In some alternative s, m is 6 (i.e., compound IV-2 or a salt thereof).

In some aspects, the polyamine compound may comprise a compound of Formula V-P or a salt thereof, wherein n is an integer from 1 to 13, and the three hydrophilic polyamine chains may be at any position on the hydrophobic phenyl group. ,- f l FT’WWEWHJB In some aspects, the polyamine compound may comprise a compound of Formula VI-P or a salt thereof, wherein each m, n and p is an independently selected integer from 1 to l3: and each R1 and R2 may independently be hydrogen or a C1—C13 alkyl group.

*RERN«-w<::u2}m--wn‘ #fN~ VI-P In some aspects, m, p, and n are 3 (e.g., compound VI-l). In some aspects, m, p, and n are 6 (e. g., compound VI-2).

[0188] In some aspects, the polyamine compound may comprise a compound of Formula VII-P or a salt thereof: foltx 'L x «\ A. §/‘\ xx {ii saw [,, ‘ NH’ “v“ “m ‘v” “ New?) ”If H 3" xxiﬁgx VII-P wherein: x is an integer from 1 to 6, y is an integer from zero to 6, each R1 and R2 is independently ed from hydrogen and C1-C13 alkyl; and wherein the substituted hydrophilic polyamine chains may be at any position of the hydrophobic phenyl group.

[0189] In some aspects, the polyamine nd may comprise a compound of Formula VII-l or a salt thereof, wherein y is an r from zero to 6, and the two hydrophilic polyamine chains may be at the ortho-, meta-, or para- position of the hydrophobic phenyl group .

VII-1

[0190] In some aspects, the polyamine compound may comprise a compound of Formula VII-2 or a salt thereof. x \f‘Ax (“xN” ﬁx. {,1«\‘c.er’ié.‘\\\II» \ .-”(\\,,xx \ /J\ “/0.

”EN N N NH f N \x XNHw H H kg? H V: VII-2 In some aspects, the ine compound may comprise a nd of Formula VII-3 or a salt thereof, wherein y is an integer from zero to 6, and the three hydrophilic polyamine chains may be at any on of the hydrophobic phenyl group. [if \f‘9"“lew”f‘v‘mwi:\’”’A\NRIR§)3 VII-3 In yet some aspects, the polyamine compound is a compound of Formula VII—4 or a salt thereof, wherein f, g and h are each an independently selected integer from zero to 6. -3mmL a» A w“ J“ ”V x 9 «~ “ii \V 14mm H j \NH”T\\ \‘NHE»!\NR‘RZ» \V’ﬁ’ LNH ﬂ\ f]05V”NR1 R2 VII-4 In some aspects, the polyamine compound is a compound of Formula VII-5 or a salt thereof: "a“ \\Niki} WM» “No, I -' ' r ”(6135533. f .~ 3x{SHEER K ”MS“ N" 2 “NR RNT H1 ‘“‘§. 3‘ i w E i ‘ E #1:: f ‘X VII-5 wherein: a is an independently selected integer from 2 to 13, x is an independently selected integer from 1 to 6, y is an independently selected integer from zero to 6, each R1 and R2 is independently ed from hydrogen and C1-C13 alkyl, and the substituted hydrophilic polyamine chains may be at any position of the hydrophobic phenyl group.

In some aspects, the polyamine compound is a compound of a VIII—P or a salt thereof: ’w‘lj"\\\ i“, ii 1 gill ‘w‘x '9‘ ‘ '“ k \ Nm-=-M~WWNR R‘ § § H 3 xxgﬁﬁﬁrw VIII-P wherein: M may be selected from: (a) C1-C3 alkyl, l or alkynyl group, (b) —(L—NH)Z—L—group, wherein L is C3-C3 alkyl, alkenyl or alkynyl group, and z is an integer from zero to 6; x is an integer from 1 to 6; and R1 and R2 each may independently be hydrogen or a C1-C1; alkyl group.

The polyamine compound of general a VIII-P may comprise a hydrophobic phenyl group and up to six hydrophilic polyamine chains, each of the hydrophilic polyamine chains connecting to the hydrophobic phenyl group Via an amide functional group. Each of the hydrophilic polyamine chains may be the same, or some of the hydrophilic polyamine chains may be same, or none of the hydrophilic polyamine chain may be the same.

Additionally, each hydrophilic polyamine chain may comprise a l amine, a cationic ammonium salt, or both.

In some aspects, the polyamine compound is a compound of Formula VIII-l or a salt thereof, wherein n is an integer from 3 to 13, and the two hydrophilic polyamine chains may be at the ortho-, meta— or para- position of the hydrophobic phenyl group.

AM”Nﬂwti. {3 "ME N " ”((3425$le is3) Ki'x j H i E: 9’13: \z’ﬁf VIII-1 In some s, the polyamine compound is a compound of formula VIII-2 or a salt thereof, wherein y is an integer from zero to 6, and the two hydrophilic ine chains may be at the 0rth0-, meta—, or para- position of the hydrophobic phenyl group. 73,,» N5; ’ i Emmi?x f» "N N” ,N “f \N3‘ \NH.::) N2 VIII-2 In some s, the polyamine compound is a compound of Formula VIII-3 or a salt thereof. {j} HN"»\W’” ” \[lfklfkg N:N ' {\N‘} x\\¢§§ {"l N VIII-3 In some aspects, the polyamine compound is a compound of Formula VIII—4 or a salt thereof: VIII-4 a is an integer from 2 to 13, x is an integer from 1 to 6, y is an integer from zero to 6, each R1 and R2 is independently selected from the group hydrogen and a C1-C13 alkyl group, and the substituted hydrophilic polyamine chains may be at any position of the hobic phenyl group.

In some apects, the polyamine compound is a compound of Formula I-P, Formula VIII-P, or a salt thereof, n M, R1 and R2 are as described , and x is an integer from 1 to 6. The polyamine nd comprises a hobic phenyl group and at least one hilic polyamine chain.

[0201] In certain aspects, the nds of the present invention are antimicrobial and provide triple action against bacteria and biofilms. Advantageously, the antimicrobial compounds of the present invention have speciﬁc activity against biofilms.

Organisms residing in ms present a complex extracellular matrix of polysaccharides (exopolysaccharides) and proteins. As a result of this complex matrix, nutrient-limiting conditions exist that alter the normal or planktonic lic state. These conditions reduce the efﬁcacy of traditional antibiotic agents, rendering them up to l,000x less active against bioﬁlms. A rk of these exopolysaccharides is the presentation of acidic residues from repeated glucoronic acid motifs and pyruvate derived acetals. A recent study by Losick and co-workers demonstrated that the simple polyamines spermine and norspermidine (A) were naturally occurring inhibitors of biofilm formation, endogenously produced at high concentrations (5 0-80 uM) in response to nutrient limiting conditions and waste accumulation in mature pellicles (Kolodkin-Gal, I. et al., A self- produced trigger for biofilm disassemby that targets exopolysaccharide. Cell 149 (2012)). In this study, they were able to demonstrate that norspermidine could inhibit bioﬁlm formation at 25 uM and showed that, at similar concentrations, it could disperse the ysaccharide component of the matrix but not the protein component. Interestingly, dine was only active at much higher concentrations (~l mM) leading them to propose a rationale for this ty in the ability of the polyamines to engage the acidic residues in the matrix at regular intervals (B).

In certain aspects, compounds of the present invention having sed numbers of chains, produce a more effective nd against A. baumanniz’. For example, compounds with four polyamine chains can be generated with Pd(II) mediated dimerization of 5- bromoisopthalaldehyde followed by reductive amination ().

[0204] In certain aspects, the compounds of present invention combine a hydrophobic backbone with a cationic tail that have the functionality to inhibit biofilm formation, disrupt established biofilms, and kill the emerging planktonic bacteria. Certain of the compounds of the invention are set forth in FIGS. 27 and 28.

In a particular embodiment, the polyamine compound may comprise a hydrophobic I5 moiety head and at least one hydrophilic moiety tail comprising a polyamine group. When the polyamine compound comprises more than one hydrophilic moiety tails, the hydrophilic moiety tails may be the same, or alternatively, the hydrophilic moiety tails may be different.

As discussed above, the exemplary polyamine compounds shown herein are not ed to be limiting.

SYNTHESIS The sis of diaminopropane substituted backbones is straightforward and s in CZ-4, 12, 32 () from the known mono-Boc ted diaminopropane and commercially available aldehydes ldehyde, isopthalaldehyde or 1,3,5— benzenetricarboxaldehyde). This three-step synthetic procedure proceeds Via reductive amination (Baxter, E. W. & Reitz, A. B. Reductive ions of Carbonyl Compounds with Borohydride and Borane ng Agents. Org Reac l, 59 (2004)) and acidic removal of the Boc group. The norspermidine series (e.g., CZ-7, CZ-25, and CZ-52) can be prepared in a similar manner from the mono-Boc protected norspermidine (). No purification is ed until a final recrystallization of the HCl salt, which has allowed easy preparation of these compounds on larger scale.

In some ments, the polyamine compound may be produced by the reductive amination method of General Synthetic Scheme 1.

General Synthetic Schemel + H N—(CR2n yH—(CRz)—nr{J—R1 —> Step1 y= 0-2 n=2-20 R1: 800, alkyl acy/ su/fony/ \ 1L Y = H, formyl R2__ H, Me, [> I,~N\ NH X = H, halo, formyl, aryl, R1’ R3 _ "" R3 = H formylphenyl, heteroaryl, 0R4 R4 = H, alkyl, benzyl, alkylaminoalkyl R1—E:(CR2)-n—N CR2)-N Hn—(CR2 yH-(CR2)—nN-—R1 Step 2 NIXCNWNN[QWNWig:1N1;.N; 0(NVNN —total # of basic amines ts: (a) MeOH, 3 A molecular sieves, rt, 1-28 h; NaBH4; (b) HCl, MeOH, rt 1-4 h.

The l Synthetic Schemes included herein set forth methods of preparing some nds within the aspects and embodiments disclosed herein. In some alternative aspects, these methods can be used to produce a compound of the one of the aspects and embodiments disclosed . In some alternative aspects, these methods can be used to produce a precursor or starting material for producing such a compound.

In General Synthetic Schemes l, 2, 3, and 5, “CR2” indicates a ene with two substituents, which may be hydrogen, methyl (or, alternatively, lower alkyl), or spirocyclopropane , the two substituents are the spirocyclic ring).

In some embodiments, the polyamine compound may be produced by the acylation method of General Synthetic Scheme 2.

General Synthetic Scheme 2 O 0 CI CI H H H a + H N CR2 N CR2 N—R1 —> n " Step1 x y = 0-2 n = 2-20 R1 = Boc, alkyl, acyl, sulfonyl X = H, haloaryl, R2 = H’ Me, [> heteroar R3 = H yI, OR4 R4 = H, alkyl, benzyl, alkylaminoalkyl O O H H H H b n H H n n Step2 O O H H H H R1—N CR2 N CR N N CR2 N CR2 N-R1 '(HCl)z n H H n n z = O — W W = total # of basic amines ts: (a) TEA, CHzClz, rt, 1-28 h; (b) HCl, MeOH, rt 1-4 h.

In some embodiments, the polyamine compound may be produced by the ive amination method of General Synthetic Scheme 3.

General Synthetic Scheme 3 H H H H a H2N CR2 N CR2 N N CR2 N CR2 NH2 _> n n n n Step 3 y y H H H H H H b R1HZC_N CR2 N CR2 N N CR2 N CR2 N—CH2R1 —> n n n n Step4 y y H H H H H H R1HZC-N CR2 N CR2 N N CR2 N CR2 N-CH2R1-(HCI)Z n n n n y y z = O - W X W = total # of basic amines Reagents: (a) R1CHO, MeOH, 3 A molecular sieves, rt, 1-28 h; NaBH4; (b) HCl, MeOH, rt 1- 4 h.

In some embodiments, the polyamine nd may be produced by the ive amination method of General Synthetic Scheme 4.

Ge_v—neralS nthetic Scheme 4 l\a/R4 OH Step 1 Step 2 O‘R4 H H —> _> + H Step 3 N‘(CR2H’:H4(CR2>—nN——R1 Step 4 0eR4 y= 0-2 n= 2-20 R1 = Boc, alkyl, acyI, sulfonyl R2=H,Me, [> R3=H R4 = H, alkyl, benzyl, alkylaminoalkyl H H H H H H e R1—N CR2 N CR N N CR2 N CR2 N—R1 —> n n n Step5 y y R1_N CR2 nN CR2 CR2” CR2 nN—R1 o (HC|)Z Z=0-W W= total # of basic amines Reagents: (a) CH3CN, Cs2C03, rt, 16 h; (b) THF, LiAlH4, rt, 8 h; (c) PCC, CH2C12, rt 1-4 h; (d) MeOH, 3 A molecular sieves, rt, 1-28 h; NaBH4; (e) HCl, MeOH, rt 1—4 h.

[0214] In some aspects, the R4CH21 can include a ent g group for the nucleophilic substitution reaction of (a). Other leaving groups useful for phenol alkylation include Br, Cl, tosylate, mesylate, triﬂate, and the like.

The precursor compounds shown below were made by the method of Steps 1 to 3 in the General Synthetic Scheme 4 above. 5-(Cyclohexylmethoxy)isophthalaldehyde: 1H NMR (300 MHz, CDC13) 8 ppm .02 (s, 2H), 7.91 (s, 1H), 7.61 (s, 2H), 3.84 (d, J: 6.0 Hz, 2H), 1.87-1.67 (m, 6H), 1.31- 1.03 (m, 5H). 13C NMR (75 MHz, CDC13)8 ppm 191.4, 160.9, 138.6, 124.3, 120.2, 74.6, 37.9, 30.1, 26.7, 26.0. 0 0 \OJ\/ 5-(2-Ethy1butoxy)1sophtha1aldehyde: 1H NMR (500 MHz, CDC13) 8 ppm 10.01 (s, 2H), 7.90 (s, 1H), 7.62 (s, 2H), 3.94 (d, J= 5.5 Hz, 2H), 1.68 (hept, J= 6.5 Hz, 1H), 1.46 (dec, .1: 6.5 Hz, 4H), 0.91 (t, J = 7.5 Hz, 6H). 13(2 NMR (125 MHz, CDC13) 8 ppm 191.1, 160.7, 138.5, 124.1, 120.0, 71.1, 40.9, 23.5, 11.3. o’ \0 5-(Hydroxymethy1)1sophthalaldehyde: 1H NMR (500 MHZ, CDC13) 8 ppm 10.09 (s, 2H), 8.27 (s, 1H), 8.15 (s, 2H), 4.88 (s, 2H), 2.54 (br s, 1H). 13c NMR (125 MHz, CDC13)8 ppm 191.4, 143.7, 137.4, 132.8, 130.3, 63.9.

[0219] In some embodiments, the polyamine nd may be produced by the method of Scheme 5, wherein R1 may be hydrogen or a C1-C13 alkyl group.

Scheme 5 HzN“"'WMW‘-NHR" {‘1} C} l(:BQC]20 ’x’N\ f H ‘ ‘ k“ ”ii-“ME: CH) + HRNMN]2...... {x} R 3806 ,i ' 3‘ {2] ~55” “i MeOH. moiacuiar sieve 24 News“ fe/‘ctw‘ 1” psi \Nmmwwwgm]” ’f%\ w [ Acid, MeoH waif ”‘x A - .............................

K J J, H x ] NMM»-NHR j- (g; (A H k \frr \Wgw {4] LA The method of Scheme 6 may include reacting a polyamine of formula [1] with di-t- butyldicarbonate compound [(Boc)20] to protect at least one terminal amine group of the polyamine [1], while leaving at least one terminal amine group of the polyamine [1] unprotected. The resulting polyamine of formula [2] having at least one Boc-protected terminal amine is reacted With a tuted benzadehyde of formula [3]. Then, the resulting product is reduced, such as by a hydride reducing agent (e. g., NaBH4 or ) to provide a corresponding polyamine conjugate of formula [4] having the al amine group on at least one hydrophilic polyamine chain Boc-protected. The Boc-protected terminal amine group is then deprotected, such as by acid hydrolysis, to provide the polyamine compound of Formula I-A comprising a hobic phenyl group and at least one hydrophilic polyamine chain.

In some ments, the polyamine compound of formula VIII-P may be produced by the method of Scheme 6, wherein R1 may independently be hydrogen or C1-C13 alkyl group.

Scheme 6 HgﬂmeNHz gqancho (I: i v” $yﬁ(,g~—cg) l HENMMWNRT‘BDC fl " X {2} \5?’ E5} NEt3, salami E «j—’i’c\gwwgum-mm“Bo;x ‘v [6] Acid, MeOH fffkkt§w :"{3‘\\.

Ll xl ““NwM-~«~«N2—§R*)H ‘ijgﬁf VIII-P The method of Scheme 7 may include reacting the substituted benzoyl chloride of formula [5] with a ine of formula [2] having at least one terminal amine group protected with a Boc-protecting group, in a presence of base such as triethylamine (NEt3) to provide a corresponding substituted benzyl amide of formula [6]. The compound of a comprises a hydrophobic phenyl group and at least one hydrophilic polyamine chain having a Boc-protected terminal amine group. Then, the Boc-protected terminal amine group ofthe hydrophilic polyamine chain is deprotected, such as by acid hydrolysis, to provide the polyamine nd of Formula VIII-P.

The polyamine compounds exhibit unexpectedly superior ability in treating ms, compared to most known antimicrobial compounds. The microorganisms in the bioﬁlm community may be eradicated effectively and rapidly such that they have minimum, if any, opportunity to upregulate their defense mechanism and develop resistance against the polyamine compound. Without being bound by any theory, it is believed that the unexpectedly superior bioﬁlms treatment is due to the beneﬁcial synergistic of hydrophobicity and hydrophilicity of the polyamine compound. It is ed that the hydrophobic moiety of the polyamine compound tates the dispersion of the microorganisms in biofilms, while the hydrophilic polyamine moiety provides an crobial effect on the dispersed microorganisms.

The ine compounds may ate the bioﬁlms, reduce the formation of biofilms, or t the formation of biofilms. The hydrophobicity and hydrophilicity of the ine compounds may be designed by tailoring the hydrophobic moieties and hydrophilic moieties of the polyamine compounds such that the desired level of antimicrobial effect on the biofilms may be achieved. One skilled in the arts recognizes the ters controlling the hydrophobicity/hydrophilicity effect, and, therefore, may readily modify the teachings of present sure to produce various polyamine compounds without departing from the scope of present disclosure. As non-limiting examples, the hydrophilicity effect of the polyamine compound may be modified by varying numbers of the hydrophilic polyamine chains in the polyamine compound, s of amine groups in the hydrophilic polyamine chains, numbers of carbons between amine groups in the hilic polyamine chains, etc.

As non-limiting examples, the hydrophobicity effect of the polyamine compound may be modiﬁed by varying positions of the hydrophilic polyamine chains on the hobic groups, altering chemical structures of the hydrophobic groups to other known non-polar functional group, etc.

The polyamine compounds may exhibit enhanced antimicrobial effect on biofilms comprised of Gram-negative or Gram-positive ia. The polyamine compounds may exhibit enhanced antimicrobial effect on s consisting of mycobacteria.

In one embodiment, the antimicrobial composition may comprise a polyamine compound and at least one additive. Various additives may be used for the antimicrobial composition. By way of non-limiting examples, the ves may further enhance the dispersion of microorganisms in biofilms, impart the antimicrobial effect against the dispersed microorganisms, facilitate the application/administration of the crobial composition to the bioﬁlms, improve the stability of the crobial composition, control the release/application rate of the antimicrobial composition to the biofilms, etc. Non- limiting examples of additives for further enhancing the antimicrobial effect may be biocide and other bactericide. By way of non-limiting examples, the additives for facilitating the administration of the crobial composition may include a pharmaceutically acceptable carrier typically used for medical or pharmaceutical applications, an emulsiﬁer or dispersant typically used for industrial applications.

The crobial composition may be formulated to provide the d level of antimicrobial effect on the biofilms by selecting a polyamine compound and other additives as well as by adjusting the amount of each component in the antimicrobial ition. In some embodiments, the antimicrobial composition may be formulated to inhibit the formation of ms. In some ment, the antimicrobial composition may be formulated to disrupt the bioﬁlms. In still other embodiments, the antimicrobial composition may be formulated to eradicate substantially all microorganisms in the biofilms.

Any suitable amount ofpolyamine can be used in the itions and methods of the invention. In general, the polyamines are used in concentrations ranging from about 1 ppm to about 100,000 ppm, or higher. The concentration of a polyamine used in a composition or method of the invention can be, for example, from about 1 to about 100,000 ppm, or from about 10 to about 10,000 ppm, or from about 100 to about 1,000 ppm, or from about 1 to about 100 ppm, or from about 1,000 to about 10,000 ppm, or from about 10,000 to about 100,000 ppm. The concentration of a ine can be about 1; 2; 3; 4; 5; 6; 7; 8; 9; ; 15; 20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100; 125; 150; 175; 200; 225; 250; 275; 300; 325; 350; 375; 400; 425; 450; 475; 500; 525; 550; 575; 600; 625; 650; 675; 700; 725; 750; 775; 800; 825; 850; 875; 900; 925; 950; 975; 1000; 1500; 2000; 2500; 3000; 3500; 4000; 4500; 5000; 5500; 6000; 6500; 7000; 7500; 8000; 8500; 9000; 9500; ,000; 12,500; 15,000; 17,500; 20,000; 22,500; ; 27,500; 30,000; 32,500; 35,000; 37,500; 40,000; 42,500; 45,000; 47,500; 50,000; 52,500; 55,000; 57,500; 60,000; ; 65,000; 67,500; 70,000; 72,500; 75,000; 77,500; 80,000; ; 85,000; 87,500; ; 92,500; 95,000; 97,500; or about 100,000 ppm. Other concentrations amines can be useﬁal in the compositions and methods of the invention, depending in part on factors including the speciﬁc polyamine used, the presence of potentiating agents if any, or the species ofmicroorganisms that are targeted.

In certain aspects, increasing number of ine chains on the hydrophobic backbone systematically increase the antimicrobial activity. For example, it is trated herein that with a single chain of diaminopropane added to a benzyl backbone (CZ-4, ), the MIC (as deﬁned by the Clinical and Laboratory Standards Institute (CLSI)) against MRSA is greater than 1,200 ug/mL, whereas two chains decrease the MIC to 300 ug/mL (CZ-l2) and three chains reduce it further to 45 ug/mL (CZ-32). Similarly, with a single chain ofnorspermidine attached to a benzyl ne (CZ-7, ), the MIC t MRSA is r than 1,200 ug/mL. By adding a second chain of norspermidine (CZ-25), the MIC is 45 ug/mL. With a third chain of norspermidine attached (CZ-52), the MIC becomes 3 ug/mL ().

This trend of enhancing antimicrobial activity by increasing the number of polyamine chains attached to a backbone is effective for treating MRSA, but ting this trend with increased hydrophobicity enhances the ty of CZ compounds against A. baumanm‘i. Interestingly, CZ-52, with three norspermidine chains, has greater activity against MRSA bioﬁlms than against A. baumannii, whereas CZ-58 () has greater ty (10-fold increase) t A. baumanm’z’ bioﬁlms than MRSA s (see Table 4 below).

APPLICATIONS As described herein, bioﬁlms can also affect a wide variety of biological, medical, and processing ions. Methods and treatments using a ine compound, or a combination of a polyamine compound with another compound, may include killing, dispersing, treating, reducing bioﬁlms or preventing or inhibiting bioﬁlm formation.

In one embodiment, the invention provides a method for dispersing or killing a biofilm, the method sing a step of treating the bioﬁlm with an anti-biofllm composition, thereby effectively dispersing or killing the bioﬁlm; wherein the method comprises, consists essentially of, or consists of using a polyamine compound or composition as set forth in any of the embodiments or aspects described herein.

In some aspects, the step of treating the bioﬁlm with an anti-biofilm composition effectively disperses the bioﬁlm.

[0234] In another embodiment, the ion provides a method for inhibiting formation of a biofilm, the method comprising a step of treating planktonic bacteria with a polyamine composition as set forth in any of the ments or aspects herein, thereby inhibiting incorporation of the planktonic bacteria into the biofilm.

In n aspects, the method of killing, dispersing, dislodging, treating, or reducing biofilms, or preventing or inhibiting biofilm ion, includes contacting the bioﬁlm with an effective amount of a composition of the present invention.

In some aspects, the formation of a biofilm is inhibited. In other aspects, a previously formed biofilm is dispersed. In still other aspects, substantially all of the cells comprising a biofilm are killed.

In some embodiments, the invention provides a method of killing, sing, ng, or reducing biofilms, or preventing or inhibiting biofilm formation, the method comprising contacting a bioﬁlm or a surface having a bioﬁlm disposed thereon with an effective amount of a polyamine compound.

In some aspects, a surface comprises a medical device, a wound dressing, a contact lens, or an oral device. In some aspects, the medical device is ed from a clamp, forceps, scissors, skin hook, tubing, needle, retractor, scaler, drill, chisel, rasp, saw, catheter, edic , artiﬁcial heart valve, prosthetic joint, voice etic, stent, shunt, pacemaker, surgical pin, respirator, ventilator, and an endoscope and combinations thereof.

In some aspects, the method described herein comprises, ts essentially of, or consists of using the ine compound or composition described in any of the embodiments or aspects herein.

In some aspects, the invention provides a method that comprises, consists essentially of, or consists of using a ine compound or composition from any of the embodiments or aspects described herein.

In some embodiments, the invention provides a method for sing or killing a biofilm, the method comprising a step of treating the biofilm with an anti-biofilm composition, thereby effectively dispersing or killing the m; wherein the anti—biofilm composition comprises, consists essentially of, or consists of a polyamine compound selected from A2 A3 Ra Ra Ra Y 1&A5 1 A§89 L1 A9 1\ 4 5A\\89 \8 I I l l‘ I A A I21&(L 4 ls i i \E '7 A 2A A 2A A / :A A ,A \A3 \A3 \A6 \A3 L1 A6 \AB and a salt thereof; , , , , wherein: each R21 is a member independently selected from m R10 R1a 1b R181 1b EA< RZa R1d R28 z1J<R2b R2 sz N_kR R20 ( Rm%R2d R1 ( Rm%R2d m R3 m and R3 ; A1, A2, A3, A4, A5 , A6, A7, A8, and A9 are each an A11 member independently selected from N, CRa, and CR5; or, alternatively, a pair of adjacent AI1 members join to form an independently selected aryl, cycloalkyl, heterocyclyl, or heterocycloaryl ring that is fused with an A11 ring at the pair’s AI1 ring positions; wherein at least one AI1 member and at most ﬁve AI1 members are an independently selected CR3; each R13, Rlb, R”, and R1d is a member independently selected from hydrogen, ﬂuoro, alkyl, and ﬂuoroalkyl; or, alternatively, an R1a and an R1b join to form an oxo group; each Rza, sz, R2°, de, R26, and R2f is a member independently selected from hydrogen, alkyl, ﬂuoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; alternatively, a pair of R2 members from the same Ra group independently selected from the group R2a and R21), R20 and RN, and R2‘3 and n to form a member independently ed from spirocycloalkyl, eterocycyl, and oxo; or, alternatively, an R2a and an R2c from the same Ra group join to form a ring independently selected from lkyl and heterocycyl; each Rm is a member independently selected from -CR2aR2b-, 2d-, -C(R2a)=(R2b)-, -cc-, and -C(R23)(R2b)-L2-C(R2c)(R2d)-; each m is an integer independently selected from 1 to 20; each L1 and L2 is a member independently selected from a bond, —o—, -C(O)O-, -NR4-, -NR4C(O)-, and -C(O)NR4-; each R3 is a member independently ed from —Zl-R4, -Zl-Y1-R4, —zl—Y1—Y2—R4, and —zl—Y1—Y2—Y3—R4; each R4 is a member independently ed from hydrogen, alkyl, ﬂuoroalkyl, l, alkynyl, aryl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl, and heteroarylalkyl; or, atively, for an -N(R4)2 group, one of the two R4 in the group is a member selected from -(CO)OR63-, -(CO)N(R63)(R6b), and -C(NR63)N(R6b)(R6°); or, alternatively, for an - N(R4)2 group, the two R4 groups join to form a heterocyclic ring; each R5 is a member independently selected from hydrogen, alkyl, hydroxyl, alkoxy, lkoxy, alkylamino, alkylaminoalkoxy, alkenyl, alkynyl, aryl, aryloxy, arylamino, cycloalkyl, cycloalkoxy, cycloalkylalkoxy, cycloalkylamino, cycloalkylalkylamino, heterocyclyl, heterocycyloxy, heterocycylamino, halo, haloalkyl, ﬂuoroalkyloxy, heteroaryl, aryloxy, heteroarylamino, arylalkyl, arylalkyloxy, arylalkylamino, arylalkyl, heteroarylalkyloxy, heteroarylalkylamino, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; each Y1, Y2, and Y3 is an independently selected group of Formula IA: each Z1 and Z2 is a member independently ed from —N(R4)—and —O—; and each R621, R61), and R60 is a member independently selected from hydrogen, alkyl, fluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, arylalkyl, heteroarylalkyl, and cycloalkylalkyl; or, atively, two R6n members R621 and R6b or R621 and R60 join to form a heterocycyl ring; wherein the polyamine compound comprises at least two primary or secondary amino groups.

In some s, each R4 is a member independently ed from hydrogen, alkyl, ﬂuoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or, alternatively, for a -N(R4)2 group, one of the two R4 in the group is a member selected from -(CO)OR6a-, (CO)N(R“)(R""), and —C(I~IR“)N<R6")(R6°); each R5 is a member independently selected from hydrogen, alkyl, hydroxyl, , alkylamino, alkenyl, alkynyl, aryl, aryloxy, arylamino, cycloalkyl, cycloalkoxy, cycloalkylamino, heterocyclyl, heterocycyloxy, heterocycylamino, halo, haloalkyl, ﬂuoroalkyloxy, heteroaryl, heteroaryloxy, heteroarylamino, arylalkyl, arylalkyloxy, arylalkylamino, heteroarylalkyl, heteroarylalkyloxy, heteroarylalkylamino, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; and each R6a, R61), and R60 is a member independently selected from en, alkyl, fluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, cycloalkyl, and heteroarylalkyl; or, alternatively, two R611 members R621 and R6b or R621 and R60 form a heterocycyl ring.

In some aspects, the method has the proviso that the polyamine compound is not of Formula IB: R\N/\/\N4 NMN/R4 KL F'a4 F'z4 J) R4 R4 wherein the 2 groups are tertiary .

In some aspects, the method has the proviso that the polyamine compound is not: 4 4 i R4 R4 )2 ITI/Me Mew?l 1 0 Me Me IB-2 wherein the —R4 groups are hydrogen or methyl.

In some aspects, the method has the proviso that the polyamine compound is not of formula IC: RZ-HN-(CH2)p-NH-(CH2)q-NHCH2-Y-CHzNH-(CH2)s-NH-(CH2)t-NH-RZ (IC) or a salt thereof; wherein Y is selected from anthracenyl, naphthyl, and ; wherein each RZ is independently selected from hydrogen and alkyl; and wherein p, q, t, and s are each an r independently selected from 3 and 4. In some r aspects, this proviso is combined with one or more of the prior provisos.

In some aspects, the polyamine compound is selected from Ra R8 2\A3 2‘\A3 L1 A6 , ,and a salt thereof.

In some aspects, each Ra is an independently selected . In some s, all the Ra are the same group.

In some aspects, each A11 member is independently selected from CRa and CR5.

In some aspects, exactly one AI1 member is CRa. Alternatively, exactly two AI1 members are each an independently selected CRa. In some aspects, at most two AI1 members are each an independently selected CRa. Alternatively, exactly three All members are each an independently selected CRa. In some aspects, the AII members are all the same CRa.

In some aspects, each R13, Rlb, R“, and R1d is a member independently selected from hydrogen, ﬂuoro, alkyl, and ﬂuoroalkyl. In some aspects, each R”, Rlb, R”, and R1d is a member independently selected from hydrogen and alkyl. In some aspects, each R”, Rlb, RIC, and R1d is en.

In some aspects, each R23, R2b, RZC, R”, R2 and R2f is a member independently selected from hydrogen, alkyl, lkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, or arylalkyl. In some aspects, each R”, R21), RZC, RZd, R26, and R2f is a member independently selected from hydrogen, alkyl, and ﬂuoroalkyl. In some aspects, each Rza, R21), RZC, RZd, R23, and R2f is hydrogen.

In some aspects, each m is an integer independently selected from 1 to 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 1,12,13, 14,15, or 16). In some aspects, each m is an r independently selected from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). In some aspects, at least one m is an integer independently selected from 6 to 20 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some s, at least one m is an integer independently selected from 10 to 20 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some aspects, each m is 1. In some s, at least one m is 1. In some aspects, each m is 2.

In some aspects, at least one m is 2.

[0253] In some aspects, each L1 and L2 is a member independently selected from a bond, -O-, and -NR4-. In some aspects, each L1 and L2 is a member independently selected from a bond and -O-. In some aspects, each L1 and L2 is a bond.

In some aspects, each R3 is a member independently selected from -Zl-R4 and -Zl-Y1- R4. In some aspects, each R3 is an independently selected -Z1-Y1-R4. In some aspects, each R3 is an independently ed -Z1-R4.

In some aspects, each R4 is a member independently selected from hydrogen, alkyl, ﬂuoroalkyl, alkenyl, l, aryl, cycloalkyl, heteroaryl, arylalkyl, cycloalkylalkyl, and heteroarylalkyl. In some aspects, each R4 is a member independently ed from hydrogen, alkyl, ﬂuoroalkyl, l, l, arylalkyl, and cycloalkylalkyl. In some s, each R4 is a member independently selected from hydrogen, alkyl, arylalkyl, and cycloalkylalkyl. In some aspects, R4 is hydrogen, butyl, isobutyl, hexyl, or octyl.

[0256] In some aspects, at least one R4 is a member independently selected from alkyl, ﬂuoroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, arylalkyl, cycloalkylalkyl, and heteroarylalkyl. In some s, at least one R4 is a member independently selected from alkyl, arylalkyl, and cycloalkylalkyl. In some aspects, at least one R4 is an independently selected alkyl. In some aspects, at least one R4 is not hydrogen. In some aspects, at least one R4 is butyl, isobutyl, hexyl, or octyl.

In some aspects, at least one pair of R4 are both a member independently selected from alkyl, ﬂuoroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, arylalkyl, cycloalkylalkyl, and heteroarylalkyl. In some aspects, at least one pair of R4 are both a member independently selected from alkyl, arylalkyl, and cycloalkylalkyl. In some s, at least one pair of R4 are both an alkyl. In some aspects, at least one pair of R4 is not hydrogen. In some aspects, at least one pair of R4 is butyl, isobutyl, hexyl, or octyl.

In some aspects, each R5 is a member independently selected from hydrogen, alkyl, hydroxyl, alkoxy, aminoalkoxy, alkylamino, alkylaminoalkoxy, aryl, aryloxy, cycloalkyl, cycloalkoxy, cycloalkylalkoxy, halo, ﬂuoroalkyl, lkyloxy, heteroaryl, arylalkyl, arylalkyloxy, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl. In some aspects, each R5 is a member independently selected from hydrogen, alkyl, hydroxyl, alkoxy, aminoalkoxy, alkylaminoalkoxy, aryl, y, cycloalkylalkoxy, halo, ﬂuoroalkyl, ﬂuoroalkyloxy, arylalkyloxy, and hydroxyalkyl. In some aspects, each R5 is a member independently selected from hydrogen, alkyl, hydroxyl, , aryl, aryloxy, halo, ﬂuoroalkyl, and ﬂuoroalkyloxy. In some s, R5 is hydrogen.

In some aspects, at least one R5 is a member ndently ed from hydrogen, alkyl, hydroxyl, alkoxy, aminoalkoxy, alkylamino, alkylaminoalkoxy, aryl, aryloxy, cycloalkyl, cycloalkoxy, cycloalkylalkoxy, halo, ﬂuoroalkyl, ﬂuoroalkyloxy, heteroaryl, arylalkyl, arylalkyloxy, hydroxyalkyl, lkyl, and alkylaminoalkyl. In some aspects, at least one R5 is a member independently selected from hydrogen, alkyl, hydroxyl, alkoxy, aminoalkoxy, alkylaminoalkoxy, aryl, aryloxy, cycloalkylalkoxy, halo, ﬂuoroalkyl, fluoroalkyloxy, arylalkyloxy, and hydroxyalkyl. In some aspects, at least one R5 is a member independently selected from hydrogen, alkyl, yl, alkoxy, aryl, aryloxy, halo, ﬂuoroalkyl, and ﬂuoroalkyloxy.

[0260] In some aspects, each Z1 and Z2 is an ndently selected -N(R4)-.

In some aspects, each R621, R613, and R60 is a member ndently selected from hydrogen, alkyl, ﬂuoroalkyl, l, alkynyl, aryl, heteroaryl, cycloalkyl, kyl, heteroarylalkyl, and cycloalkylalkyl. In some aspects, each R63, R61), and R60 is a member independently selected from hydrogen, alkyl, fluoroalkyl, alkenyl, alkynyl, aryl, and arylalkyl. In some aspects, each R6a, R61), and R6C is a member independently selected from hydrogen and alkyl.

In some aspects, the polyamine compound comprises at least four primary or secondary amino groups. In some aspects, the polyamine compound comprises at least six primary or secondary amino groups.

[0263] In some s, each R81 is independently a group of Formula II: each of the An members is independently selected from CR3 and CR5; or, alternatively, a pair of adjacent AI1 members join to form an independently selected cycloalkyl, aryl, heterocyclyl, or heterocycloaryl ring that is fused with an A11 ring at the pair’s A11 ring positions; wherein at least one A11 member and at most three AI1 members are independently selected CRa, each R”, Rlb, R”, and R1d is a member independently selected from hydrogen, ﬂuoro, alkyl, and ﬂuoroalkyl; for each Ra member, at most two R”1 of the Ra member are selected from - C(R2a)=(R2b)-, -cc-, and )(R2b)-L2-C(R2c)(R2d)-; and each m is an integer independently selected from 1 to 16.

In some aspects, the polyamine compound is selected from: Ra R3 ﬁl J§A5 ﬁ1J§A4 A2 l4 l2 A ﬁS’A\\AB9iv \A3. \A3 L1 AG' and a salt thereof; wherein: at least one An member and at most three A11 members are independently selected CRa, each Rza, R21), R”, R”, R23, and R2f is a member independently selected from hydrogen, alkyl, ﬂuoroalkyl, alkenyl, alkynyl, aryl, aryl, arylalkyl, and heteroarylalkyl; alternatively, a pair of R211 members from the Ra group independently selected from the group R2a and R21), R20 and R”, and R2e and R2f join to form a ring independently selected from spirocycloalkyl and spiroheterocycyl; or, alternatively, the R221 and the R20 from the R3 group join to form a ring independently selected from cycloalkyl and heterocycyl; each m is an integer independently selected from 1 to 12; each R3 is a member independently selected from -Z-Y1-R4 and -Z-Y1-Y2-R4; and each Z1 and Z2 is an independently selected NR4.

[0265] In some aspects, A1, A2, A3, A4, A5, A6, A7, A8, and A9 are each an AI1 member independently selected from CRa, and CR5; or, alternatively, a pair of nt AIl members join to form a cycloalkyl, aryl, heterocyclyl, or cycloaryl ring; each Rza, R2b, RZC, RM, R26, and R2f is a member independently selected from hydrogen, alkyl, ﬂuoroalkyl, alkenyl, alkynyl, aryl, aryl, arylalkyl, or heteroarylalkyl; each m is an integer independently selected from 1 to 10; each L1 and L2 is a member independently selected from a bond, -O-, and -NR4-; each R3 is a member independently selected from -Z-R4, R4, and —Z-Y1-Y2-R4; and each Z1 and Z2 is an independently selected NR4.

[0266] In some s, the invention provides the polyamine compound selected from: Ra R8 ﬁ/K’F P11J§A4 5’A§Ae9 ' ' A\ 3—.A4 ‘l 2 A2 ,r —.A7 A \A3 L1 A6 and a salt thereof; , , wherein: each R"1 is independently a group of Formula V: N R20 R“: ( Rm%R2d m R3 each of the An members is independently selected from CRa and CR5; or, alternatively, a pair of adjacent AIl members join to form a cycloalkyl, aryl, heterocyclyl, or heterocycloaryl ring; wherein at least one A11 member and at most three A11 members are independently selected CRa, each Rla, Rlb, R10, and R1d is a member independently selected from hydrogen, ﬂuoro, alkyl, and ﬂuoroalkyl; for each Ra member, at most two R”1 of the Ra member are selected from — =(R2b)-, —cc—, and -C(Rza)(R2b)-L2-C(R2°)(R2d)-; and each R3 is a member independently selected from -Z-Y1-R4 and -Z-Y1-Y2-R4.

[0267] In some aspects, the polyamine compound comprises at least four primary or secondary amino . In some s, the polyamine compound comprises at least six y or ary amino groups.

In some aspects, the invention provides the polyamine compound selected from Ra Ra 1km 1kg) ﬁgs/L J1 A Ra, Ra A Ra, and a salt f; wherein each A11 member is an independently selected CR5.

In some aspects, the invention provides the polyamine compound selected from Ra Ra R5 and a salt thereof.

In some aspects, Ra is an independently selected group of Formula VII: / _\ H ( Rm)-\ m R3 VII.

In some further aspects, the polyamine compound is selected from Ra Ra R5 and a salt f.

In some aspects, R5 is hydroxyl, alkoxy, cycloalkoxy, or arylalkoxy.

In some aspects, Ra is —CH2[NH(CH2)n]pNH2; each n is an integer independently selected from 3 to 12; and each p is an integer independently selected from 1 to 3.

In some aspects, Rm is —CH2-. In some aspects, m is 1. In some aspects, R5 is selected from en, phenyl, and oxy.

In some aspects, the polyamine nd is selected from Ra Ra Ra and a salt thereof.

In some aspects, the polyamine compound is selected from: H2N[(H2C)3HN]2H2C\©/CH2[NH(CH2)3]2NH2 H2'\'[(H2C3)3HNl2HzC CHleH(CH2)3]2NH2 Ph H2N[(H2C3)3HN]2|‘|2C CH2[NH(CH2)3]2NH2 H2N[(HzC)3HN]2HzC CH2)3]2NH2 CHleH(CH2)3]2NH2 and a salt thereof.

In some aspects, the polyamine compound is selected from: H2N[(H20)3HN]2H20 CHleH(CH2)3]2NH2 H2N[(H20)3HN]2H2C CHleH(CH2)3]2NH2 OPh and a salt thereof.

In some aspects, the polyamine compound is selected from Ra Ra Ra R8 AN. ”A18 kA. 5J\A31J%A4 Re SA9 RaiaM A qu\,laiiL1A 5 & L, AARa and a salt thereof; wherein each Ar1 member is an independently selected CR5.

[0278] In some aspects, the polyamine nd is selected from L1 R8 L1 L1 Ra L1 R5 URE UP? ; :RS and a salt thereof.

In some aspects, Ra is an independently ed group of Formula V11: V11.

In some s, R5 is hydrogen. In some aspects, L1 is selected from a bond and O.

In some aspects, Rm is —CH2-. In some aspects, n1 is 1.

In some aspects, the polyamine compound is selected from H2N[(H20)3HN]2H20QCHleH(CH2)3]2NH2 HzN[(HzC)3HleHzCQCHleH(CH2)3]2NH2 H2N[(H20)3HN]2H20 CH2[NH(CH2)3]2NH2 CH2[NH(CH2)3]2NH2, H2N[(H20)3HN]2HZCMMCHleH(CH2)3]2NH2 CHleH(CH2)3]2NH2 l‘lzl‘l[(HzC)3l‘lN12H2C O CHleH(CH2)3]2NH2 CH2[NH(CH2)3]2NH2 OiPr H2N[(H20)3HN]2H2C O CHleH(CH2)3]2NH2 H2N[(H20)3HN]2H2C CH2[NH(CH2)3]2NH2, and a salt thereof.

In some aspects, Ra is —CH2[NH(CH2)n]pNH2; each n is an integer independently selected from 3 to 12; and each p is an integer ndently selected from 1 to 3. In some aspects, n is 3. In some aspects, n is 4. In some s, n is 5, 6, 7, 8, 9, 10, 11, or 12.

In some aspects, the polyamine compound is H H N N R‘MNJ? WVNHmNHR4 or a salt thereof.

In some aspects, the polyamine compound is H H R4HNJ9 7\/NH NHR4 HN O O or a salt thereof.

[0285] In some s, R4 is alkyl, cycloalkyl, or arylal 1. In some asPects, R4 is alkyl.

In some aspects, R4 is isobutyl.

In some embodiments, the polyamine compound or combination of a polyamine compound and at least one other composition may be used to treat Gram negative and Gram positive ia (including strains that are ant to conventional antibiotics), mycobacteria (including Mycobacterium tuberculosis), enveloped viruses, fungi and even transformed or cancerous cells.

In some embodiments, the polyamine compounds of the present invention are used for the treatment of cancer. The method comprises administering to a subject a composition sing a polyamine nd as described herein in an amount effective to reduce cancer cell eration or differentiation. In some embodiments, the cancer is breast cancer, a leukemia, or a melanoma. In some embodiments, the method includes administering to the subject an effective amount of a composition comprising a polyamine compound as bed herein and a chemotherapeutic agent. The polyamine compounds and compositions can be administered according to known methods. Such methods are described, for e, in International Patent Application No. PCT/U820 1 3/03 1 166, Which is incorporated herein by reference in its entirety.

The compounds, compositions, and methods described herein can be used to kill, disperse, treat, reduce biofilms, or prevent or inhibit biofilm formation. In exemplary methods, the biofilms are formed by biofilm-forming bacteria. The bacteria can be a gram- negative bacterial species or a ositive bacterial s. Nonlimiting examples of such bacteria include a member of the genus Actinobacillus (such as bacillus actinomycetemcomitans), a member of the genus Acinetobacter (such as Acinetobacter baumannii), a member of the genus Aeromonas, a member of the genus Bordetella (such as Bordetella pertussis, Bordetella bronchiseptica, or ella rtussis), a member of the genus Brevibacillus, a member of the genus Brucella, a member of the genus Bacteroides (such as Bacteroidesfragilis), a member of the genus Burkholderia (such as Burkholderia cepacia or Burkholderia pseudomallei), a member of the genus B0relia (such as Borelia burgdorferi), a member of the genus Bacillus (such as Bacillus anthracis or Bacillus is), a member of the genus Campylobacter (such as Campylobacterjejuni), a member of the genus Capnocytophaga, a member of the genus Cardiobacterium (such as Cardiobacterium s), a member of the genus Citrobacter, a member of the genus Clostridium (such as Cl0stridium tetani or Clostridium dijﬁcile), a member of the genus Chlamydia (such as Chlamydia trachomatis, Chlamydia pneumoniae, or Chlamydia psiffaci), a member of the genus Eikenella (such as Eikenella corrodens), a member of the genus Enterobacter, a member of the genus Escherichia (such as Escherichia coli), a member of the genus Francisella (such as Francisella tularensis), a member of the genus Fusobacterium, a member of the genus Flavobacterium, a member of the genus Haemophilus (such as Haemophilus ducreyi or Haemophilus inﬂuenzae), a member of the genus Helicobacter (such as bacter pylori), a member of the genus Kingella (such as Kingella kingae), a member ofthe genus ella (such as Klebsiella pneumoniae), a member of the genus ella (such as Legionella pneumophila), a member of the genus Listeria (such as Listeria monocytogenes), a member of the genus Leptospirae, a member of the genus lla (such as Moraxella catarrhalis), a member of the genus Morganella, a member of the genus Mycoplasma (such as Mycoplasma hominis or Mycoplasma pneumoniae), a member of the genus Mycobacterium (such as Mycobacterium tuberculosis or Mycobacterium leprae), a member ofthe genus Neisseria (such as Neisseria hoeae or Neisseria meningitidis), a member of the genus Pasteurella (such as Pasteurella multocida), a member of the genus Proteus (such as Proteus vulgaris or Proteus mirablis), a member of the genus Prevotella, a member ofthe genus monas (such as Plesiomonas shigelloia’es), a member of the genus Pseudomonas (such as Pseudomonas aeruginosa), a member of the genus Providencia, a member of the genus tsia (such as Rickettsia rickettsii or Rickettsia typhi), a member of the genus Stenotrophomonas (such as Stenotrophomonas maltophila), a member of the genus Staphylococcus (such as Staphylococcus aureus or Staphylococcus epidermidis), a member of the genus Streptococcus (such as Streptococcus viria’ans, Streptococcus pyogenes (group A), Streptococcus agalactiae (group B), Streptococcus bovis, or Streptococcus pneumoniae), a member of the genus Streptomyces (such as Streptomyces hygroscopicus), a member of the genus Salmonella (such as Salmonella a’itis, Salmonella typhi, or Salmonella typhimurium), a member of the genus Serratia (such as Serratia marcescens), a member of the genus Shigella, a member of the genus Spirillum (such as Spirillum minus), a member of the genus Treponema (such as Treponema pallia’um), a member of the genus Veillonella, a member ofthe genus Vibrio (such as Vibrio cholerae, Vibrio emolyticus, or Vibrio cus), a member of the genus Yersinia (such as Yersinia enter ocolitica, Yersinia pestis, or Yersinia pseudotuberculosis), and a member of the genus Xanthomonas (such as Xanthomonas maltophilia).

In some embodiments, the bioﬁlm exposed to the compounds, compositions, or methods of the present ion may comprise Gram-negative or Gram-positive bacteria. In some embodiments, the bacteria are mycobacteria.

In some aspects, the bioﬁlm comprises an antibiotic-resistant bacterial species.

[0291] The antimicrobial compounds, compositions, and methods comprising a polyamine compound may be used to l, prevent or kill bioﬁlms in various environments. In some ments, they may be used for treating bioﬁlms in subjects that include human or other animals. In some ments, they may be used for treating biofilms in medical applications such as medical devices, wound ngs, contact lens, oral devices, etc. In some embodiments, they may be used for treating or preventing a biofilm-related disorder. In some embodiments, they may be used for treating bioﬁlms in industrial applications such as oil pipelines, water pipelines, water treatment at manufacturing sites, industrial ﬂush solution, industrial wash water, industrial coatings, etc. In some embodiments, they may be used for household and hygiene applications. In some embodiments, they may be used for agricultural applications, such as water remediation, crop ent, etc. In some embodiments, they may be used for food preparation applications, such as meat sprays, fruit and vegetable sanitizers.

In some aspects, the method comprises a step of coating an object with the anti- biofilm ition. In some aspects, the method ses a step of treating a contact lens with the anti-biofilm composition.

In some embodiments, the polyamine compound or combination of a ine nd and at least one other composition are directed for use in rial ations, for example oil pipelines, water treatment, water pipelines, fracking water sanitation, milk production facility pipeline ﬂush solution, oil ﬁelds, paper and pulp production, ing fluids, ship coatings, shipping, paint, il sanitizers, water filtration, biofouling and biocorrosion, natural gas pipeline treatment, HVAC units, etc.

[0294] In some embodiments, the polyamine compound or combination of a ine nd and at least one other composition are directed for use in household applications, for example, sanitizing wipes, cleansers, toilet bowl inserts, baby care products, toys, etc.

In some embodiments, the polyamine compound or combination of a polyamine compound and at least one other composition are directed for use in environmental applications, for example, agriculture, water remediation, water treatment, crop treatment, etc.

In some aspects, the method comprises a step of treating a pipe with the anti-bioﬁlm composition. In some aspects, the method comprises a step of treating a heating or cooling tower with the anti-bioﬁlm composition.

[0297] In some embodiments, the polyamine compound or combination of a polyamine compound and at least one other composition are directed for use in food production, for example, fruit and vegetable sanitizers, water systems in food tion facilities, meat sprays, cooling system sanitizers, air ion units, feed, packaging, etc.

In some aspects, the iofilm composition is a paint.

In some aspects, the method comprises a step of treating a patient with a biofilm- related disorder.

Some s of this disclosure is directed to s of treating a biofilm-related disorder in a subject in need thereof, the method comprising administering to the t an effective amount of a polyamine compound of the present invention.

In some embodiments, the ition is administered to a surface of the t selected from the group of dermal and mucosal surfaces and combinations thereof. In other embodiments, the surface is an oral surface, a skin surface, a urinary tract surface, a vaginal tract surface, or a lung surface.

In some embodiments, the composition is administered to the subject via aneous, muscular, intra-peritoneal, intravenous, oral, nasal, or topical administration, and a combination thereof.

In some aspects, a subject is treated. A subject can be a mammal including, but not limited to, a e (e.g., a monkey, such as a cynomolgous monkey, a chimpanzee, and a human). A subject can be a non-human animal such as a bird (e.g., a quail, n, or turkey), a farm animal (e.g., a cow, goat, horse, pig, or sheep), a pet (e.g., a cat, dog, or guinea pig, rat, or mouse), or laboratory animal (e.g., an animal model for a disorder). Non— limiting representative subjects can be a human infant, a pre-adolescent child, an adolescent, an adult, or a senior/elderly adult.

In some embodiments, the subject is a human.

In some instances, a subject in need of treatment can be one afﬂicted with one or more of the infections or disorders described herein. In some aspects, the subject is at risk of developing a biofilm on or in a biologically relevant surface, or already has developed such a biofilm. Such a subject at risk can be a candidate for treatment with a polyamine compound, or combination of a polyamine compound with another compound, in order to inhibit the development or onset of a bioﬁlm—production—related disorder/condition or prevent the 3O recurrence, onset, or development of one or more ms of a biofilm-related disorder or condition. Such a subject can be harboring an re bioﬁlm that is clinically evident or detectable to the skilled artisan, but that has not yet fully formed. A subject at risk of developing a bioﬁlm can also be one in which implantation of an indwelling device, such as a medical device, is scheduled. The risk of developing a bioﬁlm can also be due to a propensity of developing a bioﬁlm—related disease (such as the presence of a channel transporter mutation associated with cystic ﬁbrosis). In such subjects, a bioﬁlm-related disorder can be at an early stage, e.g. no bacterial infection or bioﬁlm formation is yet detected.

In certain ments a bioﬁlm—related disorder is selected from pneumonia, cystic ﬁbrosis, otitis media, chronic obstructive pulmonary disease, and a urinary tract infection and ations thereof. In other embodiments, the bioﬁlm-related disorder is a medical device-related infection. In further embodiments, the bioﬁlm-related er is a periodontal disease, such as gingivitis, periodontitis or breath malodor. In still further ments, the bioﬁlm—related disorder is caused by bacteria. In some embodiments, the bacteria are egative or Gram-positive bacteria. In still other embodiments, the ia are of the genus Actinobacillus, Acinetobacter, Aeromonas, Bordetella, Brevibacillus, Brucella, Bacteroia'es, Burkhola'eria, Borelia, Bacillus, Campylobacter, Capnocytophaga, Cardiobacterium, Citrobacter, Clostridium, Chlamydia, Eikenella, Enterobacter, Escherichia, acter, Francisella, Fusobacterium, acterium, Haemophilus, Helicobacter, Kingella, Klebsiella, Legionella, Listeria, Leptospirae, Moraxella, Morganella, Mycoplasma, Mycobacterium, Neisseria, Pasteurella, Proteus, Prevotella, Plesiomonas, Pseudomonas, Providencia, tsia, Stenotrophomonas, Staphylococcus, Streptococcus, Streptomyces, Salmonella, Serratia, Shigella, Spirillum, Treponema, nella, Vibrio, Yersinia, or Xanthomonas.

[0307] miting examples of bioﬁlm-related disorders include otitis media, prostatitis, cystitis, iectasis, bacterial rditis, osteomyelitis, dental caries, periodontal disease, infectious kidney stones, acne, Legionnaire's disease, chronic obstructive pulmonary disease (COPD), and cystic s. In one c example, subjects with cystic ﬁbrosis display an accumulation of bioﬁlm in the lungs and digestive tract. Subjects afﬂicted with COPD, such as emphysema and chronic bronchitis, display a characteristic inﬂammation of the s wherein airﬂow through such airways, and subsequently out of the lungs, is chronically obstructed. m-related disorders can also ass infections derived from ted/inserted devices, medical device-related infections, such as infections from biliary stents, orthopedic t infections, and catheter-related infections (kidney, vascular, peritoneal). An infection can also originate from sites where the integrity of the skin or soft tissue has been mised. Non-limiting examples e dermatitis, ulcers from peripheral vascular disease, a burn injury, and trauma. For example, a Gram-positive bacterium, such as S. pneumoniae, can cause opportunistic infections in such tissues. The ability of S. pneumoniae to infect burn wound sites, e. g., is enhanced due to the breakdown of the skin, burn-related immune defects, and antibiotic selection.

[0309] In yet other embodiments, a biofilm-related disorder is pneumonia, cystic ﬁbrosis, otitis media, chronic obstructive pulmonary disease, or a urinary tract infection. In some embodiments, the biofllm-related er is a l device-related infection.

In other aspects, this disclosure features compounds, compositions, or methods, such as industrial, therapeutic or pharmaceutical compositions, comprising polyamine compounds in combination with one or more additional active compositions.

In some instances a polyamine compound can be administered alone or in combination with a second agent, 6.g. a biocide, an antibiotic, or an antimicrobial agent, to thereby kill, disperse, treat, reduce prevent, or inhibit bacterial bioﬁlms. An antibiotic can be co-administered with the polyamine compound either sequentially or simultaneously.

[0312] The otic can be any compound known to one of ordinary skill in the art that can inhibit the growth of, or kill, bacteria. UseﬁJl, non-limiting examples of antibiotics include lincosamides (clindomycin); chloramphenicols; tetracyclines (such as tetracycline, chlortetracycline, demeclocycline, methacycline, cline, minocycline); aminoglycosides (such as gentamicin, tobramycin, netilmicin, smikacin, kanamycin, streptomycin, neomycin); beta-lactams (such as penicillins, osporins, em, aztreonam); glycopeptide antibiotics (such as vancomycin); polypeptide antibiotics (such as bacitracin); macrolides (erythromycins), ericins; sulfonamides (such as sulfanilamide, sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole, sulfacytine, sulfadoxine, mafenide, p-aminobenzoic acid, hoprim-sulfamethoxazole); methenamin; nitroﬁJrantoin; phenazopyridine; hoprim; rifampicins; metronidazoles; cefazolins; lincomycin; spectinomycin; mupirocins; ones (such as nalidixic acid, cinoxacin, norﬂoxacin, ciproﬂoxacin, acin, oﬂoxacin, enoxacin, ﬂeroxacin, levoﬂoxacin); novobiocins; polymixins; gramicidins; and antipseudomonals (such as carbenicillin, carbenicillin l, ticarcillin, azlocillin, mezlocillin, piperacillin) or any salts or variants thereof. Such antibiotics are commercially available, e.g., from Daiichi Sankyo, Inc.

(Parsipanny, NJ), Merck (Whitehouse Station, NJ), Pﬁzer (New York, NY), Glaxo Smith Kline (Research Triangle Park, NC), Johnson & Johnson (New Brunswick, NJ), AstraZeneca (Wilmington, DE), Novartis (East Hanover, NJ), and Sanofi-Aventis (Bridgewater, NJ). The antibiotic used will depend on the type of bacterial infection.

Additional known biocides include biguanide, chlorhexidine, triclosan, chlorine e, and the like.

[0314] Useful examples of antimicrobial agents include, but are not limited to, Pyrithiones, especially the zinc complex (ZPT); Octopirox®; dimethyldimethylol hydantoin (Glydant®); methylchloroisothiazolinone/methylisothiazolinone (Kathon CG®); sodium e; sodium ite; imidazolidinyl urea (Germall llS®), diazolidinyl urea (Germaill II®); benzyl alcohol; 2-bromonitropropane-l,3-diol (Bronopol®); formalin (formaldehyde); iodopropenyl arbamate (Polyphase PI 00®); chloroacetamide; methanamine; methyldibromonitrile glutaronitrile (1,2-dibromo—2,4-dicyanobutane or Tektamer®); glutaraldehyde; 5-bromonitro-l,3-dioxane (Bronidox®); phenethyl alcohol; 0- phenylphenol/sodium 0-phenylphenol; sodium hydroxymethylglycinate (Suttocide A®); polymethoxy bicyclic oxazolidine pt C®); dimethoxane; thimersal; dichlorobenzyl alcohol; captan; chlorphenenesin; dichlorophene; chlorbutanol; glyceryl laurate; halogenated diphenyl ethers; 2,4,4'-trichloro-2'-hydroxy-diphenyl ether (Triclosan®. or TCS); 2,2'- dihydroxy—S,5'—dibromo-diphenyl ether; phenolic compounds; phenol; 2-methylphenol; 3- methylphenol; 4-methylphenol; 4-ethylphenol; methylphenol; 2,5-dimethylphenol; 3,4- dimethylphenol; 2,6-dimethylphenol; opylphenol; 4-n-butylphenol; ylphenol; 4- tert-amylphenol; 4—n—hexylphenol; 4—n—heptylphenol; mono- and poly—alkyl and ic halophenols; p-chlorophenol; methyl p-chlorophenol; ethyl p-chlorophenol; n-propyl p- chlorophenol; n-butyl p-chlorophenol; n-amyl p-chlorophenol; sec-amyl p-chlorophenol; cyclohexyl p-chlorophenol; yl p—chlorophenol; n-octyl p—chlorophenol; rophenol; methyl o-chlorophenol; ethyl o-chlorophenol; n-propyl o-chlorophenol; n—butyl o- chlorophenol; n-amyl rophenol; tert-amyl o-chlorophenol; l o-chlorophenol; n- heptyl o-chlorophenol; yl p-chlorophenol; o—benxyl-m-methyl p-chlorophenol; o- benzyl-m,m-dimethyl-p-chlorophenol; o-phenylethyl-p-chlorophenol; o-phenylethyl-m- methyl p-chlorophenol; 3-methyl p-chlorophenol; 3,5-dimethyl p-chlorophenol; 6-ethyl-3 - methyl rophenol; 6-n—propylmethyl-p-chlorophenol; 6-isopropylmethyl-p- chlorophenol; 2-ethyl-3,5-dimethyl p-chlorophenol; 6-sec-butylmethyl p-chlorophenol; 2- isopropyl-3, 5 -dimethyl p-chlorophenol; 6-diethylmethyl-3 -methyl p-chlorophenol; 6- pylethylmethyl p-chlorophenol; 2-sec-amyl-3, 5-dimethyl p-chlorophenol; 2- diethylmethyl-3, 5 hyl p-chlorophenol; 6-sec-octylmethyl p-chlorophenol; p-chloro- m-cresol: p-bromophenol; methyl p-bromophenol; ethyl p-bromophenol; n-propyl p- bromophenol; n-butyl p-bromophenol; n-amyl p-bromophenol; sec-amyl p-bromophenol; n- hexyl p-bromophenol; cyclohexyl p-bromophenol; o-bromophenol; tert-amyl o-bromophenol; n-hexyl o-bromophenol; n-propyl-m,m-dimethyl-o-bromophenol; 2-phenylphenol; 4-chloro- 2-methylphenol; 4-chloromethyl phenol; 4-chloro-3,5-dimethyl phenol; chloro-3,5- dimethylphenol; 6-tetrabromo—2-methyl—phenol; 5-methyl—2—pentylphenol; 4-isopropyl— 3-methylphenol; p-chloro-m-xylenol (PCMX); chlorothymol; phenoxyethanol; phenoxyisopropanol; 5-chlorohydroxydiphenylmethane; resorcinol and its derivatives; resorcinol; methyl inol; ethyl resorcinol; n—propyl resorcinol; n-butyl resorcinol; n-amyl resorcinol; n-hexyl resorcinol; n-heptyl resorcinol; n-octyl resorcinol; l resorcinol; phenyl resorcinol; benzyl resorcinol; phenylethyl resorcinol; phenylpropyl resorcinol; p- chlorobenzyl resorcinol; 5-chloro 2,4-dihydroxydiphenyl methane; 4'-chloro 2,4- dihydroxydiphenyl methane; o hydroxydiphenyl methane; 4'-bromo 2,4- dihydroxydiphenyl methane; bisphenolic compounds; 2,2'-methylene bis-(4-chlorophenol); 2,2‘—methylene bis-(3,4,6-trichlorophenol); ethylene bis(4-chlorobromophenol); bis(2-hydroxy-3,5-dichlorophenyl)sulﬁde; bis(2-hydroxychlorobenzyl)sulﬁde; benzoic esters (parabens); methylparaben; propylparaben; araben; ethylparaben; pylparaben; isobutylparaben; benzylparaben; sodium methylparaben; sodium propylparaben; halogenated carbanilides; 3,4,4'-trichlorocarbanilides (e.g., Triclocarban® or TCC); 3-triﬂuoromethyl-4,4'-dichlorocarbanilide; 3,3',4-trichlorocarbanilide; chlorohexidine and its digluconate; diacetate and dihydrochloride; undecenoic acid; thiabendazole, hexetidine; and poly(hexamethylenebiguanide) hloride (Cosmocil®).

In some embodiments of any methods described herein, the method further comprises administering a biocide. In some ments, the biocide is an antibiotic.

[0316] In instances Where a polyamine compound, or combination of a polyamine compound with another compound, is to be administered to a subject, the compound or composition herein can be incorporated into pharmaceutical compositions. The ine compound, or combination of a polyamine compound with another compound, can be incorporated into pharmaceutical compositions as pharmaceutically acceptable salts or derivatives. Some pharmaceutically acceptable derivatives of the ine compounds of the present invention may include a chemical group, which increases aqueous solubility. As used herein, a “pharmaceutically acceptable carrier” means a carrier that can be administered to a subject together with a polyamine compound, or combination of a polyamine compound with another compound, bed herein, which does not destroy the pharmacological activity thereof. ceutically acceptable rs include, for example, ts, binders, dispersion media, coatings, preservatives, colorants, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

Non-limiting examples ofpharmaceutically able carriers that can be used e poly(ethylene-co-Vinyl acetate), PVA, partially hydrolyzed poly(ethylene-co-vinyl acetate), poly(ethylene-co-Vinyl acetate-co-vinyl alcohol), a cross-linked poly(ethylene-co- Vinyl acetate), a cross-linked partially hydrolyzed poly(ethylene-co-vinyl acetate), a cross- linked poly(ethylene—co-vinyl acetate-co—vinyl alcohol), poly-D,L-lactic acid, poly—L-lactic acid, polyglycolic acid, PGA, copolymers of lactic acid and glycolic acid (PLGA), polycaprolactone, polyvalerolactone, poly (anhydrides), copolymers of polycaprolactone with polyethylene glycol, copolymers of polylactic acid with hylene , polyethylene glycol; and combinations and blends thereof.

[0318] Other carriers include, e. g., an aqueous gelatin, an aqueous n, a polymeric carrier, a cross-linking agent, or a combination thereof. In other instances, the carrier is a matrix. In yet another ces, the r es water, a pharmaceutically acceptable buffer salt, a pharmaceutically acceptable buffer solution, a pharmaceutically acceptable antioxidant, ascorbic acid, one or more low molecular weight pharmaceutically acceptable ptides, a e comprising about 2 to about 10 amino acid residues, one or more pharmaceutically able proteins, one or more pharmaceutically acceptable amino acids, an essential-to-human amino acid, one or more pharmaceutically acceptable carbohydrates, one or more pharmaceutically acceptable carbohydrate-derived materials, a non—reducing sugar, glucose, sucrose, sorbitol, trehalose, mannitol, maltodextrin, dextrins, cyclodextrin, a pharmaceutically acceptable chelating agent, EDTA, DTP A, a chelating agent for a divalent metal ion, a chelating agent for a trivalent metal ion, glutathione, pharmaceutically acceptable nonspeciﬁc serum albumin, or combinations thereof.

In other embodiments, the compositions can also comprise a pharmaceutically acceptable carrier. In still other embodiments the effective amount is an amount effective to treat or prevent a biofilm-related disorder. In some ments, an effective amount comprises and amount effective to treat or prevent a m on a surface.

In some embodiments, the compositions discussed herein further comprises an agent suitable for application to the surface. In other embodiments, the composition is formulated as a wash solution, a dressing, a wound gel, or a synthetic tissue. In further ments, the composition is formulated as tablets, pills, troches, capsules, aerosol spray, solutions, suspensions, gels, pastes, creams, or foams. In some embodiments, the composition is formulated for parenteral (e.g., intravenous), intradermal, aneous, oral (e.g. inhalation), transdermal (topical), transmucosal, vaginal, or rectal administration.

Another aspect of this disclosure is directed to biofilm resistant medical devices, comprising a surface likely to contact a biological ﬂuid and a ine compound. In some embodiments, the medical device further comprises a polyamine compound, or combinations of a polyamine compound and at least one other composition, that is coated on or impregnated into said surface.

In some embodiments, the polyamine compound or combination of a polyamine compound and at least one other composition is formulated as a slow-release formulation.

In some embodiments, the polyamine compound or combination of a polyamine compound and at least one other composition are directed for use in l applications, for example, active release or passive antimicrobial gs for medical devices, lavage solutions for open wounds, oral mouthwashes, toothpaste additives, hand sanitizers, systemic prophylactic antibiotics, lock solutions for catheters, eye drop ons for tion and contact lens cleaners, prophylactic dental inserts, high level ectants, gastrointestinal (GI) tract oral medications for the treatment of ions such as those caused by Shigella, Cryptosporz'dz'um, Vibrio cholerae, or Clostrz'dz'um dz'jj‘icz'le, cancer treatment including multiple myeloma, osteosarcoma, lymphoma or other forms of cancer, topical ointrnents to treat dermatological cations including infection, canker sores, psoriasis, , chronic wounds, diaper rash, mycosis (athletes foot), tinea unguium (toenail fungus), ulcers, or acne, etc.

In some embodiments, the base is selected from a , gel, paste, or powder. In further embodiments, the ition is selected from shampoos, bath additives, hair care preparations, soaps, s, , ants, skin-care ations, cosmetic personal care preparations, intimate hygiene preparations, foot care preparations, light protective preparations, skin tanning ations, insect ants, antiperspirants, shaving preparations, hair removal preparations, fragrance preparations, dental care, denture care and mouth care preparations and combinations thereof.

A pharmaceutical composition containing a polyamine compound, or combination of a polyamine compound with another compound, can be formulated to be compatible with its intended route of administration as known by those of ry skill in the art. Nonlimiting examples of routes of administration include parenteral, e. g., enous, intradermal, subcutaneous, oral {e.g., inhalation), transdermal (topical), transmucosal, vaginal and rectal administration. ons or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for ion, saline solution, ﬁxed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable es or multiple dose Vials made of glass or plastic.

Pharmaceutical compositions suitable for able use e sterile aqueous solutions (where water —soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile inj ectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, pany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition can be e and can be ﬂuid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved t the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, ol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.

The proper ﬂuidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for e, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. It may be desirable to include isotonic agents, for example, sugars, polyalcohols such as ol, sorbitol, or sodium chloride in the composition.

Prolonged absorption of the injectable compositions can be accomplished by including in the ition an agent that delays absorption, for example, um monostearate and gelatin (see, e. g., Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Gennaro, ed. (2006)).

Sterile inj e solutions can be prepared by incorporating a polyamine compound, or combination of a polyamine compound with r compound, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by ﬁltered ization. lly, sions are prepared by incorporating an active compound into a sterile vehicle that contains a basic sion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile inj ectable solutions, the methods of preparation include, without limitation, vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-ﬁltered solution f.

Oral compositions may e an inert t or an edible carrier or binders. For the purpose of oral therapeutic administration, a polyamine, or a combination of a polyamine compound, or combination of a polyamine compound with another compound, can be incorporated with excipients and used in the form of tablets, pills, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be ed using a ﬂuid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, or adjuvant materials can be ed as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a t such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a ﬂavoring agent such as peppermint, methyl late, or orange ﬂavoring.

For administration by inhalation, polyamine compound, or combination of a polyamine compound with another compound, can be delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g. a gas such as carbon dioxide, or a nebulizer.

Systemic stration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, but are not limited to, for example, for ucosal administration, detergents, bile salts, and c acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds and compositions are formulated into pharmaceutically acceptable formulation embodiments, such as ointments, salves, gels, or creams as generally known in the art.

For treatment of acute or chronic wounds, polyamine compound, or combination of a polyamine compound with another compound, can be formulated as a dressing, a wash solution, gel, or a tic tissue, etc.

The pharmaceutical compositions containing a polyamine compound, or combination of a polyamine compound with r compound, can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

Some pharmaceutical compositions containing a polyamine compound, or combination of a ine compound with another compound, can be ed with a carrier that protects the polyamine compound, or combination of a polyamine compound with another compound, against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems as described, e.g., in Tan et al., Pharm. Res. 24:2297-2308 (2007).

Additionally, biodegradable, biocompatible polymers can be used, such as ne vinyl acetate, polyanhydrides, ycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations are apparent to those skilled in the art.

The materials can also be obtained commercially (e.g., from Alza Corp, Mountain View, Calif). Liposomal suspensions (including liposomes ed to particular cells with onal antibodies to cell e antigens) can also be used as pharmaceutically acceptable rs. These can be ed according to s known to those skilled in the art, e.g., as described in US. Pat. No. 4,522,811.

Toxicity and therapeutic efﬁcacy of such compounds and compositions can be determined by rd pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50%> of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDSQ/EDso.

While compounds and compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets active components to the site of affected tissue in order to minimize potential damage to normal cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in ating a range of dosage for use in humans. The dosage of such compounds and compositions lies lly within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compounds or compositions used in the methods described herein, the therapeutically ive dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that es the ICso (i.e., the concentration of the test compound or ition that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for e, by high mance liquid chromatography. ation for preparing and testing such compositions are known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, o, ed. (2006).

A physician will appreciate that certain factors may ce the dosage required to effectively treat a subject, including but not limited to the severity of the e or disorder, previous treatments, the general health or age of the subject, and other diseases present.

Moreover, treatment of a t with a therapeutically effective amount of a polyamine compound, or combination of a polyamine compound with another compound, can include a single ent or a series of treatments.

The compounds or pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. A person of ordinary skill in the art will appreciate that the compounds or pharmaceutical compositions described herein can be formulated as single—dose vials.

Polyamine compounds, or combination of a polyamine nd with another compound, may be le as antibiofilm active substances in personal care preparations, for example shampoos, bath additives, hair care preparations, liquid and solid soaps (based on synthetic surfactants and salts of saturated or rated fatty acids), lotions and creams, deodorants, other aqueous or alcoholic solutions, e. g. cleansing ons for the skin, moist cleaning cloths, oils or powders.

Any suitable amount ofpolyamine can be used in the compositions and methods of the invention. In general, the polyamines are used in concentrations ranging from about 1 ppm to about 100,000 ppm, or higher. The concentration of a polyamine used in a composition or method of the invention can be, for example, from about 1 to about 100,000 ppm, or from about 10 to about 10,000 ppm, or from about 100 to about 1,000 ppm, or from about 1 to about 100 ppm, or from about 1,000 to about 10,000 ppm, or from about 10,000 to about 100,000 ppm. The concentration ofa polyamine can be about 1; 2; 3; 4; 5; 6; 7; 8; 9; ; 15; 20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100; 125; 150; 175; 200; 225; 250; 275; 300; 325; 350; 375; 400; 425; 450; 475; 500; 525; 550; 575; 600; 625; 650; 675; 700; 725; 750; 775; 800; 825; 850; 875; 900; 925; 950; 975; 1000; 1500; 2000; 2500; 3000; 3500; 4000; 4500; 5000; 5500; 6000; 6500; 7000; 7500; 8000; 8500; 9000; 9500; ,000; 12,500; 15,000; 17,500; 20,000; 22,500; 25,000; 27,500; 30,000; 32,500; ; 37,500; 40,000; 42,500; 45,000; 47,500; ; ; 55,000; 57,500; 60,000; 62,500; 65,000; 67,500; 70,000; 72,500; 75,000; 77,500; 80,000; 82,500; 85,000; 87,500; 90,000; 92,500; 95,000; 97,500; or about 0 ppm. Other concentrations amines can be useﬁJl in the compositions and methods of the invention, depending in part on factors including the speciﬁc polyamine used, the presence of other active agents if any, or the species of microorganisms that are targeted.

[0341] There is thus disclosed compounds, compositions, or methods comprising novel polyamine compounds, or combinations of polyamine compounds with other compounds, that have antimicrobial activity and dispersing activity against a variety of bacterial strains capable of forming bioﬁlms, and methods of using the same.

EXAMPLES

[0342] The ing es serve to explain embodiments of the present disclosure in more detail. These examples should not be construed as being exhaustive or exclusive as to the scope of this disclosure.

The following material and methods were used.

Bacterial strains. A clinical strain of MRSA, isolated from a patient who underwent arthroscopic knee surgery and characterized by ARUP Laboratories, Salt Lake City, UT, was used for this study in addition to Pseudomonas aeruginosa ATCC 27853 and Alcanivorax ensz's ATCC 700651. Both ATCC strains were purchased as -dried pellets from ATCC. P. aeruginosa was ended in BHI broth, grown overnight at 37° C and transferred to fresh BHI with 30% glycerol for storage at -80° C. The MRSA isolate was likewise stored in BHI with 30% glycerol at -80° C. Notably, the clinical MRSA isolate was not passaged more than three times prior to or during the study. Prior to ming MIC analysis and bioﬁlm experiments, the frozen stocks ofMRSA and P. aeruginosa were streaked onto Columbia blood agar plates and grown ght at 37° C. A. borkumensz's ATCC 70065] was resuspended from a lyophilized pellet into marine broth, grown overnight at 30° C and passaged on marine agar plates prior to experimentation.

Example 1

[0345] MIC Determination. To determine the MIC of the ine chains alone and the synthesized compounds against MRSA and P. aeruginosa ATCC 27853, a modiﬁed protocol from the Clinical and Laboratory Standards Institute (CLSI) guideline M26-A was used.

Consistent with this guideline, the MIC was deﬁned as the tration of crobial that was required to reduce approximately 5 x 105 cells per mL to approximately 5 x 102 per mL in a 24-hour period. Each MIC ment was performed n=10 times for each antimicrobial.

All data was collected using cation adjusted MHB for standardization.

As mentioned, MRSA and P. aeruginosa ATCC 27853 were grown on ia blood agar plates prior to experimentation. From the blood agar plates, a 0.5 McFarland standard was made of each isolate. This equated to approximately 5 x 107 cells/mL for the MRSA isolate and approximately 3 x 107 cells/mL for the P. aeruginosa ATCC 27853 isolate. Of the 0.5 McFarland stock solution, 50 [LL were added to MHB. All of the antimicrobials were kept in PBS stock solutions at 10 mg/mL and diluted in the MHB to reach the desired concentration that would determine the MIC value. All tests were performed such that a ﬁnal volume of 5 mL ofMHB were obtained. This provided a ﬁnal bacterial concentration of approximately 5 x 105 cells/mL.

Each sample was incubated in glass test tubes at 37° C for 24 hours, then plated in duplicate on tryptic soy agar (TSA) using a 10-fold dilution series. TSA plates were further ted 24 hours at 37° C. The number of colony forming units (CFU) that grew were counted in order to calculate the number of bacteria that were present per mL in the original MHB solution. The concentration of compound that ed in a reduction of approximately x 105 cells/mL to approximately 5 x 102 cells/mL in a 24-hour period was deﬁned as the MIC.

For ison to a current standard of care, the MICs of vancomycin against the MRSA isolate and tobramycin against the P. aeruginosa ATCC 27853 isolate were also determined. MIC results for various compounds are shown in MRSA and P. aeruginosa Bioﬁlm Eradication. Bioﬁlms ofMRSA and P. aeruginosa ATCC 27853 were grown on the surface of PEEK membranes using a ne bioﬁlm reactor. Data demonstrating the design and repeatability of this reactor have been published previously. Williams et al., In Vivo Efficacy ofa Silicone - Cationic Steroid Antimicrobial g to Prevent Implant-Related Infection, erials 33, 8641—8656 ; Williams et al., Use in plastic in a modified CDC biofilm reactor, Res J Microbiol 6, 425-429 ; Williams et al, Experimental model ofbiofilm implant-related osteomyelitis to test combination biomaterials using biofilms as initial inocula, J Biomed Mat Res A 100, 1888-1900 (2012); Williams et al., A modified CDC biofilm reactor to e mature biofilms on the surface ofPEEK membranesfor an in vivo animal model application, Curr Microbiol 62, 1657—1663 (2011). Prior to g the bioﬁlms, PEEK membranes were ﬁrst sonicated for 10 min in medical grade detergent, rinsed with running reverse osmosis water for 10 min, sonicated in reverse osmosis water for 10 min and rinsed once again using 70% ethanol.

Four guillotine—like holders were designed to hold eight PEEK membranes (two per holder). All ents were washed, assembled and the reactor autoclaved prior to each use. Following American Society for Testing and Materials (ASTM) standard E2562-07, the modiﬁed reactor was run under the following conditions: approximately 5 x 107 bacterial cells were ated into 500 mL of BHI in the bioﬁlm reactor. A paddle in the base of the reactor was stirred at 130 rpm. The unit was placed on a hot plate set at 33° C for 24 hrs. A % BHI broth solution was then ﬁowed through the reactor at 6.94 mL/min for an additional 24 hrs.

Following the 48-hour growth period, six of the PEEK membranes were aseptically removed and placed into 5 mL of MHB that contained varying concentrations of the synthesized compounds. For ison, vancomycin was also tested against MRSA bioﬁlms and tobramycin was tested against P. aeruginosa ATCC 27853 bioﬁlms. The membranes and broth were incubated in glass test tubes at 370 C for 24 hours. The test tubes containing membranes were vortexed for 1 minute, sonicated at 42 kHz for 10 minutes and plated in duplicate on TSA using a 10-fold dilution series and incubated at 37° C for 24 hours. This process allowed for the determination ofwhat was deﬁned as the effective bioﬁlm eradication concentration (EBEC) of the selected compounds. In this case, the EBEC was deﬁned as the concentration of compound required to reduce the number of cells in the bioﬁlms from approximately 109 cells/PEEK membrane to 105 cells/membrane. A level of 109 bacteria in the bioﬁlms represented an amount of bacteria that may be present in one gram of soil in a natural ecosystem. See e.g., Bakken, Separation and Puriﬁcation of Bacteriafrom Soil, Appl Environ Microbiol 49, 487 (1985); Torsvik et al., High Diversity in DNA ofSoil Bacteria, Applied and Environmental Microbiology 56, 782-787 (1990).

[0352] Two membranes from each run of the reactor were aseptically removed and ﬁed to serve as ve controls of bioﬁlm growth. All of the bioﬁlm eradication studies were repeated n=5 times. EBEC results are summarized in FIGs. 12A and 12B.

Model oil pipeline contamination using Alcanivorax ensis. shows an example ofmicrobial ination in the oil and gas industry. To test the ability and y of the novel sized compounds to disperse and kill bioﬁlms that may exist in a variety of settings, a model oil pipeline system was prepared to grow s ofA. borkumensis, a egative organism that metabolizes alkane chains. In cases of oil spills, this organism may be beneﬁcial for bioremediation, but in this instance, it was used to ent bacteria in an oil ﬁeld or pipeline that may have adverse or unwanted effects of oil degradation.

B shows a representative bacteria bioﬁlm, such as the bioﬁlm shown in FIG. l4A, treated according to certain standards in the oil and gas industry. The bioﬁlm shown in B was treated with a solution comprising 0.25% aldehyde. A similar representative bioﬁlm was treated with a solution comprising 0.5% of a polyamine compound described herein, and is shown in C. shows the results of treating bioﬁlms of Alcam'vorax borkumensz's grown on the surface of galvanized steel with a solution comprising 0.25% of a polyamine compound ofthe present invention. ation treatment. Data collected with benzylnorspermidine (CZ-7; PBC-7) at 0.25% mixed with chlorhexidine ate at 0.25% has shown signiﬁcant dispersal of a bioﬁlm ofMRSA that ted of 1011 cells within one hour.

Example 2 A compound was screened for its ability to create zones of inhibition on cation adjusted Mueller Hinton agar. It is believed that the compound of Example 2 may be ented by the structure disclosed in . To perform this test, lawns of methicillin— resistant Staphylococcus aureus (MRSA) or Pseudomonas aerugz'nosa ATCC 27853 were made on cation adjusted Mueller Hinton agar using standard microbiological ques.

Immediately after plating ia on agar, a 50 [LL drop of the compound was placed onto the agar surface. The compound was tested in duplicate at 100 ug/mL, 50 ug/mL, 25 ug/mL and 10 ug/mL. All plates were incubated at 370 C for 24 hours. Results indicated that total clearing was seen against MRSA at 10 ug/mL. Partial ng was seen against P. aerugz'nosa at 25 ug/mL and total clearing at 50 ug/mL.

The second test determined the minimum inhibitory concentration (MIC) of the compound against MRSA and P. aerugz'nosa ATCC 27853. In this instance, the MIC was deﬁned as the amount of antimicrobial required to reduce 105 mL to 102 cells/mL in a 24-hour period. This deﬁnition was consistent with the Clinical and Laboratory Standards Institute (CLSI). More speciﬁcally, the microdilution method as ed in the M26-A CLSI guideline was used. All tests were performed with cation adjusted Mueller Hinton broth.

MIC data are presented in Table l.

[0359] In addition to determining the MIC, the minimum bioﬁlm eradication concentration (MBEC) of PBC-51 was also ined. The MBEC was determined using the MBEC assay, ly known as the Calgary Bioﬁlm Device, developed by tech. In this instance, the MBEC was deﬁned as the amount of antimicrobial required to eradicate 100% of detectable bioﬁlm after being grown in the MBEC device. To grow the bioﬁlms within this device, 150 uL of solution containing a concentration of 105 cells/mL were inoculated into each well of a 96-well plate. A lid containing 96 pegs was placed onto the plate and incubated for 24 hours at 370 C while being shaken at 110 rpm. Bioﬁlms developed on the surface of the yrene pegs within the 96-well plate. After 24 hours, each peg contained between 105 and 106 cells in stablished biofilms. The lid with pegs was transferred to a 96-well plate that contained 200 uL of varying concentrations of the compound and was incubated at 37° C for an additional 24 hours. The plate was then sonicated, and the bioﬁlms quantified using a 10-fold dilution series to determine the MBEC. Data are presented in Table 1.

Table 1: MIC, MBEC and EBEC of PBC-51 Against MRSA and P. aeruginosa ATCC 27853. Data are presented in ug/mL and uM concentrations.

EOrganism I IC (ug/mL / uM) MBEC (ug/mL / uM) EBEC (pg/mL/ EMRSA 8 25/<47 250/~470.88 EP. aeruginosa 6/~11.30 35/~65.92 100/<188.33

[0360] The fourth test that was performed was the ive m eradication concentration . To determine the EBEC, bioﬁlms ofMRSA and P. aeruginosa ATCC 27853 were grown on the surface of PEEK membranes using a previously established protocol. Brieﬂy, 500 mL of brain heart on (BHI) broth were inoculated with 105 cells/mL of MRSA or P. aeruginosa. BHI broth was placed into a membrane bioﬁlm reactor and placed on a hot plate set at 34° C for 24 hours with a stir bar rotating at 130 rpm.

Following the initial 24-hour growth period, a 10% BHI broth solution was ﬂowed through the reactor at a rate of 6.944 mL/min for r 24 hours. After 48 hours of growth, each PEEK membrane had bioﬂlm grth on it. In this experiment, there were ~2 x 1011 cells of MRSA per PEEK membrane and ~2 x 1010 cells ofP. aeruginosa per PEEK membrane. The EBEC was deﬁned as the concentration of the compound that was required to eradicate all detectable s (100%) of ia within the bioﬁlms. Data are presented in Table 1.

In addition to determining the antimicrobial efﬁcacy of the compound, an initial test was performed to determine its hemolytic nature. To do so, the compound was suspended in concentrations of 100 ug/mL, 50 ug/mL and 25 ug/mL. Twenty uL of each concentration was placed onto the surface of Columbia blood agar and incubated at 37° C for 24 hours. s indicated that none of these concentrations displayed hemolytic activity.

Initial tests based on visual observation were performed to determine the ability of the compound to disperse bioﬂlms. This was done by observing the bioﬁlms ofMRSA and P. nosa that had grown on the surface ofPEEK membranes after they had been placed in three separate concentrations of the compound (300 ug/mL, 200 ug/mL and 100 ug/mL).

Bioﬁlms were observed every 30 minutes to see if they ted from the PEEK membrane.

Within 2 hours, bioﬁlms began to separate from the PEEK nes, by 4 hours imately half of the bioﬁlms had separated and by 24 hours, all detectable amounts of bioﬁlm had separated from the PEEK membrane. For comparison, it is the understanding of the ors hereof that this dispersal effect is not seen with other antimicrobials such as chlorhexidine gluconate, glutaraldehyde, or benzethonium chloride.

The MIC, MBEC and EBEC ofvancomycin were also determined for comparison, and the results are presented in Table 2.

Table 2: MIC, MBEC and EBEC of Vancomycin Against MRSA.

Organism [MIC (ug/mL / uM) MBEC (ug/mL / uM) EBEC (ug/mL / uM) MRSA [10/673 >500/>336.50 >20,000/>l3,460 Example 3 MIC Analysis

[0364] To determine the MIC of polyamine compounds, the protocol described herein was used. The MIC is deﬁned as being the concentration of antimicrobial (in ug/mL) required to reduce the number of bacteria in a solution from 105 colony forming units (CFU)/mL to 102 CFU/mL in a 24-hour period.

In brief, a 0.5 McFarland of each bacterial isolate was made. A 0.5 McFarland is a measure of turbidity in a liquid sample that contains approximately 1 x 10A8 CFU/mL. The 0.5 McFarland standard was diluted in cation adjusted r Hinton Broth (CAMHB), and 50 [LL of broth were added to a well of a 96-well plate. In addition, 50 uL of CAMHB that contained a desired concentration of antimicrobial were also added to the well for a ﬁnal volume of 100 [LL and a ﬁnal concentration of approximately 5 x 104 CFU/well (which equated to approximately 5 x 105 CFU/mL). Each well ned a d amount of polyamine compound in order to experimentally determine the MIC. Each 96-well plate was incubated at 37° C for 24 hours. The contents of each well were plated on tryptic soy agar (TSA). TSA plates were incubated for 37° C for 24 hours after which the number of CFU were counted and used to calculate the CFU/mL that remained after re to varying concentrations of compound. This ure was repeated n=8 times for each concentration of antimicrobial. The concentration of polyamine compound that reduced bacteria from 105 CFU/mL to 102 CFU/mL in 24 hours was considered the MIC.

MICs from representative polyamine compounds as well as select antibiotics are provided in Table 3A and 3B. Consistent with what was mentioned previously, these data indicate that with an increase in the number of polyamine chains attached to a lipophilic backbone, the MIC is lowered, indicating it has greater antimicrobial potential. CZ-7 has one chain ofnorspermidine ed, CZ-25 has two chains ofnorspermidine and CZ-51 and CZ- 52 have three chains ed.

Table 3A: MICs of Pol amine Com ounds and Select Traditional otics /mL MRSA P. aeruginosa .. MRSA — baumanmz — aeru; znosa CZ-52 3 17 ~25 400 Vancomycin 10 , , Tobramycin 7* 2 , Polymyxin B , , 2 sulfate *Not yet determined **No change Table 3B: MICs of Pol amine Com ounds and Select Traditional otics /mL 11 1i ﬂ A nd .. MRSA baumannzz . aeru mosa baumannzz..

CZ-25 450 >5,000 >5,000 >5,000 CZ-52 300 250 >5,000 Vancomycin , , >25,000 sulfate *Not yet determined **No change To determine the MBEC of each polyamine compound, the MBEC ation Tray by Innovotech, formerly known as the Calgary bioﬁlm device, was used. Within this device, biofilms grow on the surface of polystyrene pegs, 96 of which are attached to a lid. These pegs are inserted into a ﬂat bottom 96-well plate. In this instance, the MBEC of a molecule was deﬁned as the concentration of compound (in ug/mL) required to reduce 105 or 106 CFU/peg m levels varied by isolate) to 102 CFU/peg in a 24-hour period.

Following the manufacturer's guidelines, bioﬁlms were grown on the surface of each peg by ﬁrst making a 0.5 McFarland of each isolate. The 0.5 McFarland was diluted 1:100 in CAMHB. Into each well of a ﬂat bottom 96-well plate, 150 uL of broth were pipetted. The plate was shaken at 100 rpm for 24 hours (P. aeruginosa and A. baumannii) or 48 hours (MRSA). The pegs were then placed into a separate ﬂat bottom 96-well plate for 10 seconds with 200 uL of phosphate buffered saline (PBS) in each well to remove nonadherent cells. The lid was then placed into a l plate that contained varying concentrations of antimicrobial with 200 uL per well. The plate was incubated for 24 hours at 37° C after which time 100 uL of broth were plated on TSA. TSA plates were incubated 24 hours at 370 C and the number of CFU counted to calculate the CFU/peg. In this instance, the MBEC was deﬁned as the concentration of crobial required to reduce 105 or 106 CFU/peg to 102 g in a 24 hour period.

[0369] MBEC data are presented in Table 4. Similar to the MIC data, the trend of increasing antimicrobial activity by increasing the number ofpolyamine chains ed to a backbone was conﬁrmed against low number bioﬁlms.

EBECAnalysis In addition to ining the MIC and the MBEC ofpolyamine compounds against planktonic bacteria and low number bioﬁlms, our group wanted to determine the efﬁcacy of polyamine compounds against high number bioﬁlms. To do so, bioﬁlms were grown on the surface of polyetheretherketone (PEEK) membranes using a membrane bioﬁlm reactor. This reactor is similar to the CDC bioﬁlm reactor, but rather than growing bioﬁlms on coupon surfaces, the reactor was modiﬁed to hold PEEK membranes. In short, to grow bioﬁlms within this system, 500 mL of brain heart infusion (BHI) broth were inoculated with 1 mL of a 0.5 McFarland. The reactor was placed on a hot plate set at 34° C and the bacteria were grown under batch conditions for 24 hours. Following this protocol, bioﬁlms typically grow to 109 CFU/PEEK membrane. each PEEK membrane has high number s A on of 10% BHI was then ﬂowed h the reactor at a rate of 6.94 mL/min for an additional 24 hours. PEEK membranes were then removed and placed into 2 mL of CAMHB that contained a d concentration of polyamine compound or antibiotic. The EBEC was deﬁned as the concentration of antimicrobial required to reduce a bioﬁlm from approximately 109 CFU/PEEK ne to approximately 102 CFU/PEEK membrane in a 24-hour period.

EBEC data are presented in Table 4. One of the most striking results was the difference in efﬁcacy between CZ-52 and ycin. At 25,000 ug/mL, vancomycin did not have the ability to reduce bioﬁlms of MRSA by even 1 logo unit. In contrast, at 250 ug/mL CZ-52 was able to reduce MRSA bioﬁlms by greater than 7 logo units in a 24-hour period.

Example 4 Release OfPolyamz'ne ndsfrom Carrier Products

[0372] To collect proof-of-concept/preliminary data that carrier(s) will release these products in sufﬁcient amounts to have antimicrobial efﬁcacy, a polytherapy formula was prepared. A Vanicream—based cream from the University of Utah compounding pharmacy was used as the formulation base, and active ingredients were added to create a cream with ﬁnal concentrations of 0.25% CZ-52, 0.25% CZ-25 and 0.1% Polymyxin B sulfate. To test its y, 0.5 McFarland standards of MRSA, P. aeruginosa, and A. baumannii were made, and a lawn of each isolate was spread on ia blood agar. Approximately 20 mg of cream was placed in the center of each lawn. The plates were incubated 24 hours at 37° C.

Zones of clearing in each experiment provided a preliminary indication that the antimicrobials will elute out of the cream in sufﬁcient quantities to eradicate each bacterial isolate (). Notably, the cream alone (negative control) created no zones of inhibition, and polyamine compounds used alone in the cream likewise created zones of clearing. In broth samples, the erapy composition has shown r results by eradicating all detectable amounts of each bacterial isolate after they had been grown as bioﬁlms on PEEK membranes with approximately 109 CFU/PEEK membrane. In short, these data indicate that the polytherapy crobial agents will elute out of r product(s) and eradicate bacteria, including those in bioﬁlms.

Example 5 Bioﬁlm Dispersion CZ—25 and CZ-52 were tested for their ability to disperse s ofMRSA and P. aeruginosa. To test for dispersion, bioﬁlms of each isolate were grown on the surface of commercially pure titanium (Ti) coupons (l/2” diameter x l/8” height) in a CDC bioﬁlm reactor. The growth conditions for this reactor were the same as those for the membrane biofilm reactor. After bioﬁlms were grown for 48 hours, each Ti coupon was aseptically removed and placed into 2 mL of CAMHB for 2 hours. The CAMHB had a ﬁnal concentration of 0.25% (2.5 mg/mL) CZ-25 or CZ-52. Each compound was tested n=3 times.

Following the 2-hour exposure, each coupon was ﬁxed in 0.25% glutaraldehyde for 24 hours, dehydrated using ascending concentrations of ethanol (70%, 95%, 100%) with 3x min. changes and desiccated. One side of each coupon was carbon coated and imaged using a JEOL ISM-6610 SEM to directly observe the surface of the coupons and determine the ability of polyamine nds to se biofilms compared to positive controls that had no ent.

When compared to untreated controls, data have indicated that CZ-25 and CZ-52 have the ability to disperse bioﬁlms from the surface of a material () such that a monolayer of cells or reduced communities remain.

The MRSA biofilms were grown on the surface of titanium (Ti) coupons using a CDC biofilm reactor. For this reactor, an initial inoculum of N5 x 10A5 cells/mL were put into 500 mL of brain heart infusion (BHI) broth. The reactor was placed on a hot plate set at 34 C for 24 hours and the bacteria were allowed to grow in batch e. For an additional 24 hours, 10% BHI broth was ﬂowed through the reactor at a rate of 6.94 mL/min. After the r growth period, the coupons were aseptically removed and placed into 2 mL of cation adjusted Mueller Hinton broth (CAMHB) for 2 hours. Digital images of the biofilm sion process were collected after 30 minutes of exposure to ine compounds.

After 2 hours, the same procedure as above was used to fix the biofilms and image them by SEM.

Digital images of MRSA biofilms being dispersed from the surface of the Ti coupon also help to highlight the dispersive ability of CZ-25 ().

Data has also been collected to determine the dispersive capacity amine compounds against Alcanivorax ensz's, an oil-eating bacterium (FIGS. 19 and 20).

For the dispersal data with the Alcanivorax ensis, 1/2 inch diameter galvanized steel pipe that was approximately 6 inches long was purchased. Two strips of galvanized sheet metal were cut into 1/2 cm x 2 cm strips, and two of those were placed into the pipe. The pipe was connectedto silicone tubing, and marine broth was run through the pipes with an inoculum of 5 x 10A5 cells/mL. The broth was ﬂowed through the pipes at a rate of 6.94 mL/min using a peristaltic pump. s were grown on the surface of the galvanized steel for 48 hours, then exposed to polyamine compounds or glutaraldehyde for two hours. The strips of galvanized steel were then removed, ﬁxed in 0.25% glutaraldehyde for 2 hours (those that had already been exposed to glutaraldehyde were not ﬁxed twice), and dehydrated in ascending concentrations of ethanol (70%, 95%, 100%) with 3x 10 minute exchanges of each. Lastly, the strips were carbon-coated and were imaged using a JEOL JSM-6610 scanning electron microscope (SEM) with a lanthanum hexaboride (LaB6) ﬁlament.

Example 6 One of the most promising aspects of polyamine compounds is that preliminary data suggests that these compounds address the characteristics of bioﬁlms that have made them resistant to antibiotic treatment. Speciﬁcally, polyamine nds have potent crobial activity against very high inocula of bacteria and kill bacteria in a rapid n.

Without meaning to be bound by theory, initial characterization suggests that ine compounds are membrane-active, meaning they are nonspeciﬁc in nature. This nonspeciﬁc action reduces the potential for bacteria to upregulate their e mechanisms and reduces the risk of resistance development. From the data collected to date, it does not appear that polyamine compounds are limited to eradicating bacteria that are in ase growth as indicated by their activity t bacteria in the bioﬁlm phenotype. Bacteria in a bioﬁlm have reduced metabolic activity, which is one of the primary contributing factors that allows bioﬁlms to resist traditional antibiotic y. Typically, otics affect bacteria in log- phase growth. Finally, polyamine compounds have the ability to disperse bioﬁlms while demonstrating antimicrobial kill. By dispersing and killing bacteria in a bioﬁlm, it is hypothesized that polyamine compounds interrupt water channels and the community as a whole, allowing for distribution of polyamine compounds to a greater number, if not all, of the cells within a bioﬁlm.

A key aspect ofpolyamine compounds is the process by which they are synthesized to provide triple action against ia and bioﬁlms. The ability to size antimicrobial compounds with c activity against bioﬁlms is signiﬁcant. ional approaches to discovering or developing antibiotics has primarily been done through some, high— throughput screening methods with limited results wherein perhaps 1 in 1,000 compounds may be of interest. Using the below method, our group was able to e a lead product after synthesizing approximately 20 compounds.

Organisms residing in biofilms present a complex extracellular matrix of polysaccharides (exopolysaccharides) and proteins. As a result of this x matrix, nutrient limiting ions may exist that alter the normal or planktonic metabolic state. As stated above, this produces to a reduced efficacy of traditional antibiotic agents, rendering them up to 1,000 times less active. A hallmark of these ysachharides is the presentation of acidic residues from repeated glucoronic acid motifs and pyruvate derived acetals. A recent study by Losick and kers demonstrated that the simple polyamines spermine and norspermidine were naturally occurring tors of biofilm formation, nously produced at high concentrations (5 0-80 uM) in response to nutrient limiting conditions and waste accumulation in mature pellicles. In this study they were able to demonstrate that norspermidine could inhibit bioﬁlm formation at 25 uM and showed that at similar concentrations it could disperse the exopolysaccharide component of the matrix but I5 not the protein component. This does suggest, however, that these agents might be capable of disassembling established bioﬁlms. Interestingly, spermidine was only active at much higher concentrations (~ 1 mM) leading them to propose a ale for this activity in the ability of the polyamines to engage the acidic residues in the matrix at regular intervals.

Given the lack of activity for spermidine vs. norspermidine, it is interesting to note that many secondary metabolites ning the polyamine frameworks have been identified as potent antimicrobials against planktonic bacteria. Most notably mine, which contains a spermidine tail, and the related natural product incorporating a spermine tail show potent antimicrobial activity against Gram-positive and Gram-negative bacteria (). -rich ic peptides such as magainin and pexiganin are also potent anti-microbials against a wide range of organisms and have been developed as topical antimicrobials for the treatment of diabetic foot ulcers.

Ofnote is the incorporation of hydrophobic and cationic es that are present in these antimicrobial “kill” compounds, without adhering to the strict hydrogen bond donor- acceptor pattern of the m disruptors. Thus, an l study was made to investigate the utility of combining a simpliﬁed hydrophobic backbone with a cationic tail that might impart molecules with the ability to inhibit m formation, disrupt established bioﬁlms and kill the emerging planktonic bacteria. The study began with the synthesis of benzylic substituted ines (). The synthesis of diaminopropane substituted backbones is straightforward to provide CZ-4, 12,32 from the known mono-Boc ted diaminopropane and the commercially available aldehydes (benzaldehyde, isopthalaldehyde or 1,3,5- etricarboxaldehyde). This three-step synthetic procedure proceeds via reductive amination and acidic removal of the Boc group. Of note is that no puriﬁcation is required until a ﬁnal recrystallization of the HCl salt. The norspermidine series (CZ-7,25,52) can be prepared in a similar manner from the mono-Boc protected norspermidine. Of note is that no puriﬁcation is required until a ﬁnal recrystallization of the HCl salt, which allows easy preparation of these compounds on at least a ~ 10 g scale.

[0386] In some cases, increasing number of polyamine chains on the hobic backbone systematically ses activity. For example, with a single chain of diaminopropane added to a benzyl backbone (CZ-4, ), the minimum inhibitory concentration (MIC; as deﬁned by the Clinical and Laboratory Standards ute (CLSI)) against MRSA is greater than 1,200 ug/mL, whereas two chains decrease the MIC to 300 ug/mL (CZ—12) and three chains reduce it further to 45 ug/mL (CZ-32). Similarly, with a single chain of norspermidine attached to a benzyl backbone (CZ-7, ), the MIC against MRSA is greater than 1,200 ug/mL. By adding a second chain of norspermidine (CZ-25), the MIC is 45 ug/mL. With a third chain of norspermidine attached (CZ-52), the MIC s 3 ug/mL.

[0387] Concurrently to the other goals of this project, a focused synthetic effort will continue to exploit this trend and generate related chemotypes with increased efﬁcacy (e. g., compounds with four polyamine chains). This is ed to be lished, for example, via Pd(II) mediated dimerization of oisopthalaldehyde followed by reductive amination ().

Example 7 Evaluation ofBioﬁlm Remediation and Removal Chemistries A series of experiments is conducted to igate the impact ofbioﬁlm remediation and removal chemistries on mimetic multispecies bioﬁlms commonly associated with anthropogenic heating and cooling water systems.

[0389] The criteria for success is to demonstrate that these chemistries: (1) Remove a multispecies biofilm from a copper, mild steel, galvanized steel, and stainless steel substrate to a r extent than that which can be achieved by the existing water treatment chemistries. (2) Do not aggressively corrode the metal substrates when used in an m concentration. (3) Avoid material compatibility issues with the existing treatment als used to minimize scale formation and prevent corrosion of the metal parts of the anthropogenic heating and g system.

Bioﬁlm Generation on the Metal s

[0390] Normally, monoculture bacteria belonging to the Pseudomonas genus have been used to investigate the physical and mechanical properties of biofilm on different heat exchanger surfaces and substrates under changing ﬂow ions. However, to more accurately represent conditions that would be found in the real environment, a mixture of Pseudomonasfluorescens and monas putida together with a 0.1% e ofNCH 3010 Bacillus subtilis, NCH 3032 Bacillus subtilis, NCH 3016 Bacillus licheniformis, and NCH 3040 Bacillus thuringiensis all of which are naturally occurring soil bacteria is used.

The microorganisms will be provided in pellet form from Eco-Bionics, and each pellet contains a starter e and appropriate nutrients that produce actively growing bacteria with the addition of water.

[0391] The experimental test procedure is a modiﬁed version of the single tube method.

Biofilm are grown on the metal s using a biofilm reactor designed around a uous stir tank r (). Prior to operation the reaction chamber and the individual components are disassembled, soaked in a 10% bleach solution, then scrubbed in hot soapy water and rinsed in distilled water. Once cleaned, the stir tank r is charged with 2 liters of DI water, and 20 g of the Free Flow pellets will be added to the water.

Coupons are placed onto the coupon holding rods, which is then inserted into the Free Flow pellet solution. The reactor runs for 6 days with the Free Flow solution being replaced every 2 days.

Biofilm growth isvisually observed and documented photographically. To quantify the presence of viable microorganisms within the biofilm structure formed on the metal coupons (see for an example of the typical biofilm formed on the substrate) the bioﬁlm covered coupons is removed from the reactor and lightly rinsed with sterile buffer to remove any loose debris from the surface. The coupons are then transferred to a container with 40 ml of Phosphate buffer solution (PBS) and ed for 30 sec then placed in a sonication bath (Branson model 3200) for 30 sec. After sonication, the container with the buffer and coupon is removed and vortexed again for 30 sec before being returned to the sonication bath for an additional 30 sec of processing. A third and final vortexing step ensures that the adhered biofilm will be thoroughly removed. After processing to remove the bioﬂim the PBS solution will be ly diluted onto tryptic soy agar (TSA) plates which will be incubated at 30 0C for up to 36 hours before being enumerated.

The biofilm coupons are exposed to a high, medium, and low concentration of the t (based on the recommended dosing rates) for a prescribed time. After treatment, the coupons are removed, and the eluant and treatment solutions are assayed to enumerate viable bacteria A standard dispersant used for bioﬁlm removal, together with a standard biocide (oxidizing and non-oxidizing) used at the recommended label use concentrations, are used as controls.

Corrosion Testing Treatment solution of the same concentrations used in the experiments above is prepared with standard corrosion inhibitor products and calcium deposit products in regular tap water and tested in a standard corrosion test. The solution is added to a treatment tank that is stirred ually for two weeks, after which the coupons are removed and ed for corrosion using standard corrosion test methods. ion rates of less than 3.0 mpy for mild steel and 0.2 mpy for copper are considered non corrosive. A standard sant used for bioﬁlm removal together with a standard biocide (oxidizing and non-oxidizing) used at the ended label use trations are used as controls.

Conclusions

[0395] We have been able to determine m amounts of our polyamine compounds that will have activity against bioﬁlms of P. aeruginosa. In general, the polyamine compounds do better t Bacilli than P. aeruginosa, so what is active t P. aeruginosa is likely active against Bacilli.

These formulas are made in PBS, water or broth.

Example 8 Methodsfor MIC, MBEC andEBECAnalysis To determine the MIC of the compounds of the present invention the following procedure was used.

A 0.5 McFarland of A. baumanniz’ is made. A 0.5 McFarland is a e of turbidity in a liquid sample that ns approximately 7.5 x 107 CFU/mL. The 0.5 and standard is diluted in CAMHB and 50 [LL of broth is added to a well of a 96-well plate. In addition, 50 [LL of CAMHB that contains a desired concentration of crobial is added to the well for a ﬁnal volume of 100 uL and a ﬁnal concentration of approximately 5 x 104 CFU/well (which s to approximately 5 x 105 CFU/mL). Each well contains a desired amount of CZ compound in order to experimentally determine the MIC. Each 96- well plate is incubated at 37° C for 24 hours. The contents of each well is plated on tryptic soy agar (TSA). TSA plates is incubated at 37° C for 24 hours after which the number of CFUs is counted and used to calculate the CFU/mL that remain after exposure to varying concentrations of compound. This procedure is ed n=8 times for each concentration of antimicrobial. The concentration of CZ compound that reduces bacteria from 105 CFU/mL to 102 CFU/mL in 24 hours is considered the MIC.

To determine the MBEC, the MBEC Inoculation Tray by tech, formerly known as the Calgary bioﬁlm device, is used. Within this device, bioﬁlms grow on the surface of polystyrene pegs, 96 of which are attached to a lid. These pegs are inserted into a ﬂat bottom 96—well plate. In this instance, the MBEC of a molecule is defined as the concentration of compound required to reduce approximately 106 CFU/peg to approximately 102 CFU/peg in a 24-hour period.

Following the manufacturer's guidelines, biofilms are grown on the surface of each peg by first making a 0.5 McFarland ofA. baumanm’z‘. The 0.5 and is diluted 1:100 in CAMHB broth. Into each well of a flat bottom 96-well plate, 150 uL of broth is pipetted.

The plate is shaken at 100 rpm for 24 hours. The pegs are placed into a separate ﬂat bottom 96—well plate for 10 seconds with 200 uL ofphosphate buffered saline (PBS) in each well to remove nonadherent cells. The lid is placed into a 96-well plate that contains varying concentrations of crobial with 200 uL of broth per well. The plate is incubated for 24 hours at 37° C after which time 100 uL of broth is plated on TSA. TSA plates is incubated 24 hours at 37° C and the number of CFU counted to ate the CFU/peg.

In addition to determining the MIC and the MBEC of compounds against planktonic bacteria and low number s, the efficacy of the compounds of the invention against high number bioﬁlms is determined. To do so, bioﬁlms are grown on the surface ofPEEK membranes using a membrane bioﬁlm reactor (Williams, D. L., Haymond, B. S. & Bloebaum, R. D. Use of delrin plastic in a modiﬁed CDC bioﬁlm reactor. Res biol 6, 425-429 (2011); Williams, D. L., Woodbury, K. L., Haymond, B. S., , A. E. & Bloebaum, R. D. A modiﬁed CDC bioﬁlm reactor to produce mature bioﬁlms on the surface ofPEEK membranes for an in vivo animal model application. Curr Microbiol 62, 1657-1663 (2011); Williams, D. L. et al., In Vivo Efﬁcacy of a Silicone - Cationic Steroid Antimicrobial Coating to Prevent Implant-Related ion. Biomaterials 33, 8641-8656 (2012); Williams, D. L. et al., Experimental model ofbioﬁlm implant-related osteomyelitis to test combination biomaterials using bioﬁlms as initial inocula. J Biomed Mat Res A 100, 900 (2012)).

This reactor is similar to the CDC bioﬁlm reactor, but rather than growing bioﬁlms on coupon surfaces, the reactor was modiﬁed to hold PEEK membranes. In short, to grow bioﬁlms within this system, 500 mL of brain heart lOl’l (BHI) broth is inoculated with 1 mL of a 0.5 McFarland. The reactor is placed on a hot plate set at 340 C and the bacteria grown under batch conditions for 24 hours. A solution of 10% BHI will then be ﬂowed through the reactor at a rate of 6.94 mL/min for an additional 24 hours. Following this ol, bioﬁlms typically grow to 109 CFU/PEEK membrane. PEEK membranes is removed and placed into 2 mL of CAMHB that contain a desired concentration of the test compound. The EBEC is deﬁned as the concentration of antimicrobial required to reduce a bioﬁlm from approximately 109 EK membrane to approximately 102 CFU/PEEK membrane in a 24-hour period.

MIC, MBEC and EBECAnalysis To determine the MIC of the test compounds and traditional antibiotics, a protocol which deﬁnes the MIC as being the concentration of antimicrobial ed to reduce the number of bacteria in a solution from 105 colony forming units (CFU)/mL to 102 CFU/mL in a 24-hour period. MICs for tests compounds as well as for select antibiotics are provided in Table 4. Consistent with what was mentioned usly, these data te that, with an increase in the number of polyamine chains attached to a lipophilic backbone, the MIC is lowered, indicating it has greater crobial ial. CZ-7 has one chain of norspermidine attached, CZ-25 has two chains of norspermidine, and CZ-52 has three chains attached ().

The trend of increasing antimicrobial activity by increasing the number of polyamine chains attached to a backbone while also modulating the hydrophobicity of that backbone shows promising activity t A. baumannii, more speciﬁcally in the case of CZ-58 (). The analysis below shows that compounds of the invention have sed y relative to current therapies (e. g., polymyxin B).

MBEC data were collected using the MBEC Inoculation Tray by Innovotech. The MBEC was deﬁned in these preliminary results as the concentration of antimicrobial required to reduce 105 or 106 CFU/peg to 102 CFU/peg in a 24-hour . MBEC data are presented in Table 4.

—MIC ug/mL (uM) MBEC ug/mL (uM) EBEC ug/mL (uM) i 4 i Compound MRSA .. MRSA .. MRSA baumannll baumannll baumannll.. >500 >500 >500 >500 >5,000 >10,000 CZ—7 (>2,259) (>2,259) 9) (>2,259) (>22,590) (>45,180) 40 150 25 450 >5,000 cz-25 ~7,500 (110) (411) (69) (1234) (13,715) 3 400 20 >500 250 ~7,000 CZ-52 (6) (800) (40) (995) (500) (~14,000) 6 30 30 >50 1,500 400 CZ-58 (13.6) (68) (68) (>113) (3,403) (908) >500 >>25,000 Vancomycin (6.9) (>345) (>> 17,225) 1 50 Polymyxin B 1,000 sulfate (0.7) (36) (722) Table 4: MICs, MBECs and EBECs of CZ compounds and ional antibiotics. Data reported in ug/mL and uM concentrations.

EBEC data were collected to determine the efﬁcacy of CZ-52, CZ-58 and traditional antibiotics against high number bioﬁlms containing approximately 109 CFU grown on the surface of a polyetheretherketone (PEEK) membrane. Results are shown in Table 4.

EBEC data showed ng differences in efﬁcacy between CZ-52 and vancomycin (Table 4). At 25,000 ug/mL, vancomycin did not have the y to reduce bioﬁlms ofMRSA by even 1 logo unit. In contrast, at 250 ug/mL CZ-52 was able to reduce MRSA s by greater than 7 loglo units in a 24-hour period. Although the MIC of CZ—58 was lower against MRSA compared to A. baumannz’z’, CZ-58 had greater activity against bioﬁlms ofA. baumanm‘i than against MRSA bioﬁlms in the EBEC assay. The EBEC ofpolymyxin B againstA. baumanm‘z‘ was 1,000); r than the MIC, whereas the EBEC for CZ-58 was only ~l3x greater than the MIC. Although the MIC of CZ-58 was “high” ed to MICs of traditional antibiotics, the efﬁcacy against bioﬁlms was promising.

Table 5 provides MIC results for a variety of compounds against MRSA, Pseudomonas aeruginosa, and Acinetobacter baumannii.

CZ MIC MIC (P. MIC (A.

No. Compound (MRSA) aeruginosa) bumannii) CZ-3 N/\/\/NH2 H + - 2 HCI CZ-4 CZ-5 CZ—6 CZ-7 CZ—8 CZ-9 CZ MIC MIC (P. MIC (A.

No. nd (MRSA) aeruginosa) bumannii) + ++ 14 + 21 +++ + MIC MIC (P. MIC (A.

Compound (MRSA) nosa) bumannii) Nl/NHHN\)/Nl-l2 LALNH NHBoc ++ ++ MIC MIC (P. MIC (A.

. (MRSA) nosa) bumannii) MIC (P. MIC (A. nosa) bumannii) +++ +++ +++ +++ ++ ++ t0 +++ CZ MIC MIC (P. MIC (A.

No. Compound (MRSA) aeruginosa) ii) kNH NHBoc +++ +++ +++ +++ NH NH; 'x decanoic acid +++ + +++ ++ t0 +++ ++ MIC (P. MIC (A. nosa) bumannii) +++ +++ +++ +++ +++ +++ ++ +++ +++ MIC MIC (P. MIC (A.

. (MRSA) nosa) bumannii) +++ +++ +++ +++ +++ MIC MIC (P. MIC (A. . nd (MRSA) aeruginosa) bumannii) +++ + +++ +++ +++ ++ CZ MIC MIC (P. MIC (A.

No. Compound (MRSA) nosa) bumannii) +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ MIC MIC (P. MIC (A.

. (MRSA) nosa) bumannii) +++ +++ : : t0 : : : Free Base MIC (P. MIC (A. nosa) bumannii) +++ +++ +++ +++ +++ +++ ++ +++ . Compound (MRSA) nosa) bumannii) +++ +++ +++ +++ MIC (P. MIC (A. nosa) bumannii) ++ to +++ MIC (P. MIC (A. nosa) bumannii) +++ +++ +++ +++ +++ +++ ++ ++ +++ +++ MIC (P. MIC (A. nosa) bumannii) +++ ++ to +++ ++ ++ MIC (P. MIC (A. nosa) bumannii) +++ +++ +++ +++ +++ +++ +++ +++ +++ MIC (P. MIC (A. nosa) bumannii) +++ +++ +++ +++ +++ +++ ++ +++ MIC (P. MIC (A. nosa) bumannii) —— = MIC >100 uM. = 100 uM Z MIC Z 50 uM. MIC results of “>n uM” or “<n uM” will also be designated as --+ if 100 Z n 2 50. : 50 [1M > MIC. MIC results of “>n ”M” or “<n HM” will also be designated as +++ if n < 50.

Table 5: MICs of CZ compounds.

Example 9 Bioﬁlm Dispersion The compounds of the present invention such as CZ 25, CS—58 and CS-52 have excellent dispersal characteristics. To test for dispersion, biofilms of each isolate were grown on the surface of commercially pure titanium (Ti) coupons (1/2” er x 1/8” height) in a CDC bioﬁlm reactor. The growth conditions for this reactor were the same as those for the membrane bioﬁlm reactor. After bioﬁlms were grown for 48 hours, each Ti coupon was aseptically removed and placed into 2 mL of cation ed Mueller Hinton broth (CAMHB) for 2 hours. The CAMHB had a ﬁnal concentration of 0.25% (2.5 mg/mL) CZ-25 or CZ-52.

Each compound was tested n=3 times.

Following the 2-hour exposure, each coupon was ﬁxed in 0.25% glutaraldehyde for 24 hours, dehydrated using ascending concentrations of l (70%, 95%, 100%) with 3x min. changes and desiccated. One side of each coupon was carbon coated and imaged using a JEOL ISM-6610 SEM to directly observe the surface of the coupons and determine the ability of CZ compounds to disperse bioﬁlms compared to positive controls that had no treatment. When compared to ted controls, data have indicated that CZ—25, CZ—52 and CZ-58 have the ability to disperse bioﬁlms from the surface of a material () such that a monolayer of cells or reduced communities remain.

[0409] provides SEM images of Ti s with MRSA bioﬁlms exposed to CZ- (left), CZ—52 (middle) or water only ). CZ-25 and CZ—52 demonstrated the ability to disperse the majority of bioﬁlms such that a monolayer of cells ed on the surface of the Ti. Those samples d with water only had bioﬁlm communities that remained in all areas of the coupons.

Example 10 Preparation 1 V—(I, 3-phenylenebis(methylenej)bis(N3-(3-amin0pr0pyl)pr0pane-I, 3- diamz'ne), hydrachlaride salt[C0mp0und VII-2] The polyamine compound (VII-2) may be synthesized as shown in Scheme 7. rmidine was reacted with utyl dicarbonate compound to provide N—Boc protected norspermidine using the following procedure: To a solution of norspermidine (27.2 g, 207.7 mmol, 3.0 equiv.) in THF solvent (1000 mL) at 00C was added di-t-butyl dicarbonate (18.1 g, 83.1 mmol, 1 equiv.) dropwise over a period of two hours. During the period of addition, the solution went from clear to a cloudy white. Following addition, the reaction e was allowed to stir at room temperature for a period of 12 hours. The THF solvent was evaporated, and the residue was dissolved in CH2C12 (250 mL) and washed with water (250 mL). The resultant aqueous layer was washed with CH2C12 (4 x 100 mL). The organic layer was dried with Na2804 and evaporated to afford the Boo-protected amine as a clear viscous liquid.

Scheme 7 y{5.1qmvxxﬁAVANHﬁ: i {gﬂﬂj‘go MeOH. 3; A ms, than 8.133%; N’N’”‘§”‘\X‘§’ ' {’N”\§’\f\§“\f\m~;am.E 'n L' £30mpwnd ‘33an Acid. MESH \VA§\Qr\Vx\’N”r\-ﬁ% $NN*N*ANWTNNHQ H H El {f H H nd w-z Benzene-1,3-dicarboxyaldehyde was reacted with at least two equivalents of the N- Boc protected rmidine in the presence of molecular sieve and methanol solvent to provide a corresponding polyamide product, which was then d by sodium borohydride (NaBH4) to produce the N—Boc protected polyamine compound VII-2a using following procedure: To a stirring solution of 4 A molecular sieve (100 mg) in MeOH solvent (25 mL) was added benzene-1,3-dicarboxyaldehyde (0.78 g, 5.87 mmol, 1 equiv.) followed by the Boo-protected amine (2.71 g, 11.74 mmol, 2 equiv.). The solution was stirred for 12 hours at room temperature. The newly formed imine was quenched at room temperature by the addition ofNaBH4 (0.89 g, 23.48 mmol, 4 equiv.) after which the solution was stirred for an additional one hour. The solution was d through a pad of celite, and the solvent evaporated in vacuo to afford brown oil. The crude mixture was dissolved in EtOAc (100 mL) and washed with 10% NaOH (100 mL): The resulting NaOH was further washed with EtOAc (50 mL x l). The organic layer was dried with Na2SO4 and concentrated under reduced pressure to provide a crude Boc-protected product.

The Boc-protecting group on the terminal amine of the polyamine compound VII- Za was deprotected by subjecting compound VII-23 to acid ysis to e the polyamine compound (VII-2) using the following procedure: The crude Boc-protected product was added to a stirring solution of HCl in MeOH (100 mL) and left for one hours.

The solvent was evaporated in vacuo to yield a white to ite solid which was recrystallized from MeOH to e the polyamine compound (VII-2) as white solid to off- white solid (30% yield). 1H NMR (500 MHZ, D20): 7.60 (s, 4H), 4.33 (s, 4H), 3.26-3.18 (m, 12H), 3.12 (t, J=8Hz, 4H), 2.21-2.08 (m, 8H).

Example 11 Preparation ofNI,NI '— (((1,3-phenylenebis(methylene))bis(azanediyl))bz’sQaropane-j’, I— diyl))bis(N3,N3-dz'methylpropane-I,3-dz'amz'ne), hydrochloride salt [Compound ] The polyamine compound (VII-2C) may be synthesized as shown in Scheme 8.

Benzene-l ,3-dicarboxyaldehyde was reacted with at least two equivalents ofN, N- dimethylnorspermidine in the presence of molecular sieve and methanol solvent to provide a corresponding polyamide product, which was then reduced by sodium borohydride (NaBH4) to produce the ine compound VII-2B using the following procedure: To a stirring solution of 4 A molecular sieve (100 mg) in MeOH solvent (25 mL) was added benzene-1,3- oxyaldehyde (0.78 g, 5.87 mmol, 1 equiv.) followed by N, N—dimethylnorspermidine (2.71 g, 11.74 mmol, 2 equiv). The on was stirred for 12 hours at room temperature.

The newly formed imine was quenched at room temperature by the on ofNaBH4 (0.89 g, 23.48 mmol, 4 equiv.) after which the solution was stirred for an additional one hour. The solution was ﬁltered through a pad of , and the solvent evaporated in vacuo to afford brown oil. The crude mixture was dissolved in EtOAc (100 mL) and washed with 10% NaOH (100 mL). The resulting NaOH solution was further washed with EtOAc (50 mL X 1).

The organic layer was dried with Na2804 and concentrated under reduced pressure to provide the polyamine compound VII-ZB.

Scheme 8 {3 H Mia [“1" \lLg;\ ’9 ‘i- \’%’,4\MG, MGUH, 4 A ms. than NaBm MENN,J"\\VM\N.A\~,AN9+ Me: K “NAVANMN‘X H a l H H Me M£2. w Compound VME ; Acid, MesﬂH xx “ck f\Nx\,~v’\ N“’«V‘N A ,MES H H '3‘ Me: ‘ ' of Me Compound Vii-2C3 The crude the ine compound VII-ZB was added to a stirring on of HCl in MeOH solvent (100 mL) and left for one hour. The solvent was evaporated in vacuo to yield a white to off-white solid, which was recrystallized from MeOH to afford the polyamine compound VII-2C at about 30% yield as white solid to off-white solid. 1H NMR (500 MHZ, D20): 7.59 (s, 4H), 4.24 (s, 4H), 3.29-3.15 (m, 16H), 2.91 (s, 12H), 2.21-2.15 (m, 8H).

Example 12 Preparation ofN-benzyl—I,12-d0decyldiamine (V-I) The polyamine compound (V-l) may be synthesized as shown in Scheme 9 from 1,12-dodecyldiamine, either via synthetic route [A] or [B].

HZNWNHZ l (800)20 H2N/\/\/\/\/\/\/NHB0c (DPhCHO,MeOH, [A] [B] BnCLEgN 4 A mol. sieves (ii)NaBH4 BnHN’“\¢/\\/”\V/A\v/\\//\\/NHBOC Compound V-1a l HCI, MeOH BnHNWVVWNHZ Compound V-1 In the synthetic route [A], N—Boc protected—1,12—dodecyldiamine was d with benzyl de in the presence of base and solvent to provide a corresponding polyamide compound V-la. In the synthetic route [B], N-Boc protected-1,12-dodecyldiamine, ed as described in Example 10 as following: To a solution of amine (27.2 g, 207.7 mmol, 3.0 equiv.) in THF solvent (1000 mL) at 0°C was added di-tert—butyl dicarbonate (18.1 g, 83.1 mmol, 1 equiv.) dropwise over a period of two hours. During the period of addition, the solution went from clear to a cloudy white. ing addition, the reaction mixture was allowed to stir at room temperature for a period of 12 hours. The THF solvent was evaporated, and the residue was dissolved in CH2C12 (250 mL) and washed with water (250 mL). The resultant aqueous layer was washed with CH2C12 (4 x 100 mL). The c layer was dried with Na2804 and ated to afford the N—Boc protected-l,12-dodecyldiamine amine as a clear viscous liquid.

[0417] The N-Boc protected-1,12-dodecyldiamine was reacted with benzaldehyde in the presence of molecular sieve and methanol solvent to provide a corresponding polyamide product, which was then reduced by sodium borohydride ) to produce the N—Boc protected polyamine nd V-la using the following procedure: To a stirring solution of 4 A molecular sieve (100 mg) in MeOH solvent (25 mL) was added the benzaldehyde (0.78 g, 5.87 mol, 1 equiv.) followed by the N—Boc protected-1,12-dodecyldiamine (1.36 g, 5.87 mmol, 1 equiv.). The solution was stirred for 12 hours at room temperature. The newly formed imine was quenched at room temperature by the addition 4 (0.89 g, 23.48 mmol, 4 equiv.) after which the solution was d for an additional one hour. The solution was ﬁltered h a pad of celite, and the solvent evaporated in vacuo to afford brown oil.

The crude mixture was dissolved in EtOAc (100 mL) and washed with 10% NaOH (100 mL).

The resulting NaOH solution was further washed with EtOAc (50 mL X 1). The organic layer was dried with NaZSO4 and concentrated under reduced pressure to provide the N—Boc protected polyamine compound V-la.

The Boc protecting group on the terminal amine of the polyamine compound V-la was deprotected by treating compound V-la with acid to produce the polyamine compound (V-l) using following procedure: The crude N—Boc protected polyamine compound V-la was added to a stirring on of HCl in MeOH solvent (100 mL) and left for one hour. The solvent was evaporated in vacuo to yield a white to off-white solid, which was recrystallized from MeOH to afford the polyamine compound (V-l) at about 30% yield as a white solid to off-white solid. 1H NMR (500 MHz, D20): 7.42 (s, 5H), 4.15 (s, 2H), 3.26 (p, J= 1.5 Hz, 2H), 2.96-2.89 (m, 4H), .56 (m, 4H), 1.28-1.21 (m, 14H).

Example 13 ation 0f],3,5—trz's-[N-(3-amin0pr0pyl)—methylamz'nej-benzene (VI-I) The polyamine compound (VI-1) may be synthesized as shown in Scheme 10. ﬁam‘pau‘nd Vida ﬁdzéd. MeQH Compound VM 1,3-Diaminopropane was reacted with di-t—butyldicarbonate compound to provide N—Boc—1,3—diaminopropane using following procedure: To a solution of amine (27.2 g, 207.7 mmol, 3.0 equiv.) in THF solvent (1000 mL) at 0°C was added di-tert—butyl dicarbonate (18.1 g, 83.1 mol, 1 equiv.) dropwise over a period of two hours. During the period of addition, the solution went from clear to a cloudy white. Following addition, the on mixture was allowed to stir at room temperature for a period of 12 hours. The THF solvent was evaporated, and the e was ved in CH2C12 (250 mL) and washed with water (250 mL). The resultant aqueous layer was backwashed with CH2C12 (4 x 100 mL). The organic layer was dried with Na2SO4 and evaporated to afford the N-Boc-1,3-diaminopropane as a clear s liquid.

Benzene-1,3,5-tricarboxyaldehyde was reacted with at least three equivalents of three equivalents of the N-Boc protected 1,3-diaminopropane in the presence of molecular sieve and methanol t to provide a corresponding polyamide t, which was then reduced by sodium borohydride (NaBH4) to e the N-Boc protected polyamine compound VI-la using following procedure: To a stirring solution of 4 A molecular seive (100 mg) in MeOH solvent (20 mL) was added benzene-1,3,5-tricarboxyaldehyde (64 mg, 0.39 mmol, 1 equiv.) followed by N -Boc protected 1,3-diaminopropane (222 mg, 1.19 mmol, 3 equiv.). The solution was stirred for 12 hours at room temperature. The newly formed imine was quenched at room temperature by the addition ofNaBH4 (90 mg, 2.38 mol, 6 equiv.) after which the solution was stirred for an additional one hour. The solution was ﬁltered through a pad of celite, and the solvent evaporated in vacuo to afford brown oil. The crude mixture was dissolved in EtOAc (100 mL) and washed with 10% NaOH (100 mL).

The resulting NaOH was further backwashed with EtOAc (50 mL x 1). The organic layer was dried with Na2804 and concentrated under reduced pressure to provide the N-Boc protected polyamine compound VI—la.

The Boc protecting group on the terminal amine of the polyamine compound VI-la was deprotected by treating compound VI-la with acid to produce the ine compound (VI-1) using following procedure: The crude polyamine compound VI—la was added to a stirring solution of HCl in MeOH solvent (100 mL) and left for one hour. The solvent was evaporated in vacuo to yield a white to off—white solid, which was recrystallized from MeOH solvent to afford the ine compound VI—l at about 30% yield as white solid to off- white solid.

Example 14 ation 0fNI,N i,NIH—(benzene—I,3,5—trz'yltrz's(methylene))trz'sW3—is0pr0pylpr0pane—I,3— diamz'ne) (HCZ) (VII-4C) The polyamine nd (VII-4C) may be synthesized as shown in Scheme 11.

G 8 HEMW G "3' ““1“ i Q HEN’NI’A"N Me M53011, 4 ix ms, than Nam, i‘iNﬁNfNNf frﬁe’Lﬁe R {jigHr“MW“\Nx‘Lm”‘1’ Ema“.

Mei‘Me Compound “£48 Acid Mei:H “1.3me x ”N”WN Me Me Me Ciammund V348 Benzene-1,3,5-tricarboxyaldehyde was reacted with at least three lents of N,N-dimethylnorspermidine in the presence of molecular sieve and methanol solvent to provide a corresponding polyamide product, which was then reduced by sodium borohydride (NaBH4) to produce the N-Boc protected polyamine compound VII-4B using the following procedure: To a ng solution of 4 A molecular sieve (100 mg) in MeOH t (20 mL) was added e-1,3,5-tricarboxyaldehyde (64 mg, 0.39 mmol, 1 equiv.) followed by N, N-dimethylnorspermidine (222 mg, 1.19 mol, 3 equiv.). The solution was stirred for 12 hours at room temperature. The newly formed imine was quenched at room temperature by the addition ofNaBH4 (90 mg, 2.38 mmol, 6 equiv.) after which the solution was stirred for an additional one hour. The solution was ﬁltered through a pad of , and the solvent evaporated in vacuo to afford brown oil. The crude mixture was dissolved in EtOAc (100 mL) and washed with 10% NaOH (100 mL). The resulting NaOH was r backwashed with EtOAc (50 mL x 1). The organic layer was dried with NaZSO4 and concentrated under reduced pressure to provide the crude N—Boc protected polyamine compound VII-4B.

The polyamine compound VII-4B was added to a stirring on of HCl in MeOH solvent (100 mL) and left for one hour. The solvent was ated in vacuo to afford the polyamine compound VII-4C as viscous liquid at about 30% yield. 1H NMR (500 MHz, D20): 7.68 (s, 3H), 4.35 (s, 6H), 3.41 (p, J: 6.5 Hz, 3H), 3.23 (t, J: 7.5 Hz, 6H), 3.14-3.07 (m, 6H), 2.14-2.11 (m, 6H), 1.30 (d, J: 6.5 Hz, 18H).

Example 15 Preparation 0f],3-bz's-[N—(NL3-amin0pr0pyDpropyl—3-amine)-methylamine]-benzene (VII-2) The polyamine nd (VII-2) may be synthesized as shown in Scheme 12. N- (3 -Aminopropyl)propane-l,3-diamine (i.e., norspermidine) was reacted with di-t— butyldicarbonate compound to provide a N—Boc protected norspermidine compound.

Benzene-1,3,5-tricarboxyaldehyde was reacted with at least three equivalents of three equivalents of the N—Boc protected norspermidine compound in the presence of molecular sieve and methanol solvent to provide a ponding polyamide product, which was then reduced by sodium borohydride CNaBH4) to produce the N-Boc protected polyamine compound VII-2:1. using the following procedure: To a stirring solution of 4 A molecular sieve (100 mg) in MeOH solvent (20 mL) was added benzene—l,3,5—tricarboxyaldehyde (64 mg, 0.39 mmol, 1 ) followed by the N-Boc protected norspermidine compound (222 mg, 1.19 mmol, 3 equiv.). The solution was stirred for 12 hours at room temperature. The newly formed imine was quenched at room temperature by the on of NaBH4 (90 mg, 2.38 mmol, 6 equiv.) after which the solution was stirred for an additional one hour. The solution was filtered through a pad of , and the solvent evaporated in vacuo to afford brown oil. The crude mixture was dissolved in EtOAc (100 mL) and washed with 10% NaOH (100 mL). The ing NaOH was further backwashed with EtOAc (50 mL x l).

The organic layer was dried with NaZSO4 and concentrated under reduced pressure to provide the crude N—Boc ted polyamine compound VII-2a.

Mrs-OH, 4 ﬁx ms, than Naam BOCHNMm. Mw‘ M A f\ .M. . . i3! 3 i“ ill gm3’\ l NHaoc ' x i‘imN‘meHaoc N H Compound Viina Acid: MeOH ’8. X ny-n r—h ‘ A" {gmxﬂgmmNHa Compound Wk? The Boc ting group on the terminal amine of the polyamine compound VII- Za was deprotected by ng compound VII-23 with acid to produce the ine compound (VII-2) using following procedure: The crude polyamine compound VII-23 was added to a stirring solution of HCl in MeOH solvent (100 mL) and left for one hour. The solvent was evaporated in vacuo to yield a white to off-white solid, which was recrystallized from MeOH to afford the polyamine compound VII-2 as white solid to off-white solid at % yield.

Example 16 Preparation of],3-bis-[N—3-amin0pr0pyl]benzenediamide (VIII-3) The polyamine compound (VIII-3) may be synthesized as shown in Scheme 13.

+ BOC was, (3.14205 Compound Vina—33 Acid, Meﬂﬁ {:1 C) RQNMN NMNHQ H H Compound Wild Benzene-l,3-dicarbonylchloride was reacted with at least two equivalents of the N- Boc protected aminopropane (prepared as bed in Example 4) in the presence of triethylamine (NEtg) and dichloromethane solvent to provide a corresponding N-Boc protected polyamide nd VIII-3a. The Boc protecting group on the terminal amine of the polyamine compound VIII-3a was deprotected by treating compound VIII-3a with acid, such as acetyl chloride, in methanol to produce the polyamine compound (VIII-3).

Example 17 General for preparation ofa’iala’ehya'e aryl starting material An aldehyde precursor to the polyamine compound may be produced by the palladium coupling method of General Synthetic Scheme 14. l Synthetic Scheme 14 O O O O O-R5 a H H H H + X-B: O-R5 Step 1 Br X X = aryl, alkyl, vinyl, heteroaryl Reagents: (a) 5:1 DME/HZO, 3)4, K2C03 Speciﬁc examples of this method are set forth in the following Examples, such as Example 68.

Example 18 Preparation 0fN1,N] '—(1,3—phenylenebis(methyleneﬂbis(N3—(3—amm0ni0pr0pyl)pr0pane—1,3— diamz'm'um) chloride (CZ-25) < NHZ I"'\‘_\_/NH 3—} - 6 HCI HZN HN

[0432] Step 1: Di—tert—butyl ,3—phenylenebis(methylene))bz's(azanediyl))bz's(pr0pane— 3,1-diyll)l)bis(azanediyl)‘)bis(propane-3,1—diyl))dicarbamate. Isophthalaldehyde (1.07 g, 7.99 mmol) and MeOH (50 mL) were added to a round-bottom ﬂask. To the solution was added tert—butyl (3-((3 -arninopropyl)arnino)propyl)carbamate (3.69 g, 16.0 mmol) and the on mixture was stirred for 24 h. Sodium borohydride (1.21 g, 32.0 mmol) was added portionwise and stirred for 1 h. The reaction mixture was concentrated under reduced pressure, and aq. NaOH (10%, 150 mL) and EtOAc (150 mL) were added. The layers were separated, and the s layer was extracted with EtOAc (150 mL). The organic layers were combined, dried over NaZSO4, ﬁltered, and concentrated under reduced pressure to afford the desired product as a clear oil. The intermediate was carried forward without further ation.

Step 2: N1,NI '-(I,3-Phenylenebis(methylene))bis(N3-(3-amin0pr0pyl)pr0pane—1,3- diamz'ne): To the crude di-tert-butyl (((((1,3-phenylenebis(methylene))bis(azanediyl))- bis(propane-3,1-diyl))bis(azanediyl))bis(propane-3,1-diyl))dicarbamate from Step 1 was added methanolic HCl (150 mL, 1.0 M). The reaction mixture was stirred for 2 h and concentrated under reduced pressure. The solid was collected by vacuum ﬁltration, and it was washed with Et20 (30 mL) and hot MeOH (30 mL) to afford the desired product (1.1 g, 56%) as a white solid. 1H NMR (500 MHz, D20) 8 ppm 7.62 (s, 4H), 4.36 (s, 4H), .21 (m, 12H), 3.15 (t, J: 8.0 Hz, 4H), 2.23-2.11 (m, 8H). 13C NMR (125 MHz, D20) 131.4, 131.3, 131.1, 130.2, 50.8, 44.7, 44.6, 44.1, 36.5, 23.6, 22.6. LRMS [M+H]+365.3.

Example 19 Preparation ofNI-benzylbutane-1,4-a’iaminium de (CZ-3) N/\/\/NH2 - 2 HCI Example 19 was prepared in a similar fashion to Example 18 (CZ-25) from benzaldehyde and tert—butyl (4-aminobutyl)carbamate. 1H NMR (500 MHZ, D20) 5 7.52 (s, 5H), 4.26 (s, 2H), 3.15 (t, J= 8 Hz, 2H), 3.07 (t, J: 7.5 Hz, 2H), .74 (m, 4H). 13C NMR (125 MHz, D20) 130.6, 129.9, 129.7, 129.2, 51.0, 46.3, 38.8, 23.9, 22.7. LRMS [M+H]+ 179.2.

Example 20 Preparation ofNI-benzylpropane-I, 3-a’iamine (CZ-4) Example 20 was prepared in a similar fashion to Example 18 (CZ-25) from benzaldehyde and tert—butyl (3-aminopropyl)carbamate. 1H NMR (500 MHz, D20) 8 7.49 (quint, J: 3 Hz, 5H), 4.26 (s, 2H), 3.17 (t, J = 8 Hz, 2H), 3.07 (t, J = 8 Hz, 2H), 2.09 (quint, J = 7.5 Hz, 2H). 13C NMR (125 MHz, D20) 5 130.6, 129.9, 129.8, 129.4, 51.4, 44.1, 36.7, 23.9. LRMS [M+H]+291.3. e 21 Preparation enzyla'oa'ecane-IJZ-a'iamine, hydrochloride salt (CZ-5) (3‘4 NH2 '2 HCI

[0436] Example 21 was prepared in a similar fashion to Example 18 (CZ-25) from benzaldehyde and tert—butyl (12-aminododecyl)carbamate.

Example 22 Preparation ofNI-(3-(benzylamino)propyl)-N3,N3-dimethylpropane-I, 3-diamine, hydrochloride salt (CZ-6) ©/\N/\/\N/\/\N/H H | °3HCI e 22 was prepared in a similar fashion to Example 18 (CZ-25) from benzaldehyde and aminopropyl)—N3,N3-dimethylpropane-l,3-diamine. 1H NMR (500 MHz, D20) 8 7.41 (s, 5H), 4.18 (s, 2H), 3.20-3.08 (m, 8 H), 2.83 (s, 6H), 2.15-2.03 (m, 4H). 13C NMR (125 MHz, D20) 131.0, 130.5, 130.4, 129.9, 54.8, 51.8, 45.3, 45.1, 44.5, 43.5, 23.2, 21.8.

Example 23 Preparation ofNI-(3-aminopropyl)-N3-benzylpropane-I, 3-diamine, hydrochloride salt (CZ- ©/\H/\/\H/\/\NH2 '3HCI Example 23 was prepared in a similar fashion to Example 18 ) from benzaldehyde and tert-butyl (3-((3-aminopropyl)amino)propyl)carbamate. 1H NMR (500 MHz, D20) 5 7.49 (s, 5H), 4.25 (s, 2H), 3.20—3.15 (m, 6H), 3.11 (t, J = 8 Hz, 2H), 2.17—2.07 (m, 4H). 13(3 NMR (125 MHz, D20) 5 130.3, 129.8, 129.7, 129.2, 51.2, 44.6, 44.6, 43.8, 36.5, 23.7, 22.6. LRMS [M+H]+222.2.

Example 24 Preparation ofNI-benzylhexane-I, 6—diamine, hydrochloride salt (CZ-9) -2HC| Example 24 was ed in a similar fashion to Example 18 (CZ-25) from benzaldehyde and tert-butyl (6-aminohexyl)carbamate.

Example 25 Preparation ofNI-Benzyl-NI,N12,N12-trimethyldodecane-I,I2-diamine, hydrochloride salt «24m o”.N /N\ ~2Hm Example 25 was prepared in a similar fashion to Example 18 ) from benzaldehyde and N1,N1 ,N12-trimethy1dodecane-1, 12-diamine. e 26 Preparation ofNI-benzyl-N1,N12,NIZ—trimethyldodecane-I,I2-diamine, hydrochloride salt (CZ-1 1) NW”“Q ~2Hm E:j/\H Example 26 was prepared in a similar fashion to Example 18 ) from benzaldehyde and dodecane-1,12-diamine. 1H NMR (500 MHZ, D20 + CDgOD) 5 7.53 (s, 10H), 4.24 (s, 4H), 3.04 (t, J = 8.5 Hz, 4H), 1.77-1.70 (m, 4H), 1.42-1.28 (m, 16 H). 13C NMR (125 MHZ, D20 + CD3OD) 6 131.0, 129.8, 129.6, 129.3, 50.9, 47.0, 29.2, 29.1, 28.7, 26.2, 25.7.

Example 27 ation ofNI-benzyl-N1,N12,NU—trimethyldodecane-I,12-diamine, hydrochloride salt «24» HzNM”/\©/\”MNH2 °4HC|

[0442] Example 27 was prepared in a similar fashion to Example 18 (CZ-25) from isophthalaldehyde and tert—butyl (3 -aminopropy1)carbamate.

Example 28 ation ofNI-(3-methoxybenzyl)dodecane-I,12-diamine, hydrochloride salt (CZ-13) N/\/\/\/\/\/\/NHZ - 2 HCI Example 28 was prepared in a similar fashion to Example 18 (CZ-25) from 3- methoxybenzaldehyde and tert—butyl (12-aminododecyl)carbamate. 1H NMR (500 MHz, D20) 5 7.40 (t, J= 8.0 Hz, 1H), 7.06 (s, 1H), 7.05 (s, 1H), 7.03 (s, 1H), 4.16 (s, 2H), 3.82 (s, 3H), 3.00—2.93 (m, 4H). 1.66-1.58 (m, 4H), 1.32—1.22 (m, 16H). 13c NMR (125 MHz, D20) ,131.5,129.8,121.6,114.5,114.3, 54.7, 49.8, 46.1, 38.7, 27.8, 27.7, 27.4, 25.9, 24.8, 24.5.

Example 29 Preparation 3-aminopropyl)-N3-(3-(benzylamino)propyl)propane-1,3-diamine, hydrochloride salt (CZ— l 6) ©/\u/\/\u/\/\u/\/\NH2 °4HCI Example 29 was prepared in a similar fashion to Example 18 (CZ-25) from benzaldehyde and tert-butyl (3-((3-((3-aminopropyl)amino)propyl)amino)propyl)carbamate. 1H NMR (500 MHZ, D20) 5 7.49 (s, 5H), 4.27 (s, 2H), 3.51-3.49 (m, 6H), 3.27-3.14 (m, 6H), 3.12 (t, .1: 8.0 Hz,2H),2.21-2.10(m,4H). 13c NMR (125 0)5130.5, 130.0, 130.0, 129.5, 51.4, 45.3, 45.2, 43.9, 43.2, 36.6, 27.8, 23.9, 22.8.

Example 30 Preparation ofNI,N1V—((2,4,6—trimethyl—I,3-phenylene)bis(methylene))bis(propane-I,3- diaminium) de (CZ-19) HZNM” ”MNHZ Me Me - 4 HCI Example 30 was prepared in a similar fashion to Example 18 (CZ-25) from 2,4- bis(bromomethyl)- 1 ,3 ,5 -trimethylbenzene tert-butyl (3 -aminopropyl)carbamate.

Example 31 Preparation ofNI,NI -phenylenebis(methylene))bis(dodecane-I, IZ—diamine), hydrochloride salt (CZ-21) H2N\/\/\/\/\/\/\N NWNHZ H H - 4 HCI e 31 was prepared in a similar fashion to Example 18 (CZ-25) from isophthalaldehyde and tert—butyl inododecyl)carbamate. 1H NMR (500 MHZ, D20) 8 7.65 (s, 3H), 4.36 (s, 4H), 3.11 (t, J: 8.0 Hz, 4H), 3.06 (t, J: 7.5 Hz, 4H), 1.77-1.68 (m, 8H), 1.47—1.33 (m, 32H). 13c NMR (125 MHz, D20) 5 130.4, 129.9, 129.7, 128.9, 49.0, 45.6, 38.2, 27.3, 27.3, 27.2, 27.1, 26.9, 26.8, 25.4, 24.4, 24.3, 24.0. LRMS [MJrH]+ 503.5.

Example 32 Preparation of(3-(((3-((3-aminopropyl)amino)propyl)amino)methyl)phenyl)methanol, hydrochloride salt (CZ-22) HO/\©/\N/\/\NHH ° 3 HCI KL e 32 was prepared in a r fashion to Example 18 (CZ-25) from isophthalaldehyde and N1,N1-dimethylpropane-1,3-diamine.

Example 33 Preparation ofNI,NI '-(I,3-phenylenebis(methylene))bis(hexane-I, 6-diamine), hydrochloride salt (CZ-26) H2N\/\/\/\N N/\/\/\/NH2 H H ° 4 HCI

[0448] Example 33 was prepared in a similar fashion to Example 18 (CZ-25) from isophthalaldehyde and tert—butyl (6-aminohexy1)carbamate.

Example 34 Preparation ofdi-tert—butyl (((((I,3—phenylenebis(methylene))bis(azanediyl))bis(propane-3,I- diyl))bis(azanediyl))bis(propane-3,I—diyl))dicarbamate (CZ-27) BocHNJPNLNH NHBoc Example 34 was prepared in a similar fashion to Example Example 18 (CZ-25) from benzaldehyde and tert—butyl (3-((3-aminopropyl)amino)propyl)carbamate.

Example 35 Preparation ofNI,NI 2N1 ”— (benzene-I, 3,5-triyltris(methyleneUtrisCoropane-I,3-diamine) hydrochloride salt (CZ-32) HzNM” ”MNHZ f ° 6 HCI Example 35 was prepared in a similar n to Example 18 (CZ-25) from benzene-1,3,5-tricarbaldehyde and tert-butyl (3—aminopropyl)carbamate.

Example 36 Preparation ofNI,NI 2N1 ”— (benzene-I, 3, 5-triyltri's(methylene))tris(hexane-I, 6-diamine), hydrochloride salt (CZ-33) H2N\/\/\/\N NWNHZ H H '6 HCI e 36 was ed in a similar fashion to e 18 (CZ-25) from benzene-1,3,5-tricarbaldehyde and tert-butyl (6-aminohexyl)carbamate.

Example 37 Preparation ofNI,NI '-(’((I, 3-phenylenebis(n1ethylene))bis(azanediyl))bis(propane-3, I- diyl))bis(N3,Nj-dimethylpropane-I,3-diamine), hydrochloride salt (CZ-43) °6HC| Example 37 was prepared in a similar fashion to Example 18 (CZ-25) from isophthalaldehyde and N1-(3-aminopropyl)-N3 ,N3-dimethylpropane-1,3-diamine. 1H NMR (500 MHZ, D20) 8 7.57 (s, 4H), 4.31 (s, 4H), 3.28-3.14 (m, 16H), 2.91 (s, 12H), .10 (m, 8H). 13c NMR (125 MHz,D20) 5 132.1, 131.9, 131.8, 130.9, 54.8, 515,453,451, 44.8, 43.5, 23.3, 21.9.

Example 38 Preparation ofNI,NI ZN] ”— (benzene-I, 3, 5-triyltris(methylene))tris(N3,N3-dimethylpropane- 1,3-diamine), hloride salt (CZ-46) \NMN NMN/ | H H | NH ° 6 HCI Example 38 was prepared in a similar fashion to e 18 (CZ-25) from benzene-1,3,5-tricarbaldehyde and N1,N1-dimethylpropane-1,3-diamine.

Example 39 Preparation of(3, 5-bis(((3-((3-aminopropyl)amino)propyl)amino)methyl)phenyl)-methanol, hydrochloride salt (CZ-53) HZNMEME z

[0454] Example 3 was prepared in a similar fashion to Example 18 (CZ-25) from 5- (hydroxymethyl)isophthalaldehyde and utyl (3-((3- aminopropyl)amino)propyl)carbamate. 1H NMR (500 MHZ, D20) 5 7.57 (s, 2H), 7.55 (s, 1H), 4.72 (s, 2H), 4.35 (s, 4H), 3.26-3.19 (m, 12H), 3.13 (t, J: 7.5 Hz, 4H), 2.21-2.09 (m, 8H). 13C NMR (125 MHz, D20) 5 142.8, 132.0, 130.7, 130.0, 63.2, 51.0, 44.9, 44.8, 44.4, 36.7, 23.9, 22.9.

Example 40 Preparation oftri-tert—butyl— (((((benzene-1,3, 5-triyltris lene))tris (azanediyl))tris- (propane-3, ))tris(azanediyl))tris(propane-3, I -diyl))tricarbamate, hydrochloride salt (CZ-54) O O J< H H H H H H °6HC| OxJ<O

[0455] Example 40 was prepared in a similar fashion to Example 18 (CZ-25) from benzene-1,3,5-tricarbaldehyde and tert-butyl (3—((3-aminopropyl)amin0)pr0pyl)carbamate.

Example 41 ation ofNI,N1'-(1,3-phenylenebis(methylene))bis(N3-(3-((3- aminopropyl)amino)propyl)propane—1,3-diamine), hydrochloride salt (CZ-57) HZNMEMEMNUNMHMEMNHZ °8HCI Example 41 was prepared in a similar fashion to Example 18 (CZ-25) from isophthalaldehyde and tert—butyl (3-((3-((3 -amin0pr0pyl)amin0)pr0pyl)amin0)pr0pyl)- carbamate.

Example 42 Preparation of3,3 '-(isophthaloylbis(azanediyl))bis(propanaminium) de (CZ-40) Step 1: tert—BuW/(5-(3-«3-((tert—butoxycarbonyl)amin0)pr0pyl)carbamoyl)phenyl)- -oxopentyl)carbamate. Isophthaloyl chloride (344 mg, 1.70 mmol), ylamine (343 mg, 3.41 mmol), and CHzClz (10 mL) were added to a round-bottom ﬂask. To the solution was added tert-butyl (3-aminopropyl)carbamate (593 mg, 3.41 mmol), and the reaction mixture was stirred for 16 h. The e was washed with aq. NaOH (10%, 50 mL) and the layers separated. The aqueous layer was extracted with CHZCIZ (2 x 50 mL) and organic layers were combined, dried over NagSO4, ﬁltered, and concentrated under reduced pressure. Puriﬁcation by column tography (hexanes/EtOAc) afforded the desired product, which was used without further puriﬁcation.

Step 2: 3,3 '—(Isophthaloylbis(azanediyl))bisQJropan-I-aminium) chloride. To the crude tert—butyl(5 -(3-((3-((tert-butoxycarbonyl)amino)propyl)carbamoyl)phenyl) oxopentyl)carbamate from Step 1 was added methanolic HCl (150 mL, 1.0 M). The reaction mixture was stirred for 2 h and concentrated under reduced pressure. The solid was collected by vacuum ﬁltration and washed with EtzO (30 mL) and hot MeOH (30 mL) to afford the desired product (0.388 g, 56%) as a white solid.

Example 43 Preparation ofNI,NI 2N1 ”—(benzene—I, 3,5—triyltris(methylene))tris(N3—(3— aminopropyl)propane-I3-diamine) hydrochloride salt (CZ-52) H2N\V/J;:;]NLlﬁ:\lLH2 -9HCI HNVAVNH2 Step 1: Tri-tert—butyl enzene-I,3,5-triyltris(methylene))tris(azanedin)- trisCoropane-j’, ))tris (azanedin)tris(propane-3, I—din)tricarbamate. Benzene-1,3,5- tricarbaldehyde (1.80 g, 11.04 mmol) and MeOH (50 mL) were added to a bottom ﬂask. To the solution was added trimethyl orthoformate (165.6 g, 17.6 mmol) and tert-butyl (3 -((3 -aminopropyl)amino)propyl)carbamate (7.66 g, 33.1 mmol) and the reaction mixture was stirred for 24 h. Sodium borohydride (1.67 g, 44.2 mmol) was added portionwise and stirred for 1 h. The reaction mixture was concentrated under reduced pressure and aq. NaOH (10%, 150 mL) and EtOAc (150 mL) were added. The layers were separated and the aqueous layer was extracted with EtOAc (150 mL). The organic layers combined, dried over NaZSO4, ﬁltered, and concentrated under reduced pressure to afford the desired product as a clear oil.

The intermediate was carried forward without further puriﬁcation.

Step 2: N’,N’ 2N] zene-I,3,5-trz'yltrz's(methylene))tris(N3-(3- aminoproprpropane-1,3-a’z'amz'ne), hydrochloride salt. To the crude tri-tert-butyl (((((benzene- 1 ,3 ,5 tris(methylene))tris(azanediyl))tris(propane-3 ,1- tris(azanediyl))tris(propane-3,1-diyl))tricarbamate from Step 1 was added methanolic HCl (150 mL, 1.0 M). The reaction mixture was stirred for 2 h and concentrated under reduced pressure. The solid was collected by vacuum filtration and washed with EtzO (30 mL) and hot MeOH (30 mL) to afford the desired product (5.6 g, 61%) as a white solid.

LRMS [M+H]+ 508.5.

Example 44 Preparation oftert—butyl (3—((3-((3,5-bis(((3-((3-aminopropyl)amino)propyl)amz'no)methyl)- benzyl)amino)propyl)amino)propyl)carbamate ) H H HZNJg l/NH NHBOC WNH °8HCI HNWNHZ

[0461] Example 44 was prepared in a similar fashion to Example 43 (CZ—52) from e- tricarbaldehyde and tert-butyl (3-((3 -aminopropyl)amino)propyl)carbamate.

Example 45 Preparation ofN’,N”_([1,1Lbz‘phenyzj—3,5—diy1bis(methyzene))MSW—(3— ammoniapropyl)pr0pane-I,3-diaminz'um) de (CZ-58) < NH2 HN_'\_/NH 3“)”

[0462] Step 1: [1,1'-Biphenyl]-3,5-dicarbaldehyde. A solution of 5- bromoisophthaldehyde (40.0 g, 187.8 mmol) (Med. Chem. ., 2012, 3, 763—770), phenylboronic acid (22.9 g, 187.8 mmol) and potassium ate (64.8 g, 469.5 mmol) in DME/HZO (5:1, 600 mL) was purged with N2 for 5 min. Tetrakispalladium triphenylphosphine (1.1 g, 0.9 mmol) was added, and the reaction mixture was heated to 90 °C and stirred for 16 h. The reaction mixture was cooled to rt, d h a pad of Celite diatomaceous earth, and the solvent evaporated under reduced pressure. Puriﬁcation by column chromatography (10% EtOAc/hexanes) afforded the desired product (74%, 29.1 g) as an off-white solid. 1H NMR (300 MHz, CDClg) 8 ppm 10.18 (s, 2H), 8.37 (d, J: 1.2 Hz, 2H), 8.35 (t, J: 1.5 Hz, 1H), 7.70—7.66 (m, 2H), .43 (m, 3H).

[0463] Step 2: Di—tert—butyl ((((([I, I '—bz'phenyl]-3,5-dlylbis(methylene))bis(azanediy0)bis- (propane-3, I-diyl))bis(azanediywbismropane-i I—diyl))dicarbamate. [1 , 1‘-Biphenyl]—3,5- dicarbaldehyde (3.62 g, 17.24 mmol) and MeOH (100 mL) were added to a round-bottom ﬂask. To the solution was added tert-butyl (3-((3—aminopropyl)amino)propyl)carbamate (7.96 g, 34.4 mmol), and the reaction mixture was stirred for 24 h. Sodium borohydride (2.62 g, 69.0 mmol) was added, and the mixture was stirred for 1 h. The solvent was removed under reduced pressure to afford a white solid. Aqueous NaOH (10%, 200 mL) was added, and the solution was extracted with EtOAc (200 mL). The organic layer was separated, and the aqueous layer was extracted with EtOAc (200 mL). The organic layers were combined, dried over Na2S04, ﬁltered, and concentrated under reduced pressure to afford the d product as a clear oil that was used without further puriﬁcation.

Step 3: N1,NII-([I,I '-bz'phenyl]-3,5—dl'ylbis(methylene))bis(N3-(3- ammoniopropyl)pr0pane-1,3-dz'amz'm'um) chloride. To the crude -([1,l'-biphenyl]-3,5- diylbis(methylene))bis(N3—(3—ammoniopropyl)propane—1,3—diaminium) chloride from Step 2 was added olic HCl (150 mL, 1.0 M). The reaction mixture was d for 2 h and concentrated under reduced pressure. The solid was collected by vacuum ﬁltration and washed with EtzO (30 mL) and hot MeOH (30 mL) to afford the desired product (6.8 g, 60%) as a white solid. 1H NMR (D20, 500 MHZ) 5 ppm 7.85 (s, 2H), 7.74 (d, J: 7.5 HZ, 2H), 7.59-7.56 (m, 3H), 7.50-7.49 (m, 1H), 4.38 (s, 4H), 3.29 (t, J=8.0 Hz, 4H), 3.23 (q, J= 5.0 Hz, 8H), 3.14 (t, J: 8.0 Hz,4H),2.25-2.19(m,4H),2.17-2.11 (m, 4H). 13c NMR (125 MHz, D20) 142.3, 138.8, 132.1, 130.0, 129.5, 129.2, 128.4, 127.0, 50.8, 44.7, 44.6, 44.2, 36.5, 23.7, 22.7. LRMS [M+H]+441.4.

Example 46 Preparation 0fN1,N1 '—((5-(benzo[d][l,3]di0x0[—5—yl)-I,3-phenylene)bisMethylene»bis(N3- (3-amm0ni0pr0py0pr0pane-I,3-dz'amz'm'um) de (CZ-61) < NH2 HN—\_/NH HN OW j? 0 O 0 HZN HN -6HC| Example 46 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, benzo[d][1,3]dioxolylboronic acid, and tert—butyl (3 -((3- aminopropyl)amino)propyl)carbamate. N1,N1'-((5 -(benzo[d][1,3]dioxol-5 -yl)-1,3- phenylene)bis(methylene))bis(1\73-(3-aminopropyl)propane-1,3-diamine), hydrochloride salt: 1H NMR (500 MHz, D20) 8 7.72 (s, 2H), 7.49 (s, 1H), 7.15 (s, 2H), 6.93 (d, J = 8.5 Hz, 1H), .95 (s, 2H), 4.30 (s, 4H), .12 (m, 8H), 3.05 (t, J 2 8.0 Hz, 4H), 2.17—2.02 (m, 8H). 13c NMR(125 MHz, D20)8148.1, 147.6, 142.3, 133.3, 132.2, 130.8, 129.7, 129.6, 129.4, 121.1, 109.0, 107.5, 101.6, 51.0, 44.8, 44.8, 44.4, 36.6, 23.9, 22.8.

Example 47 Preparation ofNI,N1 EN] ”-([1, I '-biphenyl]-3,3 ',5-triyltris(methylene))tris(N3-(3— aminoproprpropane-1,3-diamine), hydrochloride salt (CZ-62)

[0466] Example 47 was prepared in a r fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, (3-f0rmy1phenyl)b0r0nic acid and tert-butyl (3-((3- aminopropy1)amin0)pr0pyl)carbamate. 1H NMR (400 MHZ, D20) 5 7.96 (s, 2H), 7.93—7.85 (m, 2H), 7.71-7.61 (m, J= 8.5 Hz, 3H), 4.47 (s, 4H), 4.44 (s, 2H), 3.36-3.23 (m, 18H), 3.17 (t, J: 8.0 HZ, 6H), 2.30-2.22 (m, 6H), 2.20-2.13 (m, 6H). LRMS [M+H]+ 584.5.

Example 48 ation ofN’,N’ '-((2'-methyl-[1,1 '-biphenyl]-3, 5-diyl)bis(methylene))bis(N3—(3- aminoproprpropane-1,3-diamine), hloride salt (CZ-63) HN_\_/NH e 48 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5— bromoisophthaldehyde, o-tolylboronic acid and tert—butyl (3-((3- aminopr0py1)amino)propyl)carbamate. 1H NMR (500 MHZ, D20) 5 7.59 (s, 1H), 7.56(d, J = 1.0 HZ, 2H), 7.41—7.40 (m, 2H), 7.38—7.35 (m, 1H), 7.32—7.31 (m, 1H), 4.38 (s, 4H), 3.25 (t, J =7.5 Hz, 4H), 3.20-3.18 (m, 8H), 3.11 (t, J = 8.0 HZ, 4H), 2.25 (m, 3H), 2.20-2.15 (m, 4H), 2.14—2.07 (m, 4H) ). 13c NMR (125 MHZ,D2O)8143.3,139.9,135.6,131.8,131.7,131.5, 131.0, 130.5, 129.9, 129.7, 128.2, 126.1, 50.8, 44.7, 44.6, 44.2, 36.6, 23.7, 22.7. LRMS [M+H]+455.4.

Example 49 Preparation ofNI,N1 '-('(4 '-morpholino-[1, 1 '-biphenyl]-3,5-diyl)bis(methylene))bis(N3- (3- aminoproprpropane-I,3-diamine), hydrochloride salt (CZ-64) MP 6km"...

Example 49 was prepared in a similar fashion to Example 45 (CZ-58) from 5- bromoisophthaldehyde, (4-m0rpholin0phenyl)b0r0nic acid and utyl (3-((3- aminopropy1)am1n0)propyl)carbamate.

Example 50 Preparation ofN],NI',N1"-([1, 1'-biphenyl]-3,4C5-triyltris(methylene))tris(N3-(3— roprpropane-1,3-diamine), hydrochloride salt (CZ-65) < NH2 l'm_\_/NH HZNjHM? 0 O NH -9HC| (—/ HN NH2

[0469] e 50 was prepared in a similar fashion to e 45 (CZ-5 8) from 5- bromoisophthaldehyde, (4-f0rmy1phenyl)boronic acid and tert-butyl (3-((3- aminopropy1)amino)propyl)carbamate. 1H NMR (500 MHZ, D20) 5 7.92 (s, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 3H), 4.43 (s, 4H), 4.37 (s, 2H), 3.31-3.28 (m, 6H), 3.26-3.20 (m, 12H), 3.14 (t, J = 8.0 Hz, 6H),2.25—2.17 (m, 6H), 2.16-2.10(m, 6H). 13C NMR (125 MHz, D20) 8 141.6, 140.1, 132.2, 130.6, 130.4, 129.7, 127.8, 50.8, 44.7, 44.6, 44.2, 36.5, 23.7, 22.7, 22.6. LRMS [M+H]+584.5.

Example 51 Preparation ofN’,N’ 1N1 ("-(4'-l'sopropoxy-[I,I '-bz'phenyl]-3,3 ', 5-trz'yl)tris(methylene))- tris(N3-(3-aminopropyl)propane-I,3-diamine), hydrochloride salt (CZ-66)

[0470] Example 51 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, (3-f0rmylisopropoxyphenyl)boronic acid and tert-butyl - ammopropyl)amin0)pr0pyl)carbamate. 1H NMR (500 MHZ, D20) 5 7.93 (s, 2H), 7.88-7.82 (m, 2H), 7.65 (s, 1H), 7.35 (d, J = 9.0 Hz, 1H), 4.94-4.87 (m, 1H), 4.47 (s, 4H), 4.43 (s, 2H), 3.39-3.28 (m, 18H), 3.22 (t, J = 8.0 Hz, 6H), 2.33-2.18 (m, 12H), 1.48 (d, J = 6.5 Hz, 6H). 13C NMR (125 MHz, D20)8158.9,143.9,134.8,133.8,132.9,132.6,132.3,131.8,122.7, 117.0, 84.4, 74.5, 53.5, 49.6, 47.3, 47.3, 47.0, 46.9, 46.7, 39.2, 26.3, 25.3, 25.2, 23.9. LRMS [M+H]+642.5.

Example 52 Preparation I '-(’(4 '-z'sopropoxy-[I, I '-biphenyl]-3,5-dz'yl)bis(Methylene))bis(N3-(3- aminoproprpropane-1,3-dz'amz'ne), hydrochloride salt ) {NHNHZ HN—\_/ M)—Me H2N3—?0000 -6HC| Example 52 was prepared in a similar fashion to e 45 (CZ-5 8) from 5- bromoisophthaldehyde, (4-isopropoxyphenyl)boronic acid, and tert-butyl (3-((3- aminopropyl)amino)propyl)carbamate. 1H NMR (500 MHz, D20) 5 7.81 (s, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.54 (s, 1H), 7.12 (d, J: 8.5 Hz, 2H), 4.78-4.72 (m, 1H), 4.37 (s, 4H), 3.28 (t, J = 8.0 Hz, 4H), .19 (m, 8H), 3.13 (t, J= 8.0 Hz, 4H), 2.24-2.19 (m, 4H), 2.17-2.09 (m, 4H), 1.35 (d, J= 6.0 Hz, 6H). 13C NMR (125 MHz,D20)6157.0, 141.8, 132.0, 131.8, 129.4, 129.0, 128.3, 116.8, 71.6, 50.8, 44.6, 44.6, 44.1, 36.4, 23.6, 22.6, 21.0.

Example 53 Preparation 0fN1,NI '-((5-(thz'0phenyl)-1,3-phenylene)bis(methylene))bisﬂ\73-(3- ropyl)pr0pane-1,3-a’z'amz'ne), hydrochlarz'de salt (CZ-68) < NH2 l"N_\_/NH )7HN S HZN HN '6HCI Example 53 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, (4-(thi0phenyl)phenyl)boronic acid and tert-butyl - aminopropyl)amino)propyl)carbamate. 1H NMR (500 MHz, D20) 8 7.90 (s, 2H), 7.83 (t, J = 1.0 Hz, 1H), 7.63-7.61 (m, 1H), 7.60-7.57 (m, 1H), 7.53 (s, 1H), 4.38 (s, 4H), 3.27 (t, J = 8.0 Hz, 4H), 3.23-3.19 (m, 8H), 3.13 (t, J = 8.0 Hz, 4H), 2.23-2.17 (m, 4H), 2.15-2.09 (m, 4H). 13c NMR(125 MHz,D20)8139.9, 137.1, 1322,1296, 128.7, 127.7, 125.9, 122.3, 50.8, 44.7, 44.6, 44.1, 36.5, 23.7, 22.6. LRMS [M+H]+ 447.3.

Example 54 Preparation ofN’,N’ 2N1"—((4Larmuoromethyu—[L1 '-biphenyl]-3,3',5- triyl)tris(methylene))tris(N3-(3-aminopropyl)propane-I,3-dz'aml'ne), hydrochloride salt (CZ- -9HC| g e 54 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, myl(triﬂuoromethy1)phenyl)boronic acid and tert-butyl (3- ((3-aminopropyl)amino)propyl)carbamate. 1H NMR (500 MHZ, D20) 8 7.93 (s, 2H), 7.87 (s, 2H), 7.61 (s, 2H), 4.45 (s, 2H), 4.38 (s, 4H), 3.27-3.14 (m, 16H), 3.10-06 (m, 8H), 2.25-2.05 (m, 12H). e 55 Preparation ofNI,NI '-((325 '-bis(trzﬂuoromethyl)-[I,1 '-bl'phenyl]-3,5- diyl)bis(methylene))bis(N3—(3-aminopropyl)pr0pane-I,3-dz'amz'ne), hydrochloride salt (CZ- < ‘NHZ HN—\_/NH H2N4§““1 O OHN CF3 Example 55 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, (3,5-bis(triﬂuoromethyl)phenyl)boronic acid and tert-butyl (3 -((3- aminopropy1)amino)propyl)carbamate. 1H NMR (500 MHz, D20) 5 8.20 (s, 2H), 8.07 (s, 1H), 7.91 (s, 2H), 7.69 (s, 1H), 4.41 (s, 4H), 3.30 (t, J = 7.5 Hz, 4H), 3.26-3.21(m, 8H), 3.14 (t, J = 8.0 Hz, 4H), 2.23-2.19 (m, 4H), 2.17—2.10 (m, 4H). 13C NMR (125 MHz, D20) 5 140.6, 139.7, 132.4, 131.5, 131.2, 129.9, 127.5, 124.4, 122.2, 50.7, 44.7, 44.6, 44.3, 36.5, 23.7, 22.6. LRMS [M+H]+577.3.

Example 56 Preparation ofNI,NI -(pyridinyl)-I,3-phenylene)bis(methylene))bis(N3-(3- aminopropyl)propane-1,3-diamine), hloride salt (CZ-71) < NH2 HN_\_/NH HN _ HZN HN - 6 HCI Example 56 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5— bromoisophthaldehyde, pyridin—4-ylboronic acid and tert-butyl (3 -((3- aminopropyl)amin0)pr0pyl)carbamate. 1H NMR (500 MHZ, D20) 5 8.87 (d, J = 6.0 HZ, 2H), 8.39 (d, J = 6.5 Hz, 2H), 8.15 (s, 2H), 7.88 (s, 1H), 4.47 (s, 4H), 3.30 (t, J = 8.0 Hz, 4H), 3.24-3.19(m, 8H), 3.12 (t, J = 8.0 Hz, 4H), 2.24—2.19 (m, 4H), 2.15-2.08 (m, 4H). 13c NMR (125 MHZ, D20)8156.6, 141.6, 1368, 1344,1332, 131.0, 125.2, 50.8, 44.9, 44.8, 44.6, 36.7, 23.9, 22.9. LRMS [M+H]+442.4.

Example 57 Preparation ofNI,NI -(6—methoxypyridinyl)-I,3-phenylene)bis (methylene))bis(N3-(3- aminopropyl)propane-1,3-diamine), hydrochloride salt (CZ-72) HN—\_/NH HN —N § \ / 0Me H2N HN -6HC| Example 57 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, (6-methoxypyridin-3 -yl)boronic acid and utyl (3-((3- aminopropyl)amino)propyl)carbamate. LRMS [M+H]+ 472.4.

Example 58 Preparation ofNI,N1 '_([1,1 '-biphenyl]-3, 5-dz'ylbz's (methylene))bz’s(N3—(3_((3_ aminopropyl)amino)propyl)propane—I,3-dz'amine), hydrochloride salt (CZ-73) < HN HN—\_/NH —\—\ [\N -8HC| Example 58 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, phenylboronic acid and tert—butyl (3-((3-((3- aminopropyl)amino)propyl)amino)propyl)carbamate.

Example 59 Preparation ofNI,NI phenoxy-I,3-phenylene)bis(methylene))bis(N3-(3- aminopropyl)propane-1,3-dz'amz'ne), hydrochloride salt (CZ-74) .3” Q

[0478] Step 1: Dimethyl 5-phenoxyts0phthalate. 5-Hydroxyisophthalate (210 mg, 1.00 mmol), phenylboronic acid (0.244 g, 2.0 mmol), copper(Il) acetate (0.182 mg, 1.0 mmol) and triethylamine (0.303 mg, 3.0 mmol) in CH2C12 (20 mL) was d at rt in the open air for 38 h. onal phenylboronic acid (0.061 mg, 0.50 mmol) was added, and the reaction mixture was stirred at rt in the open air. After 24 h, additional phenylboronic acid (0.061 g, 0.50 mmol) and copper acetate (0.020 g, 0.11 mmol) were added to the reaction mixture, and the e was stirred for 6 h. The mixture was diluted with CHClz (20 mL) and aqueous HCl (10 mL). The organic layer was separated, washed with aq HCl, aq NaHCOg, dried over MgSO4, and concentrated under reduced pressure. Puriﬁcation by column chromatography (10% EtOAc/hexanes) afforded the desired product (0.200 g 70 %) as a white solid. 1H NMR (300 MHz, CDClg) 8 ppm 8.42-8.41 (m, 1H), 7.85 (d, J= 1.5 Hz, 2H), 7.42-7.36 (m, 2H), .16 (m, 1H), 7.05-7.01 (m, 2H), 3.93 (s, 6H).

Step 2: 5—Phen0xyz's0phthalaldehyde. To a solution of dimethyl 5- yisophthalate (0.143 g, 0.50 mmol) in toluene (12 mL) was added ise a solution of Red-Al (60% in toluene, 0.673 mL, 2.0 mmol) and ylpiperazine (0.243 mL, 2.2 mmol) in THF at 5 °C, and the resulting mixture was stirred for 2 h. The reaction was quenched with H20 (4 mL), and then extracted with EtOAc (20 mL). The organic layer was washed with H20 (10 mL), dried over anhydrous NazSO4, and then concentrated under reduced pressure. Puriﬁcation by ﬂash column chromatography (10% hexanes) afforded the desired product (0.094 g, 83 %) as a white solid. 1H NMR (300 MHz, CDCl3) 5 ppm 10.04 (s, 2 H), 8.08 (s, 1 H), 7.72 (t, J = 1.5 Hz, 2H), 7.46-7.40 (m, 2H), 7.28-7.24 (m, 1H), 7.09-7.06 (m, 2H).

Step 3: Di—tert—butyl ((((((5-phen0xy—I,3-phenylene)bis(methylene))- bis(azanediyl))bis (propane-3,1-diyl))bis(azanediyl)) bis (propane-3,1-diyl))dicarbamate. To a on of 5-phenoxyisophthalaldehyde (0.060 g, 0.27 mmol) and 3 A mol. sieves in MeOH, was added N1 -(3-aminopropyl)-N3-isobutylpropane-1,3-diamine (0.122 g, 0.53 mmol). The resultant mixture was stirred for 18 h. Sodium borohydride (0.020 g, 0.53 mmol) was added portionwise, and the reaction mixture was d for l h. The MeOH was evaporated, and the white solid was taken up in EtOAc (10 mL) and washed with aq. NaOH (10%, 4 mL). The aqueous layer was back—extracted with EtOAc (10 mL). The organic layers were combined, dried over Na2SO4, ﬁltered and evaporated to afford clear oil. olic HCl (30 mL, 1.0M) was added to the resultant oil, and the reaction mixture was stirred for 1 h. Evaporation and collection by vacuum ﬁltration afforded a white solid, which was washed hot MeOH (25 mL) to give the desired product (0.064 g, 52%) as a white solid. 1H NMR (500 MHz, D20) 6 7.50 (t, J: 7.5 Hz, 2H), 7.36 (S, 1H), 7.30 (d, J: 7.5 Hz, 1H), 7.24 (s, 2H), 7.17 (d, J: 8.0 Hz, 2H), 4.29 (s, 4H), 3.25—3.18 (m, 12H), 3.14 (t, J: 8.0 Hz, 4H),2.21-2.10 (m, 8H). 13c NMR(125 MHz,D20)6158.2,155.6,133.3,130.3,124.8, 120.5, 119.7, 119.3, 50.5, 44.7, 44.6, 44.2, 36.5, 23.7, 22.6. LRMS [M+H]+457.4.

Example 60 Preparation ofNI,N1'—((4 '-ﬂuoro-[1,1 '-biphenyl]-3,5—diyl)bis(methylene))bis(N3-(3- aminoproprpropane-I,3-diamine), hydrochloride salt (CZ-75) < NH2 |""‘_\_/NH HZNj—liHN O O F '6HCI

[0481] Example 60 was prepared in a similar fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, rophenyl)boronie acid and tert—butyl (3-((3- aminopropyl)amin0)pr0pyl)carbamate. 1H NMR (500 MHZ, D20) 5 7.80 (S, 2H), 7.71-7.68 (m, 2H), 7.56 (s, 1H), 7.25 (t, J = 9 Hz, 2H), 4.37 (s, 4H), 3.26 (t, J = 8 Hz, 4H), 3.19 (q, J = 8 Hz, 8H), 3.11 (t, J = 7.5 Hz, 4H), 2.22—2.07 (m, 8H). 13c NMR (125 MHz, D20) 8 162.9 (d, 244 Hz), 135.3(d, 3 2.3,130.1,129.7,129.1(d,8 Hz), 116.1 (d, 21 Hz), 51.1, 44.9, 44.8, 44.4, 36.7, 23.9, 22.9. LRMS [M+H]+ 459.4.

Example 61 Preparation ofNI,N1’—([I,I'-biphenyl]-3,5-diylbis(methylene))bis(propane-I,3-diamine), hloride salt (CZ-77) HN—\_/NH2 “2“) O O -4HCI Example 61 was prepared in a r fashion to Example 45 (CZ-5 8) from 5- bromoisophthaldehyde, phenylboronic acid and tert—butyl (3-amin0propyl)carbamate. N1,N1'- ([1,1'-biphenyl]—3,5—diylbis(methylene))bis(propane-1,3-diamine). 1H NMR (300 MHZ, D20) 8 7.82 (s, 2H), 7.71-7.64 (m, 2H), 7.54-7.41 (m, 4H), 4.34 (s, 4H), 3.22 (t, J: 7.8 Hz, 4H), 3.08 (t, J: 7.5 Hz, 4H), 2.11 (quint, J: 7.5 Hz, 4H).

Example 62 Preparation ofNI,N1 '—((5-butoxy-I,3-phenylene)bis(methylene))bis(N3-(3- aminopropybpropane-1,3-a’z'amz'ne), hydrochloride salt (CZ-76) < NH2 HN—\_/NH HN_? /_/_ - 6 HCI HZN HN

[0483] Step 1: Dimethyl 5-butoxyz'sophthalate. Dimethyl oxyisophthalate (0.40 g, 1.90 mmol), cesium ate (1.30 g, 3.80 mmol) and CH3CN (12 mL) were added to a round-bottom ﬂask and d for 15-30 min. Iodobutane (0.42 g, 2.28 mmol) was added,and the reaction was stirred for 16 h. The solvent was concentrated under reduced pressure, and the reaction mixture was partitioned between EtOAc (50 mL) and H20 (50 mL). The organic layer was ted and the aqueous layer extracted with EtOAc (50 mL).

The ed organics were dried over NaZSO4, ﬁltered, and concentrated under reduced pressure to afford the desired product which was used t further puriﬁcation.

Step 2: (5-Butoxy-I,3-phenylene)a’imethanol. To a solution of dimethyl 5- butoxyisophthalate (0.51 g, 1.90 mmol) in THF (16 mL) was slowly added LiAlH4 (0.40 g, 10.5 mmol). The on mixture was stirred for 8 h. The reaction was quenched by the addition of 2N aq. HCl (5 mL). The mixture was extracted with EtZO (2 x 25 mL) and EtOAc (2 x 25 mL), the organics layers combined, dried over Na2S04, ﬁltered, and concentrated under reduced pressure to afford the desired product (0.24 g) as an oil, which was used without further puriﬁcation.

[0485] Step 3: 5-Butoxyisophthalala’ehya’e. (5-Butoxy-l,3-pheny1ene)dimethanol (0.24 g, 1.15 mmol) and CH2C12 (10 mL) were added to a round-bottom ﬂask. To the solution was added PCC (0.74 g, 3.45 mmol), and the reaction was stirred at rt for 16 h. The reaction mixture was trated under reduced pressure. Puriﬁcation by column chromatography (90% hexanes/EtOAc) afforded the desired product (0.18 g, 44%, 3 steps) as a white semi- solid. 1H NMR (300 MHz, CDC13) 5 ppm 9.99 (s, 2H), 7.89 (s, 1H), 7.58 (s, 2H), 4.03 (t, J: 6.6 Hz, 2H), 1.77 (quint, J= 6.6 Hz, 2H), 1.47 (sext, J= 7.2 Hz, 2H), 0.94 (t, J= 7.5 Hz, 3H). 13C NMR (75 MHz, CDC13)5 ppm191.3,160.6,138.6,124.2,120.1, 68.9, 313,194,141.

Step 4: Di-tert—butyl ((((((5-butoxy-1,3-phenylene)bis(methylene))bis(azanediyl))- bis(propane-3, I—diyl))bis(azanediyl)) bis(propane—3, I—diyl))dicarbamate. 5- Butoxyisophthalaldehyde (0.80 g, 0.85 mmol) and MeOH (15 mL) were added to a round- bottom ﬂask. To the solution was added tert-butyl (3-((3-aminopropyl)amino)propyl)— carbamate (0.39 g, 1.70 mmol), and the reaction mixture was stirred at rt for 24 h. Sodium borohydride (0.13 g, 3.40 mmol) was added and the reaction mixture stirred for l h. The reaction e was concentrated under d pressure and aq. NaOH (10%, 50 mL) and EtOAc (50 mL) were added. The layers were separated and the s layer was extracted with EtOAc (50 mL). The organic layers combined, dried over Na2804, ﬁltered, and concentrated under reduced pressure to afford the desired product as a clear oil which was used without further puriﬁcation.

Step 5: N1,NI'-((5—Butoxy—I,3-phenylene)bis(methylene))bis(N3-(3- aminopropyl)propane-1,3-diamine), hydrochloride salt. To the crude di-tert—butyl 5- butoxy- l ,3 -phenylene)bis(methylene))bis(azanediyl))bis(propane-3 , l -diyl))bis(azanediyl))- bis(propane-3,1-diyl))dicarbamate from Step 4 was added methanolic HCl (50 mL, 1.0 M).

The reaction mixture was stirred for 2 h and concentrated under reduced re. The solid was collected by vacuum ﬁltration and washed with Et20 (10 mL) and hot MeOH (10 mL) to afford the desired product (0.27 g, 48%) as a white solid. 1H NMR (500 MHz, D20) 8 ppm 7.20 (s, 3H), 4.30 (s, 4H), 4.15 (t, J= 6.5 Hz, 2H), 3.26-3.20 (m, 12H), 3.14 (t, J= 7.5 Hz, 4H), 2.22-2.10 (m, 8H), 1.79 (quint, J: 6.5 Hz, 2H), 1.48 (sext, J: 7.5 Hz, 2H), 0.96 (t, J = 7 Hz, 3H). 13C NMR (125 MHz, D20) 159.2, 132.9, 123.5,117.3, 68.8, 50.8, 44.7, 44.6, 44.1, 36.5, 30.3, 23.7, 22.6, 18.5, 13.0. LRMS 437.4.

Example 63 Preparation ofN],NI '-((5-((2-ethylhexyl)oxy)-1, 3-phenylene)bis(methylene))bis(N3-(3- aminopropyl)propane—1,3—diamine), hydrochloride salt (CZ—8 l) < NH2 HN_\_/NH j?HN 0 Me H2N HN - 6 HCI Me e 63 was prepared in a similar fashion to Example 62 (CZ-76) from dimethyl xyisophthalate, 3-(bromomethyl)heptane and tert-butyl (3 -((3- aminopropyl)amino)propyl)carbamate. LRMS [M+H]+ 493.5.

Example 64 Preparation ofN1,Nl'-(’(5-(2-ethylbutoxy)-1, 3-phenylene)bis(Methylene))bis(N3-(3- aminoproprpropane-I,3-diamine), hydrochloride salt (CZ-90) < NH2 HN_\_/NH RHN we HZN HN Me - 6 HCI Example 64 was prepared in a similar fashion to Example 62 (CZ-76) from dimethyl 5-butoxyisophthalate, 3-(bromomethyl)pentane and tert-butyl (3 -((3- aminopropyl)amino)propyl)carbamate. 1H NMR (500 MHZ, D20) 8 7.23 (s, 2H), 7.22 (s, 1H), 4.32 (s, 4H), 4.06 (t, J: 7.0 HZ, 2H), 3.27-3.18 (m, 12H), 3.15 (t, J: 8.0 HZ, 4H), 2.24- 2.12 (m, 8H), 1.72 (m .1: 6.0 HZ, 1H), 1.52—1.44 (m, 4H), .91 (m, 6H). 13C NMR (125 MHZ, D20) 8 159.6, 132.9, 123.5, 117.5, 71.43, 50.8, 44.7, 44.6, 44.1, 40.1, 36.5, 23.7, 22.6, 10.3. LRMS [M+H]+465.4.

Example 65 Preparation ofNI,N1'-(’(5-(Benzyloxy)-1,3-phenylene)bis(methylene))bis(N3-(3— aminopropyl)propane-1,3-a’iamine), hydrochloride salt (CZ-95) < NH2 l""l_\_/NH §lHN °L© H2” HN - 6 HCI Example 65 was prepared in a similar fashion to Example 62 ) from dimethyl 5-butoxyisophthalate, benzyl bromide and tert—butyl (3-((3- aminopropyl)amino)propyl)carbamate. 1H NMR (500 MHZ, D20) 8 7.52 (d, J = 7.0 HZ, 2H), 7.49—7.41 (m, 3H), 7.23 (s, 2H), 7.20 (s, 1H), 5.23 (s, 2H), 4.29 (s, 4H), 3.22—3.15 (m, 12H), 3.13 (t, J = 8.0 Hz, 4H), 2.20—2.09 (m, 8H). 13c NMR (125 MHz, D20) 8 158.7, 136.0, 133.0, 132.5, 128.8, 128.5, 128.0, 123.9, 117.7, 70.4, 50.7, 44.7, 44.6, 44.3, 44.0, 36.5, 23.7, 22.6.

LRMS [M+H]+ 471.4.

Example 66 Preparation ofNI,N1 '—('(5-(cyclohexylmethoxy)-1,3-phenylene)bis(methylene))bis (N3-(3- aminopropyl)propane-1,3-diamine), hydrochloride salt (CZ-101) < NH2 HN_\_/NH HZN HN ' 6 HCI Example 66 was prepared in a similar fashion to Example 62 (CZ-76) from dimethyl 5-butoxyisophthalate, (bromomethyl)cyclohexane and tert—butyl (3-((3- aminopropy1)amin0)propyl)carbamate. 1H NMR (500 MHZ, D20) 8 7.16 (d, J = 7.5 Hz, 3H), 4.27 (s, 4H), 3.93 (t, J = 6.0 Hz, 2H), 3.23-3.09 (m, 16H), 2.19-2.07 (m, 8H), 1.83-1.66 (m, 6H), 1.31-1.17 (m, 3H), 1.10-1.05 (m, 2H). 13C NMR (125 MHz,DzO)8131.4, 131.2, 131.1,130.2, 50.8, 47.9, 44.6, 446,442, 44.1, 30.9, 28.1, 28.1, 25.6, 25.4, 22.6, 21.9, 13.3.

Example 67 ation ofNI,N1 '-([1, I ’-biphenyl]-3, bis lene))bis(N3-(3- (butylamino)propyl)propane-1,3-a’iamine), hloride salt (CZ-83) 6...my”)...0 .0 Step 1: [1,1'-Biphenyl]-3,5-dicarbala’ehya’e. A solution of 5- bromoisophthaldehyde (40.0 g, 187.8 mmol), phenylboronic acid (22.9 g, 187.8) and potassium carbonate (64.8 g, 469.5 mmol) in DME/HZO (5:1 600 mL) was purged with N2 for 5 min. Tetrakispalladium triphenylphosphine (1.1 g mg, 0.9 mmol) was added and the reaction mixture was heated to 90 CC and stirred for 16 h. The on mixture was cooled, d through a pad of celite and the solvent evaporated under reduced pressure.

Puriﬁcation by column chromatography (10% EtOAc/hexanes) afforded the desired product (74%, 29.1 g) as a tan solid. 1H NMR (300 MHz, CDClg) 6 ppm 10.18 (s, 2H), 8.37 (d, J= 1.2 Hz, 2H), 8.35 (t, J: 1.5 Hz, 1H), 7.70-7.66 (m, 2H), 7.55-7.43 (m, 3H).

Step 2: N1,N1'-([1,1'-biphenyl]-3,5-diylbis(methylene))bis(N3-(3- (bugzlamino)pr0pyl)pr0pane-I,3-a’z‘amz’ne). Biphenyl]—3,5-dicarbaldehyde (0.66 g, 3.15 mmol) and MeOH (50 mL) were added to a round-bottom ﬂask. To the solution was added N1-(3-aminopropyl)-Z\73-butylpropane-l,3-diamine (1.18 g, 6.31 mmol) and the reaction mixture was stirred at rt for 24 h. Sodium borohydride (0.48 g, 12.62 mmol) was added and the reaction mixture stirred for 1 h. The reaction mixture was concentrated under reduced pressure to afford a white solid. Aq. NaOH (10%, 100 mL) and EtOAc (100 mL) were added and the reaction mixture stirred for 1 h. The layers were separated and the aqueous layer was extracted with EtOAc (100 mL). The organic layers were combined, dried over NaZSO4, ﬁltered, and concentrated under reduced pressure to afford a clear oil which was used without further puriﬁcation.

Step 3: N’,N’ '_([1,1 '-bz'phenyl]-3, 5-diylbis(methylene))bz’s(N3-(3- amin0)pr0pyl)pr0pane-I,3-dz'amz'ne) hloride salt. To the crude N1,N1'—([1,1‘— biphenyl]—3,5-diylbis(methylene))bis(Z\73-(3-(butylamino)propyl)propane-1,3-diamine) from step 2 was subjected to acidiﬁcation with methanolic HCl (100 mL, 1.0M). The reaction mixture stirred at rt for 2 h. The reaction e was concentrated under reduced pressure and the solid was ted by vacuum ﬁltration and washed with Et20 (50 mL) and hot MeOH (50 mL) to afford the desired product (1.24 g, 51%) as a white solid. 1H NMR (500 MHz, D20) 5 7.83 (s, 2H), 7.71 (d, J = 7.5 Hz, 2H), .52(m, 3H), 7.49—7.45 (m, 1H), 4.35 (s, 4H), 3.26 (t, J = 7.5 Hz, 4H), 3.21-3.16 (m, 8H), 3.14 (t, J = 8.0 Hz, 4H), 3.05 (t, J = 8.0 Hz, 4H), 2.21-2.10 (m, 8H), 1.65 (quint, J = 7.0 Hz, 4H), 1.37 (sext, J = 8.0 Hz, 4H), 0.91 (t, J = 7.0 Hz, 6H). 13C NMR (125 MHz, D20) 5 142.5, 138.9, 132.3, 130.3, 129.8, 129.5, 128.7, 127.3, 51.0, 47.8, 44.9, 44.5, 44.4, 27.7, 22.9, 22.9, 19.3, 13.0. LRMS [MJrH]+ 553.5.

Example 68 Preparation ofN’Jv’ 1 '-bz'phenyl]-3, 5-dz'ylbz's (methylene))bz's(N3-(3— (isobutylamindpropybpropane-I,3-dz'amine), hydrochloride salt ) HNLQNy 3"? O O l-gli HN ' 6 HCI Method 1 Step 1: [1,1 '—Bz‘phenyl]—3,5—dicarbaldehyde. A solution of 5— bromoisophthaldehyde (40.0 g, 187.8 mmol), phenylboronic acid (22.9 g, 187.8) and potassium carbonate (64.8 g, 469.5 mmol) in DME/HZO (5:1 600 mL) was purged with N2 for 5 min. Tetrakispalladium triphenylphosphine (1.1 g mg, 0.9 mmol) was added and the reaction mixture was heated to 90 CC and stirred for 16 h. The reaction mixture was cooled, ﬁltered through a pad of celite and the solvent evaporated under reduced pressure.

Puriﬁcation by column chromatography (10% hexanes) afforded the desired product (74%, 29.1 g) as a tan solid. 1H NMR (300 MHZ, CDClg) 8 ppm 10.18 (s, 2H), 8.37 (d, J= 1.2 Hz, 2H), 8.35 (t, J: 1.5 Hz, 1H), 7.70-7.66 (m, 2H), 7.55-7.43 (m, 3H).

[0496] Step 2: Di—tert—butyl 1,1 '-bz'phenyl]-3,5-dl'ylbl's(methylene))bis(azanediy0)- bis(pr0pane-3, l-dbzwbismzanediyl»bis(pr0pane—3, 1-diyl))dicarbamate. [1 1 '-Biphenyl]- 3,5-dicarbaldehyde (3.62 g, 17.2 mmol, 1 ) and MeOH (100 mL) were added to a round- bottom ﬂask. To the on was added tert—butyl (3-((3-aminopropyl)amino)propyl)- carbamate (7.96 g, 34.4 mmol), and the reaction mixture was stirred at rt for 24 h. Sodium borohydride (2.62 g, 69.0 mmol) was added and the reaction mixture stirred for 1 h. The reaction mixture was concentrated under reduced pressure to afford a white solid. Aqueous NaOH (10%, 200 mL) and EtOAc (200 mL) were added, and the reaction mixture was stirred for 1 h. The layers were separated, and the aqueous layer was extracted with EtOAc (200 mL). The organic layers were combined, dried over , ﬁltered, and concentrated under d re to afford a clear oil that was used without further puriﬁcation.

Step 3: N1,N1'-([1,1'-bz'phenyl]-3,5-dl'ylbz's(methylene))bis(N3-(3- aminopropyl)pr0pane-1,3-diamine), hydrochloride salt. To the crude di-tert—butyl ((((([1,1'- biphenyl]-3 ,5-diylbis(methylene))bis(azanediyl))bis(propane-3 ,1-diyl))bis(azanediyl))- bis(propane-3,1-diyl))dicarbamate from Step 2 was subjected to acidiﬁcation with methanolic HCl (200 mL, 1.0M). The reaction mixture d at rt for 2 h. The reaction e was concentrated under reduced re, and the solid was ted by vacuum ﬁltration and washed with Et20 (50 mL) and hot MeOH (50 mL) to afford the desired product (6.8 g, 60%) as a white solid. 1H NMR (500 MHZ, D20) 8 7.85 (s, 2H), 7.74 (d, J : 7.5 Hz, 2H), 7.59-7.56 (m, 3H), 7.50-7.49 (m, 1H), 4.38 (s, 4H), 3.29 (t, J = 8.0 Hz, 4H), 3.23 (q, J = 5.0 Hz, 8H), 3.14 (t, J = 8.0 Hz, 4H), 2.25-2.19 (m, 4H), 2.17-2.11 (m, 4H). LRMS [M+H]+ 441.4.

Step 4: N1,N1'-([1,1'-Bz‘phenyl]-3,5-dz'ylbz's(methylene))bis(N3-(3- (isobutylamz'no)pr0pyl)pr0pane-I,3-diamine). To N1,N1'-([1,1‘-biphenyl]-3,5- diylbis(methylene))bis(1\73-(3-aminopropyl)propane-1,3-diamine), hydrochloride salt was added aq. NaOH (10%, 100 mL) and 75% CHClg/i-propanol (100 mL). The layers were separate and the aqueous layer was extracted with 75% CHClg/i-propanol (4 x 100 mL). The organic layers were combined dried over , ﬁltered, and concentrated under reduced pressure to afford a clear oil which was used without further puriﬁcation.

To a round-bottom ﬂask was added the crude N1,N1'-([1,1'-biphenyl]-3,5- diylbis(methylene))bis(N3-(3-aminopropyl)propane—1,3—diamine) (3.5 g, 8.0 mmol) and MeOH (50 mL). To the on was added isobutyraldehyde (1.15 g, 15.9 mmol) and the reaction e was stirred at rt for 24 h. Sodium borohydride (1.2 g, 31.9 mmol) was added and the reaction mixture was stirred for 1 h. The reaction mixture was concentrated under reduced pressure to afford a white solid. Aq. NaOH (10%, 200 mL) and EtOAc (200 mL) were added, and the mixture was stirred for 1 h. The layers were separated and the aqueous layer was extracted with EtOAc (200 mL). The combined organics were dried over Na2804, ﬁltered, and trated under reduced pressure to afford the desired product as a clear oil which was used t further puriﬁcation.

Step 5: N1,N1'-([1,1'-Bl'phenyl]-3,5-dl'ylbl's(methylene))biS(N3-(3- (isobutylamz'no)pr0pyl)pr0pane—I,3—diamine), hydrochloride salt. To the crude N1,N1'—([1,1'- biphenyl]—3,5-diylbis(methylene))bis(N3-(3-(isobutylamino)propyl)propane-1,3-diamine) was added methanolic HCl (200 mL, 1.0M). The reaction mixture was stirred at rt for 1 h. The reaction mixture was concentrated under reduced pressure, and the solid was collected by vacuum ﬁltration and washed with EtzO (50 mL) and hot MeOH (50 mL) to afford the desired product as a white solid (3.07 g, 50%). 1H NMR (500 MHz, D20) 8 7.87 (s, 2H), 7.75 (d, J= 7.5, 2H), 7.59-7.48 (m, 4H), 4.39 (s, 4H), 3.26 (t, J= 8.0 Hz, 4H), 3.21-3.12 (m, 12H), 2.92 (d, J: 7.0 Hz, 4H), 2.21-2.10 (m, 8H), 2.01 (sept, J: 7.0 Hz, 2H), 0.99 (d, J: 7.0 Hz, 12H). 13C NMR(125 MHz, D20)8142.8,139.1,132.4,130.1,129.8,129.5,128.7, 127.3, 55.1, 51.1, 44.9, 44.8, 44.4, 25.8, 22.8, 22.7, 19.2. LRMS [M+H]+ 553.5.

Method 11 Step 1: [1,1’-Bz'phenyl]-3,5-dz'carbaldehyde. A solution of 5- bromoisophthaldehyde (40.0 g, 187.8 mmol), phenylboronic acid (22.9 g, 187.8) and potassium ate (64.8 g, 469.5 mmol) in DME/HzO (5:1, 600 mL) was purged with N2 for 5 min. Tetrakispalladium nylphosphine (1.1 g mg, 0.9 mmol) was added and the reaction mixture was heated to 90 OC and stirred for 16 h. The reaction mixture was , ﬁltered h a pad of Celite diatomaceous earth, and the solvent was evaporated under reduced pressure. Puriﬁcation by column chromatography (10% EtOAc/hexanes) afforded the desired product (74%, 29.1 g) as a tan solid. 1H NMR (300 MHz, CDC13) 8 ppm 10.18 (s, 2H), 8.37 (d, J= 1.2 Hz, 2H), 8.35 (t, J= 1.5 Hz, 1H), 7.70-7.66 (m, 2H), 7.55-7.43 (m, 3H).

Step 2: N1,N1'-([1,1'-Bz'phenyl]-3,5-diylbis(methylene))bis(N3-(3- (isobutylamino)pr0pyl)pr0pane-I,3-dz'amine). [1,1'-Biphenyl]—3,5-dicarbaldehyde (3.03 g, 14.4 mmol) and MeOH (60 mL) were added to a round—bottom ﬂask. To the solution was added N1-(3-aminopropyl)-]\]3-isobutylpropane-1,3-diamine (5.40 g, 28.8 mmol), and the reaction mixture was stirred at rt for 24 h. Sodium borohydride (0.55 g, 14.4 mmol) was added, and the reaction mixture was stirred for 2 h. The reaction mixture was trated under d pressure to afford a white solid. s NaOH (10%, 50 mL) was added, and the reaction mixture stirred for 30 min. The solution was extracted with CHC13 (3 x 75 mL). The organic layers were combined, dried over Na2S04, d, and concentrated under reduced pressure to afford a clear oil which was used without further puriﬁcation.

Step 3; N1,N1'-([1,1'-Biphenyl]—3,5-diylbis(methylene))bis(]\]3-(3- (isobutylamino)propyl)propane-1,3-diamine) hydrochloride salt: To the crude N1,N1'-([l,l'- biphenyl]—3,5—diylbis(methylene))bis(1\73-(3-(isobutylamino)propyl)propane—1,3—diamine) from step 2 was added methanolic HCl (75 mL, 2.0M). The resulting white solid was collected by vacuum ﬁltration and washed with minimal MeOH and EtzO and stored under high vacuum to afford the desired product (9.3 g, 89%) as a white solid.

Example 69 Preparation of3, 5-bis(((3-((3-aminopropyl)amino)propyl)amino)methyl)phenol, hloride salt (CZ-87) < NHZ HN_'\_/NH j?HN 0H . 6 HCI HZN HN

[0504] Example 69 was prepared in a similar fashion to e 62 (CZ-76) from dimethyl -butoxyis0phthalate and utyl (3 -((3-aminopropyl)amino)pr0pyl)carbamate.

Example 70 Preparation of(3 C5 '-Bis(((3- ((3-aminopropyl)amino)propyl)amino)methyl)—[I, I '-biphenyl]- 4-yl)(piperidin-I—ybmethanone, hydrochloride salt (CZ-88) < NH2 HN_\_/NH HzNj—lil"N O O 2 -6HC| O Example 70 was prepared in a similar fashion to Example 68 (CZ-86) from 5- bromoisophthaldehyde, piperidin- l -yl(4-(4,4,5 ,5 —tetramethyl— l ,3 ,2—di0xaborolan—2—yl)- phenyl)methanone and tert—butyl (3-((3-aminopr0pyl)amino)propyl)carbamate. 1H NMR (300 MHz, D20) 5 7.87 (s, 2H), 7.78 (d, J = 8.4 Hz, 2H), 7.58 (s, 1H), 7.54—7.49 (m, 2H), 4.37 (s, 4H), 3.68 (bs, 2H), 3.42—3.38 (m, 2H), .13 (m, 12H), 3.07 (t, J = 7.8 Hz, 4H), 2.20—2.01 (m, 8H), 1.71-1.64 (m, 4H), 1.58-1.49 (m, 4H). LRMS [M+H]+552.4.

Example 71 Preparation ofI, I '-((([1,1 '-biphenyl]-3,5diylbis(methylene))bis(azanediyljjbismropane-iI- diyl))bis(tetrahydropyrimidin-2(II-D-one), hydrochloride salt (CZ-89) ° 2 HCI HM "WHOO—\_/:HN—<

[0506] Example 71 was prepared in a similar n to Example 68 (CZ-86) from 5- bromoisophthaldehyde, phenyl boronic acid, 1-(3-aminopropyl)tetrahydropyrimidin—2(1H)- one and tert—butyl (3-((3-aminopropyl)amin0)pr0pyl)carbamate. 1H NMR (300 MHz, D20) 8 7.80 (d, J = 6.5 HZ, 2H), 7.71-7.68 (m, 2H), 7.56-7.45 (m, 4H), 4.30 (s, 4H), 3.34 (t, J = 6.6 Hz, 4H), 3.21 (t, J = 6.0 Hz, 4H), 3.11 (t, J = 6.0 Hz, 4H), 3.04 (t, J = 7.2 HZ, 4H), 1.97—1.88 (m, 4H), 1.78 (p, J = 6.0 Hz, 4H). LRMS [M+H]+493.4.

Example 72 Preparation I '-([I, I enyl]-3,5-diylbis(methylene))bis(N3-(3-(octylamino)propyl)- propane-1,3-diamine), hydrochloride salt (CZ-92) HN—\_/NH HNA§ H <:/—\

[0507] Example 72 was prepared in a similar fashion to e 68 (CZ-86) from 5- bromoisophthaldehyde, phenylboronic acid, tert-butyl (3 -((3-amin0propyl)amino)propyl)— carbamate, and octanal. 1H NMR (500 MHZ, D20) 8 7.88 (s, 2H), 7.75 (d, J = 8.0 Hz, 2H), 7.60-7.56(m, 3H), 7.53-7.50 (m, 1H), 4.40 (s, 4H), 3.26 (t, J = 8.0 Hz, 4H), 3.21-3.16 (m, 8H), 3.14 (t, J = 8.0 Hz, 4H), 3.06 (t, J = 8.0 Hz, 4H), 2.21-2.09 (m, 4H), 1.69-1.65 (m, 4H), 1.38—1.28 (m, 20H), 0.86 (t, J = 6.0 Hz, 4H). 13C NMR (125 MHz, D20) 8 142.5, 138.8, 132.2, 129.9, 129.5, 129.3, 128.5, 127.0, 50.8, 47.8, 44.6, 44.1, 30.9, 28.1, 28.0, 25.6, 25.4, 22.7, 22.6, 21.9, 13.3. LRMS [M+H]/2+333.3.

Example 73 ation ofN’,N’ '—([1,1 '-bz'phenyl]-3, 5-dz'ylbz's (methylene))bz's(N3-(3— (benzylamino)propyl)propane-1,3-diamine), hydrochloride salt (CZ-94) HN5mO H?NH - 6 HCI

[0508] Example 73 was prepared in a similar fashion to Example 68 (CZ-86) from 5- bromoisophthaldehyde, phenylboronic acid, tert-butyl (3-((3-aminopropyl)amin0)pr0pyl)- carbamate, and benzaldehyde. 1H NMR (300 MHZ, D20) 5 7.84 (s, 2H), 7.74-7.70 (m, 2H), 7.58-7.50 (m, 4H), 7.47 (s, 10H), 4.37 (s, 4H), 4.24 (s, 4H), 3.26-3.11 (m, 16H), .05 (m, 8H).

Example 74 ation ofNI,N1 '-([1, I '-bz'phenyl]-3,5-dl'ylbz's(methylene))bis(lV3-(3- ((Cyclohexylmethyl)amino)propyl)propane-I,3-dz'amz'ne), hydrochloride salt (CZ-96) (Tin HN—\_/NH Example 74 was prepared in a similar n to Example 68 (CZ-86) from 5- bromoisophthaldehyde, phenylboronic acid and N1—(3—amin0pr0pyl)-NS— (cyclohexylmethyl)pr0pane-l,3-diamine. 1H NMR (500 MHZ, D20) 5 7.87 (s, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.59-7.56(m, 3H), .49 (m, 1H), 4.40 (s, 4H), 3.28 (t, J = 8.0 Hz, 4H), 3.24-3.18 (m, 8H), 3.15 (t, J = 8.0 Hz, 4H), 2.93 (d, J = 7.0 Hz, 4H), 2.24—2.12 (m, 8H), 1.74- 1.60 (m, 12H), 1.30—1.14 (m, 6H), 1.05-0.98 (m, 4H). 13C NMR (125 MHz, D20) 5 142.4, 138.8, 132.1, 130.0, 129.6, 129.3, 128.5, 127.0, 53.7, 50.8, 44.7, 44.6, 44.2, 34.5, 29.7, 25.4, 24.9, 22.6, 22.5. LRMS [M+H]+633.5.

Example 75 Preparation ofNI,N1 (I-methyl—IH—pyrazol—4-yl)-I,3-phenylene)bis(methylene))bis(N3- (3-(isobutylamino)propyl)propane-I, 3—diamine), hydrochloride salt (CZ-102) (—H‘N HN—\_/NH —>— H?HN {Ill HN HN - 6 HCI

[0510] Example 75 was prepared in a r fashion to e 68 (CZ-86) from 5- bromoisophthaldehyde, (1-methyl-1H-pyrazolyl)boronic acid and tert-butyl (3-((3- aminopropyl)amino)propyl)carbamate. 1H NMR (300 MHZ, D20) 5 7.75—7.69 (m, 4H), 6.60 (d, J = 2.1 Hz, 2H), 4.38 (s, 4H), 3.92 (s, 3H), 3.27-3.09 (m, 16H), 2.89 (d, J = 7.2 Hz, 4H), 2.21-2.06 (m, 8H), 2.03-1.94 (m, 2H), 0.97 (d, J = 6.9 Hz, 12H). 1H NMR (300 MHz, D20) 5 7.75-7.69 (m, 4H), 6.60 (d, J = 2.1 Hz, 2H), 4.38 (s, 4H), 3.92 (s, 3H), 3.27-3.09 (m, 16H), 2.89 (d, J = 7.2 Hz, 4H), 2.21-2.06 (m, 8H), 2.03-1.94 (m, 2H), 0.97 (d, J = 6.9 Hz, 12H).

Example 76 Preparation ofNI,N1/—([I,I '-biphenyl]-3,5-diylbis(methylene))bis(N4-(3- (isobutylamino)propyl)butane-1,4-diamine), hydrochloride salt (CZ-1 11) .5 .. H9 § O O_\_/_ Example 76 was prepared in a similar fashion to Example 76 (CZ-86) from 5- bromoisophthaldehyde, phenylboronic acid and N1-(3-amin0pr0pyl)-]\73-is0butylpropane-1,3- diamine. 1H NMR (500 MHz, D20) 6 7.86 (s, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.58 (t, J = 8.0 Hz, 3H), 7.50 (t, J = 7.5 Hz, 1H), 4.37 (s, 4H), 3.20-3.11 (m, 16H), 2.92 (d, J = 7.5 Hz, 4H), 2.16-2.10 (m, 4H), 2.06-1.97 (m, 2H), 1.88-1.80 (m, 8H), 0.99 (d, J = 6.5 Hz, 12H). 13C NMR(125 MHz, D20) 6 142.7, 139.1, 132.6, 130.1, 129.7, 129.5, 128.7, 127.7, 55.1, 50.9, 47.2, 46.7, 44.5, 44.7, 25.8, 23.0, 22.9, 22.7, 19.2. LRMS [M+H]+ 581.5.

Example 77 Preparation 0fN1,NIV—([I,I '-bz'phenyl]-3,5-diylbz's(methylene))bz'S(N3-(3- (z'sabutylamz'no)pr0pyl)-2,2-a’imethylpr0pane-I,3-a’z'amz'ne), hydrochloride salt (CZ-112) WFQNWF x0 O >_/ -6HC| Example 77 was prepared in a r n to e 68 (CZ-86) from 5- bromoisophthaldehyde, phenylboronic acid and N1-(3-(is0butylamino)propyl)—2,2- dimethylpropane-l,3-diamine. NMR (500 MHz, D20) 8 7.89 (s, 2H), 7.71 (d, J = 7.5 HZ, 2H), 7.62 (s, 1H), 7.52 (t, J = 7.5 Hz, 2H), 7.44 (t, J = 7.5 Hz, 1H), 4.39 (s, 4H), 3.19-3.01 (m, 16H), 2.89 (d, J : 7.5 Hz, 4H), 2.21-2.15 (m, 4H), 2.03-1.96 (m, 2H), 1.18 (s, 12H), 0.97 (d, J = 6.5 Hz, 12H). 13c NMR (125 MHz, D20) 5 1422, 1388,1317, 1314, 130.2, 129.5, 128.7, 127.2, 55.6, 55.1, 4.9, 52.1, 46.3, 45.0, 33.2, 25.8, 22.5, 22.4, 219,193. LRMS [M+H]+609.5.

Example 78 Preparation ofNI,N1 '-([I, I '-bipheriyl]-3,5-diylbis(methylene))bis(lV3-(3— (hexylamino)propyl)proparie-I,3-diamine), hloride salt (CZ-l 14)

[0513] Example 78 was prepared in a similar fashion to Example 68 (CZ-86) from 5- bromoisophthaldehyde, phenylboronic acid and N1-(3-amin0propyl)-N3-hexylpropane-1,3- diamine. 1H NMR (500 MHz, D20) 8 7.83 (s, 2H), 7.71 (d, J = 7.5 Hz, 2H), 7.55-7.53 (m, 3H), 7.47 (t, J = 6.5 Hz, 1H), 4.34 (s, 4H), 3.25 (t, J =8 Hz, 4H), 3.19 (q, J = 8 Hz, 8H), 3.13 (t, J = 8 Hz, 4H), 3.04 (t, J = 8.5 Hz, 4H), 2.22-2.09 (m, 8H), 1.66 (p, J = 7.5 Hz, 4H), 1.36- 1.31 (m, 4H), 1.28-1.27 (m, 8H), 0.85 (t, J = 7 Hz, 6H). 13C NMR (125 MHz, D20) 8 142.5, 139.0, 132.3, 130.3, 129.8, 129.5, 128.7, 127.3, 51.0, 48.1, 44.9, 44.4, 44.4, 30.6, 25.6, 25.5, 22.9, 22.8, 21.9, 13.5. LRMS [M+H]+ 609.5.

Example 79 Preparation ofNI,N1 '— ([1, I '-bipheriyl]-3, 5-diylbis(methylerie))bis(dodecane-1, I2-diamine), hydrochloride salt (CZ-115) e 79 was prepared in a similar fashion to Example 68 (CZ-86) from 5- bromoisophthaldehyde, phenylboronic acid and tert—butyl (12-amin0dodecyl)carbamate. 1H NMR (500 MHZ, D20) 8 7.92 (s, 2H), 7.80 (d, J = 8.5 Hz, 2H), 7.61—7.64 (m, 3H), 7.57—7.54 (m, 1H), 4.41 (s, 4H), 3.10 (t, J =75 Hz, 4H), 3.03 (t, J = 7.5 Hz, 4H), 1.77-1.66 (m, 8H), 1.39-1.36 (m, 12H), 1.35-1.28 (m, 22H). 13c NMR (125 MHz, D20) 8 142.5, 139.0, 132.3, 130.3, 129.8, 129.5, 128.7, 127.3, 51.0, 48.1, 44.9, 44.4, 44.4, 30.6, 25.6, 25.5, 22.9, 22.8, 21.9, 13.5. LRMS [M+H]+ 579.5.

Example 80 Preparation I '—([I, I '-bz'phenyl]-3, 5-dl'ylbz's (methylene))bis(NIZ—isobugildodecane-I, 12- ne), hydrochloride salt (CZ-116) iO 11 "1 .4HCI Example 80 was prepared in a similar fashion to Example 68 (CZ-86) from 5— bromoisophthaldehyde, phenylboronic acid and N1-isobutyldodecane-1 12-diamine. 1H NMR, (500 MHz, D20 + CDgOD) 8 7.90 (s, 2H), 7.76 (d, J = 7.5 Hz, 2H), 7.60-7.56 (m, 3H), 7.52 (t, J = 7.5 Hz, 1H), 4.38 (s, 4H), 3.04 (t, J :80 Hz, 4H), 3.01 (t, J = 8.5 Hz, 4H), 2.88 (d, J = 7.5, 4H), 2.06-1.98 (m, 2H), 1.74-1.65 (m, 8H), 1.33-1.30 (m, 12H), 1.28-1.23 (m, 20H), 1.01 (d, J = 7.5, 12H). 13C NMR (125 MHz, D20 + CD30D)8142.5, 138.8, 132.4, 130.1, 129.5, 129.3, 128.5, 127.0, 51.1, 46.6, 39.5, 28.6, 28.5, 28.4, 28.2, 28.0, 26.7, 25.6, 25.6, 25.2.

LRMS [M+H]+ 691.6.

Example 81 Preparation ofNI,N1',N1H-([I, I '-biphenyl]-3,3 ',5—trz'yltris (methylene))tris(N12- z'sobulyldoa’ecane-I, I2-a’iamz'ne) (CZ-1 17) NH HN H H ”A NH ”A Y” -6HC|

[0516] Example 81 was prepared in a similar n to Example 68 (CZ-86) from 5- bromoisophthaldehyde, 3-f0rmyl-phenylb0r0nic acid and N1-is0butyld0decane-1,12-diamine. 1H NMR (500 MHZ, D20) 8 7.97 (s, 2H), 7.89 (d, J = 8.0 HZ, 2H), 7.69-7.66 (m, 3H), 4.43 (s, 4H), 4.37 (s, 2H), .05 (m, 12H), 2.94 (d, J : 7.0, 6H), 2.10-2.01 (m, 3H), 1.78-1.71 (m, 12H), 1.39-1.26 (m, 48H), 1.50 (d, J = 6.5, 18H). 13C NMR (125 MHZ, D20) 8 144.1, 142.5,135.1,133.4,133.2,133.1,132.5,132.1,130.3,130.3, 57.0, 52.9, 52.7, 50.6, 49.4, 49.3, 31.2, 31.1, 31.1, 31.0, 30.6, 28.3, 28.3, 28.2, 28.0, 27.8, 21.7. LRMS [M+H]+ 960.0.

Example 82 Preparation ofNI,N1'-([I,I’-bz‘phenyl]-3,5-a’z‘ylbz‘s(methylene))bis(N4-(3- (hexylamino)propyl)butane-I,4-a’z'amz'ne), hydrochloride salt (CZ-118) CZ: ~H a .3") e 82 was prepared in a r fashion to Example 68 (CZ-86) from 5- bromoisophthaldehyde, phenylboronic acid and N1-(3-aminopropyl)-]\73-hexylpropane-1,3- diamine. 1H NMR (500 MHz, D20) 6 7.87 (s, 2H), 7.76 (d, J = 7.5 Hz, 2H), 7.58 (t, J = 8.0 Hz, 3H), 7.51 (t, J = 7.5 Hz, 1H), 4.37 (s, 4H), 3.21-3.14 (m, 12H), 3.07 (t, J = 7.0 Hz, 4H), 2.16-2.09 (m, 4H), 1.90-1.77 (m, 10H), 1.69 (quint, J = 7 Hz, 4H), 1.39-1.37 (m, 4H), 1.32- 1.31(m, 10H), 0.86 (t, J = 7 Hz, 6H). 13C NMR (125 MHz, D20) 5 142.6, 139.1, 132.6, 130.2, 129.7, 129.5, 128.7, 127.3, 50.9, 48.1, 47.2, 46.7, 44.7, 44.5, 30.6, 25.6, 25.5, 23.0, 23.0, 22.8, 21.9, 13.4. LRMS [M+H]+ 637.6.

Example 83 Preparation 0fN1,N1'-((5-(2-ethylbut0xy)—I, 3—phenylene)bis(methylene))bis(IV3-(3- (isobutylammonio)pr0pyl)pr0pane-I,3-dz'aminium) chloride ) Step 1: Dimethyl 5—(2-ethylbut0xy)isophthalate. Dimethyl 5-hydroxyisophthalate (0.44 g, 2.10 mmol) cesium carbonate (1.37 g, 4.20 mmol) and CH3CN (20 mL) were added to a round-bottom ﬂask and stirred for 15-30 min. 3-(Bromomethyl)pentane (0.42 g, 2.52 mmol) was added and the reaction was stirred for 16 h. The t was removed under reduced pressure and the reaction mixture was partitioned between EtOAc (50 mL) and H20 (50 mL). The layers were separated and the aqueous layer was extracted with EtOAc (50 mL). The combined organics were dried over Na2S04, ﬁltered, and concentrated under reduced pressure to afford the desired product which was used without further puriﬁcation.

Step 2: (5-(2-Ethylbut0xy)-I,3-phenylene)dimethanol. To a solution of dimethyl 5- (2-ethylbutoxy)isophthalate (0.62 g, 2.10 mmol) in THF (20 mL) was added LiAlH4 (0.24 g, 6.32 mmol). The reaction e was stirred for 8 h and subsequently quenched with 2N HCl (10 mL) and extracted with EtzO (2 x 25 mL) and EtOAc (2 x 25 mL). The combined organics were dried over Nast4, ﬁltered, and concentrated under reduced pressure to afford the desired product which was used without further puriﬁcation.

Step 3: 5-(2-Ethylbut0xy)isophthalaldehyde. (5-(2-Ethylbutoxy)—l,3- phenylene)dimethanol (0.50 g, 2.10 mmol) and CH2C12 (10 mL) were added to a round- bottom ﬂask. To this solution was added PCC (1.35 g, 6.30 mmol), and the on was stirred for 16 h. The reaction mixture was concentrated under reduced re. Puriﬁcation by column chromatography (90% hexanes/EtOAc) afforded the desired t (0.13 g, 25%, 3 steps) as a white semi-solid. 1H NMR (500 MHz, CDClg) 8 ppm 10.01 (s, 2H), 7.90 (s, 1H), 7.62 (s, 2H), 3.94 (d, J: 5.5 Hz, 2H), 1.68 (hept, J: 6.5 Hz, 1H), 1.46 (dec, J: 6.5 Hz, 4H), 0.91 (t, J = 7.5 Hz, 6H). 13c NMR (125 MHz, CDC13) 5 ppm 191.1, 160.7, 138.5, 124.1, 120.0, 71.1, 40.9, 235,113.

Step 4: N1,Nli-((5-(2-Ethylbut0xy)-I,3-phenylene)bis(methylene))bis(N3-(3— (isobutylamin0)pr0pyl) propane-1,3-diamz'ne). 5-(2-Ethylbutoxy)isophthalaldehyde (0.20 g, 0.88 mmol) and MeOH (15 mL) were added to a round-bottom ﬂask. To the solution was added N1-(3-aminopropyl)—]\]3-isobutylpropane-1,3-diamine (0.33 g, 1.76 mmol) and the reaction mixture was stirred for 24 h. Sodium borohydride (0.13 g, 3.52 mmol) was added and the reaction mixture stirred for l h. The reaction mixture was concentrated under reduced pressure to afford a white solid. s NaOH (10%, 50 mL) and EtOAc (50 mL) were added and the reaction mixture stirred for l h. The layers were separated and the aqueous layer was extracted with EtOAc (50 mL). The organic layers were combined, dried over NaZSO4, ﬁltered, and concentrated under reduced re to afford the desired product a clear 011.

Step 5: N1,Nli—((5—(2—Ethylbut0xy)—I,3—phenylene)bis(methylene))bis(N3—(3— tylammonio) propprropane-I,3-dz'amim'um) chloride. To N1,Nly-((5-(2-ethylbutoxy)- 1,3-phenylene)bis(methylene))bis(]\73-(3-(isobutylamino)propyl) propane-1,3-diamine) from step 4 was which was added methanolic HCl (50 mL, 1.0M). The reaction mixture was d for 1 h, the reaction mixture was concentrated under reduced pressure and the solid collected by vacuum ﬁltration. The solid was washed with EtZO (10 mL) and hot MeOH (10 mL) to afford the desired product (0.42 g, 59%) as a white solid. 1H NMR (500 MHz, D20) 8 7.20 (s, 2H), 7.18 (s, 1H), 4.29 (s, 4H), 4.05 (d, J = 6.0 Hz, 2H), 3.25-3.15 (m, 16H), 2.94 (d, J = 7.0 Hz, 2H), 2.21-2.12 (m, 8H), 2.07-1.99 (m, 2H), 1.72 (p, J = 6.5 Hz, 1H), 1.50-1.43 (m, 4H), 1.00 (d, J = 7.0 Hz, 12H), 0.92 (t, J = 6.0 Hz, 6H). 13C NMR (125 MHz, D20) 8 159.6, 132.9, 123.4, 117.4, 71.4, 54.8, 50.8, 44.7, 44.6, 44.6, 44.1, 40.1, 25.5, 22.6, 22.5, 19.0, 10.2. LRMS [M+H]+577.6.

Example 84 Preparation ofNI,N1’—((5-isopropoxy-l,3-phenylene)bis(methylene))bis(N3-(3- aminoproprpropane-I,3-diamine), hydrochloride salt (CZ-93) < NH2 l"N_\_/NH 2.9 2 e 84 was prepared in a similar fashion to Example 83 (CZ—91) from 5- poxyisophthalaldehyde and tert—butyl (3-((3-aminopropy1)amino)propyl)carbamate. 1H NMR (300 MHZ, D20) 8 7.13 (s, 3H), 4.78-4.72 (m, 1H), 4.23 (s, 4H), 3.27-3.18 (m, 12H), 3.13 (t, J = 7.8 HZ, 4H), .18 (m, 4H), 2.18—2.10(m, 4H), 1.36 (d, J =10.2HZ, 6H).

Example 85 Preparation I ’—((5-(cyclohexylmethoxy)-I, 3-phenylene)bis(methylene))bis(N3-(3- (octylamino)propyl)propane-1,3-diamine), hydrochloride salt (CZ-103) .1144?” ..29 H;

[0524] Example 85 was prepared in a similar fashion to Example 83 (CZ-91) from dimethyl 5-butoxyisophtha1ate, (bromomethyl)cyclohexane, tert-butyl (3 -((3- aminopropyl)amino)propyl)carbamate and l. 1H NMR (500 MHZ, D20) 5 7.18 (s, 3H), 4.28 (s, 4H), 3.95 (s, 2H) 3.20-3.14 (m, 16H), 3.06 (t, J = 7.5 HZ, 4H), 2.22-2.10 (m, 8H), 1.88-1.80 (m, 3H), 1.76-1.64 (m, 8H), 1.41-1.20 (m, 22H), 1.12-1.03 (m, 2H), 0.89-0.83 (m, 6H). 13C NMR (125 MHZ, D20)8159.5,133.0, 123.4, 117.3, 74.4, 50.8, 47.8, 44.6, 44.6, 44.2, 44.1, 36.8, 30.9, 29.1, 28.1, 28.0, 25.9, 25.6, 25.4, 25.2, 22.6, 22.6, 21.9, 13.3. LRMS [M+H]+701.7.

Example 86 Preparation ofNI,N1’—((5-(cyclohexyloxy)-1,3-phenylene)bis(methylene))bis(N3-(3- (isobutylamino)propyl)propane-1,3-diamine), hydrochloride salt (CZ-104) I"'\'_\_/ Example 86 was ed in a similar n to Example 83 (CZ-91) from 5- (cyclohexyloxy)isophthalaldehyde and N1-(3-aminopropyl)-N3-isobutylpropane-1,3-diamine. 1H NMR (300 MHZ, D20) 5 7.60 (s, 2H), 7.53 (s, 1H), 4.73 (t, J = 1.2 Hz, 1H), 4.42 (s, 4H), 3.26-3.13 (m, 16H), 2.84 (d, J = 6.9 Hz, 2H), 2.23-2.08 (m, 8H), 2.06-1.96 (m, 2H), 1.87-1.78 (m, 4H), 1.65-1.36 (m, 6H), 0.99 (d, J : 6.6 Hz, 12H). LRMS [M+H]+ 575.5.

Example 87 Preparation ofNI,N1V—((5-(cyclohexyloxy)-1,3-phenylene)bis(methylene))bis(N3-(3- aminopropyl)propane-1,3-diamine), hydrochloride salt (CZ-105) < NH2 HN—\_/NH ‘23 Example 87 was prepared in a similar fashion to Example 83 ) from 5- (cyclohexyloxy)isophthalaldehyde and utyl (3-((3-am1nopr0pyl)amino)propyl)- carbamate. 1H NMR (300 MHz, D20) 5 7.22 (s, 2H), 7.18 (s, 1H), 4.68 (t, J = 1.2 Hz, 1H), 4.30 (s, 4H), 3.28-3.18 (m, 12H), 3.14 (t, J = 7.8 Hz, 4H), 2.21-2.06 (m, 8H), 1.84-1.75 (m, 4H), 1.63-1.34 (m, 6H). LRMS [M+H]+ 463.5.

Example 88 Preparation ofNI,N1V—((5-(benzyloxy)—I,3-phenylene)bis(methylene))bis(N3-(3- (isobutylamino)propyl)propane-I,3-diamine), hydrochloride salt (CZ-106) “EQU— Qi W3 $7 - 6 HCI

[0527] e 88 was prepared in a similar fashion to Example 83 (CZ-91) from dimethyl 5-butoxyisophthalate, benzyl bromide, tert-butyl (3-((3-aminopropyl)amino)- propyl)carbamate and isobutyraldehyde.

Example 89 Preparation ofNI,N1V—(I,3—phenylenebis(methylene))bis(N3—(3—((Cyclohexylmethyl)— propyl)propane-1,3-diamine), hloride salt (CZ-97) < HN HN_\_/NH Step 1: -(1,3-Phenylenebis(methylene))bis(1V3-(3-((Cyclohexylmethyl)- amino)propyl)propane-1,3-diamine). Isophthalaldehyde (0.34 g, 2.57 mmol, l) and MeOH (20 mL) were added to a round-bottom ﬂask. To the solution was added N1-(3 -aminopropyl)- N3-(cyclohexylmethyl)propane-1,3-diaminediamine (1.17 g, 5.14 mmol, 2) and the reaction mixture was stirred at rt for 24 h. Sodium borohydride (0.39 g, 10.28 mmol, 4) was added and the reaction mixture stirred for 1 h. The reaction mixture was trated under reduced pressure to afford a white solid. Aqueous NaOH (10%, 100 mL) and EtOAc (100 mL) were added and the reaction mixture stirred for l h. The layers were separated and the aqueous layer was extracted with EtOAc (100 mL). The organic layers were combined, dried over Na2S04, d, and concentrated under reduced re to afford a clear oil which was used without GI‘ puriﬁcation.

Step 2: N1,NI'-(I,3-phenylenebis(methylene))bis(N3—(3-((cyclohexylmethyl)amin0)- propyl)propane-1,3-diamine) hydrochloride salt. To the crude N1,N1‘-(1,3-phenylenebis- (methylene))bis(l\73-(3-((cyclohexylmethyl)amino)propyl)propane-1,3 -diamine) from Step 1 was added methanolic HCl (100 mL, 1.0M). The reaction mixture stirred at rt for 2 h. The reaction mixture was concentrated under d pressure and the solid was ted by vacuum ﬁltration and washed with EtZO (50 mL) and hot MeOH (50 mL) to afford the desire product (1.3 g, 67%) as a white solid. 1H NMR (500 MHz, D20) 8 7.57 (s, 4H), 4.30 (s, 4H), 3.22 (t, J = 7.5 Hz, 4H), 3.18-3.15 (m, 8H), 3.12 (t, J = 8.0 Hz, 4H), 2.91 (d, J = 7.0 Hz, 4H), 2.18-2.08 (m, 8H), 1.72-1.62 (m, 12H), 1.28-1.11 (m, 6H), 1.03-0.96 (m, 4H). 13C NMR (125 MHz, D20) 8 131.4, 131.2, 131.0, 130.2, 53.7, 50.8, 48.8, 44.7, 44.6, 44.0, 34.5, 29.7, .3, 24.8, 22.6, 22.5. LRMS [M+H]+ 557.5.

Example 90 Preparation ofNI,NI V—(I, 3-phenylenebis(methylene))bis(N3-(3-(octylamino)propyl)propane- 1,3-diamine), hydrochloride salt (CZ-100) < HN m. HN—\_/NHHN -6HC| Example 90 was prepared in a similar fashion to Example 62 (CZ-76) from isophthaldehyde, tert—butyl (3 —((3—aminopropyl)amino)propyl)carbamate and octanal. 1H NMR (500 MHZ, D20) 8 7.58 (s, 4H), 4.32 (s, 4H), 3.10-2.98 (m, 16H), 2.91 (t, J = 8.0 Hz, 4H), 2.05-1.95 (m, 8H), 1.53 (p, J = 7 Hz, 4H), 1.24-1.13 (m, 20H), 0.71 (t, J = 7.0 Hz, 6H). 13C NMR (125 MHz, D20)5131.4, 131.2, 131.1, 130.2, 50.8, 47.9, 44.6, 44.6, 44.2, 44.1, .9, 28.1, 28.1, 25.6, 25.4, 22.6, 219,133. LRMS [M+H]+ 589.5.

Example 91 Preparation ofN’,N’1(1,3—phenyzenebz‘smethylene»[mar—(3— (isobutylamino)propyl)propane-I,3-diamine), hydrochloride salt 8) HNMQNWF >_/ '6HCI

[0531] Example 91 was prepared in a similar fashion to Example 89 (CZ-97) from isophthaldehyde and N1-(3-aminopropyl)—]\73-isobutylpropane—1,3-diamine. 1H NMR (300 MHz, D20) 6 7.54 (s, 4H), 4.28 (s, 4H), 3.19-3.08 (m, 16H), 2.88 (d, J= 7.5 Hz, 4H), 2.15- 2.05 (m, 8H), 2.02-1.90 (m, 2H), 0.95 (d, J: 6.9 Hz, 12H). LRMS [M+H]+ 477.4.

Example 92 Preparation ofNI,N1V—((5-bromo-I,3-phenylene)bis(methylene))bis(N3-(3- (isobutylamino)propyl)propane-I,3-diamine), hydrochloride salt (CZ-109) HNLQN} >_/ -6HC| Example 92 was prepared in a similar fashion to Example 89 ) from 4- bromo-isophthaldehyde and aminopropyl)—N3-isobutylpropane-1,3-diamine. 1H NMR (300 MHz, D20) 8 7.76 (s, 2H), 7.51 (s, 1H), 4.27 (s, 4H), 3.21-3.08 (m, 16H), 2.88 (d, J: 7.5 Hz, 4H), 2.16-2.06 (m, 8H), 2.02-1.93 (m, 2H), 0.96 (d, J: 6.6 Hz, 12H). LRMS [M+H]+ 555.4, 557.4. e 93 Preparation ofNI,NI V—(I, 3-phenylenebis(methylenejjbis(N3-(3-(hexylaminojpropyljpropane- 1,3-diamine), hydrochloride salt (CZ-113) (—1111 HN_\_/NH -6HCI

[0533] Example 93 was prepared in a similar fashion to e 89 (CZ-97) from isophthaldehyde and N1-(3-aminopropyl)—l\73-hexylpropane-1,3-diamine. 1H NMR (500 MHZ, D20) 8 7.58 (s, 3H), 4.32 (s, 4H), 3.24-3.13 (m, 16H), 3.05 (t, J = 8.0 Hz, 4H), 2.20- 2.09 (m, 8H), 1.68—1.63 (m, 4H), 1.38—1.33 (m, 4H), 1.32—1.26 (m, 8H), 0.85 (t, J = 7.5 Hz, 6H). 13C NMR (125 MHz, D20) 8 131.6, 131.5, 131.4, 130.4, 51.1, 48.1, 44.9, 44.8, 44.5, 44.3, 30.6, 25.6, 25.5, 22.8, 21.9, 13.5. LRMS [M+H]+533.6.

Example 94 Preparation ofNI,N1'-(1,3-phenylenebis(methylene))bis(N4-(3-(hexylamino)propyl)butane- 1,4-diamine), hydrochloride salt 9) g HN—\_/—Nl-l—/—NH HN§ - 6 HCI

[0534] Example 94 was prepared in a similar fashion to Example 89 (CZ-97) from isophthaldehyde and N1-(3-aminopropyl)—N4-hexylbutane-1,4-diamine. 1H NMR (500 MHz, D20) 6 7.65 (s, 4H), 4.37 (s, 4H), 3.26-3.19 (m, 16H), 3.14 (t, J= 7.5 Hz, 4H), 2.20 (quint, J = 8.0 Hz, 4H), 1.88 (br s, 8H), 1.75 (quint, J: 8.0 Hz, 4H), .42 (m, 4H), 1.39-1.36 (m, 8H), 0.94 (t, J: 6.5 Hz, 6H).13C NMR (125 MHz, D20) 8 134.2, 133.7, 133.5, 132.7, 53.3, 50.4, 49.6, 49.1, 47.1, 46.8, 32.9, 27.9, 27.8, 25.4, 25.3, 25.2, 24.2, 15.8. LRMS [M+H]+ 561.5.

Example 95 Preparation ofNI,N1',N1”-([1, I '-bz'phenyl]-3,3 ',5-triyltris(methylene))tris(N3-(3— tylamino)pr0pyl)propane-1 amine) hydrochloride salt (CZ-99) YNMNMNQ - 9 HCIH H H Step 1: [1,1'—Bz'phenyl]—3,3Z5—tricarbaldehyde. A solution of 5— sophthaldehyde (2.13 g, 10.0 mmol), 3-formylphenylboronic acid (1.49 g, 10.0 mmol) and ium carbonate (2.76 g, 20.0 mmol) in DME/HZO (5:1 50 mL) was purged with N2 for 5 min. Tetrakispalladium triphenylphosphine (0.12 g, 0.1 mmol) was added and the reaction mixture was heated to 90 CC and stirred for 16 h. The reaction mixture was cooled, ﬁltered through a pad of Ce1ite diatomaceous earth and the solvent evaporated under reduced re. Puriﬁcation by column chromatography (10% EtOAc/hexanes) afforded the desired product (1.47 g, 62%) as a tan solid. 1H NMR (300 MHz, CDClg) 6 ppm 10.20 (s, 2H), 10.10 (s, 1H), 8.42 (s, 3H), 8.21 (s, 1H), 7.98 (t, J: 5.4 Hz, 2H), 7.72 (t, J: 5.4 Hz, 2H).

Step 2; N’,N”,N’ ”_([1,1 '—Biphenyl]—3,3 ',5—trz‘yltrz‘s (methylene))ms (N3—(3— (isobutylamino)propyl)propane-1,3-a’z'amz'ne). [1,1'—biphenyl]—3,3',5-tricarbaldehyde (0.24 g, 1.0 mmol) and MeOH (20 mL) were added to a round-bottom ﬂask. To the solution was added N1—(3—aminopropyl)—N3—isobutylpropane—1,3—diamine (570 mg, 3.0 mol, 3 equiv.) and the reaction mixture was stirred at rt for 24 h. Sodium borohydride (0.34 g, 9.0 mmol) was added and the reaction mixture stirred for 1 h. The reaction mixture was concentrated under reduced pressure to afford a white solid. s NaOH (10%, 100 mL) and EtOAc (100 mL) were added and the reaction mixture stirred for 1 h. The layers were separated and the aqueous layer was extracted with EtOAc (100 mL). The organic layers were combined, dried over Na2SO4, ﬁltered, and concentrated under reduced pressure to afford a clear oil which was used t r puriﬁcation.

Step 3: N1,N1/,N1H-([I,I'-Biphenyl]-3,3',5-triyltris(methylene))trisW3-(3- (isobutylamino)propyl)propane-I,3-diamine), hydrochloride salt. N1,N1',N1"-([1,1'- biphenyl]-3 ,3',5 -triyltris(methylene))tris(N3-(3-(isobutylamino)propyl)propane-1 ,3-diamine) from step 2 was subjected to acidiﬁcation with olic HCl (100 mL, 1.0M). The reaction mixture stirred at rt for 2 h. The reaction mixture was concentrated under reduced pressure and the solid was collected by vacuum ﬁltration and washed with Et20 (50 mL) and hot MeOH (50 mL) to afford the desired product (0.58 g, 52%) as a white solid. 1H NMR (500 MHz, D20) 8 ppm 7.91 (s, 1H), 7.85-7.82 (m, 3H), 7.65-7.56 (m, 3H), 4.42 (s, 4H), 4.39 (s, 2H), .16 (m, 24H), 2.93 (d, J = 6.0 Hz, 6H), 2.20-2.13 (m, 12H), 2.07-2.00 (m, 3H), 0.99 (d, J : 6.0 Hz, 18H). 13CNMR (125 MHz, D20) 8 ppm 141.6, 139.8, 132.2, 131.3, 130.4, 130.3, 129.7, 129.5, 128.6, 128.4, 54.8, 54.8, 51.1, 50.8, 44.7, 44.6, 44.2, 44.0, 25.5, 22.7, 22.5, 19.0. LRMS [M+2H+]2+ 376.8.

Example 96 Preparation ofNI,NI '—((5-(3-(isoButylammonio)propoxy)-1, 3-phenylene)bis(methylene))— bis(N3-(3-ammoniopropyl)propane-1,3-diaminium) chloride (CZ-107) ( NH2 \_/NH H 2 0\—\—th Step 1: tert-Butyl (3-hydroxypropyl) (isobutyl)carbamate. - Butylamino)propan—1—ol (7.92 g, 60.5 mmol) and THF (100 mL) were added to a round— bottom ﬂask. To the solution was added aqueous NaOH (10%, 100 mL) followed by the slow addition of di-tert-butyl dicarbonate (11.86 g, 54.4 mmol). The reaction mixture was stirred for 12 h. The reaction mixture was extracted with CHzClz (3 x 100 mL). The combined organics were dried over Na2SO4, ﬁltered, and concentrated under reduced pressure to afford the desired product (12.0 g, 86%) which was used without further puriﬁcation.

Step 2: 3-((tert—But0xycarb0nyl) (isobutybamz‘nomropyl methanesulfonate. tert- Butyl (3-hydroxypropyl)(isobutyl)carbamate (0.73 g, 3.15 ), triethylamine (0.64 g, 6.30 mmol) and CH2C12 (30 mL) were added to a round-bottom ﬂask. To the solution was added methanesulfonyl chloride (0.54 g, 4.72 mmol) and the reaction mixture was stirred for 5 h.

The reaction mixture was concentrated under reduced pressure and puriﬁed by column chromatography (hexanes/EtOAC) to afford the desired product (063 g, 64%) as a yellow oil.

Step 3: Dimethyl 5-(3-((tert—but0xycarb0nyl)(isobutyl)amz'n0)pr0p0xy)is0phthalate.

Dimethyl 5-hydroxyisophthalate (0.30 g, 1.44 mmol) cesium carbonate (0.95 g, 2.88 mmol) and CH3CN (25 mL) were stirred for 30 min. 3-((tert—Butoxycarbonyl)(isobutyl)amino)- propyl methanesulfonate (0.63 g, 2.02 mmol) was added and the reaction mixture was stirred for 16 h. The reaction mixture was concentrated under reduced pressure and partitioned between EtOAc (50 mL) and H20 (50 mL). The layers were separated and the aqueous layer was extracted with EtOAc (50 mL). The combined organics were dried over NazSO4, ﬁltered, and trated under d pressure to afford the d product, which was used without further puriﬁcation.

Step 4: tert—Butyl (3-(3,5-bis(hydroxymethyl)phen0xy)pr0pyl)(isobutybcarbamate.

To a on of dimethyl 5-(3-((tert-butoxycarbonyl)(isobutyl)amino)propoxy)isophthalate (0.60 g, 1.44 mmol) in THF (10 mL) was added LiAlH4 (0.30 g, 7.89 mmol). The reaction mixture was d for 8 h and uently quenched with 2N HCl (10 mL) and extracted with Et20 (2 x 25 mL) and EtOAc (2 x 25 mL). The combined organic layers were dried over , ﬁltered, and concentrated under reduced pressure to afford the d product (0.28 g, 54%) as an oil, which was used without further puriﬁcation.

Step 5: tert—Butyl (3-(3,5-dl'f0rmylphen0xy)pr0pyl) (isobutyl)carbamate. tert—Butyl (3-(3,5-bis(hydroxymethyl)phenoxy)propyl)(isobutyl)carbamate (0.29 g, 0.78 mmol) and CH2C12 (20 mL) were added to a round—bottom ﬂask. To the solution was added PCC (0.42 g, 1.94 mmol) and the reaction was stirred for 16 h. The reaction mixture was concentrated under reduced pressure. Puriﬁcation by column tography (90% Hexanes/EtOAc) afforded the desired product (0.15 g, 20% 3 steps) as a white semi—solid, which was used without further puriﬁcation.

Step 6: N1,N1/-((5-(3-(iso-Butylaminwpropoxy}I,3-phenylene)bis(methylene))- bis(N3-(3-amin0pr0pyl)pr0pane-1,3-a’z'amz'ne). utyl (3-(3,5-diformylphenoxy)propyl)- (isobutyl)carbamate (0.15 g, 0.42 mmol) and MeOH (10 mL) were added to a round-bottom ﬂask. To the solution was added tert-butyl (3-((3-aminopropyl)amino)propyl)carbamate (0.20 g, 0.84 mmol) and the reaction mixture was stirred for 24 h. Sodium borohydride (0.06 g, 1.69 mmol) was added and the reaction mixture stirred for 1 h. The on mixture was concentrated under reduced pressure to afford a white solid. Aqueous NaOH (10%, 50 mL) and EtOAc (50 mL) were added, the layers ted and the aqueous layer was ted with EtOAc (50 mL). The organic layers were combined, dried over NaZSO4, ﬁltered, and trated under reduced pressure to afford the desired product as clear oil which was used without further ation.

[0544] Step 7: N1,N1/-((5-(3-(iso-Batylammonio)propoxy)-I,3-phenylene)bis(methylene))- bis(N3-(3-ammoniopropyl)propane-1,3-diaminiam) chloride. To N1,N1'-((5-(3-(iso- butylamino)propoxy)—1 ,3 -phenylene)bis(methylene))bis(l\73-(3 -an11nopropyl)propane- 1 ,3 - diamine) from step 6 was added methanolic HCl (50 mL, 1.0M). The reaction mixture was stirred for 1 h, the reaction mixture was concentrated under reduced pressure and the solid ted by vacuum ﬁltration. The solid was washed with EtZO (10 mL) and hot MeOH (10 mL) to afford the desired product (0.14 g, 43%) as a white solid. 1H NMR (500 MHz, D20) 8 ppm 7.21 (s, 1H), 7.19 (s, 2H), 4.30 (s, 4H), 4.23 (t, J: 5.5 Hz, 2H), 3.29 (t, J: 7 Hz, 2H), 3.25—3.19(m, 12H), 3.12 (t, J: 7.5 Hz, 4H), 2.95 (d, J: 7 Hz, 2H), 2.25—2.03 (m, 11H), 1.01 (d, J= 7.5 Hz, 6H). 13c NMR (125 MHz, D20) 158.9, 133.0, 123.7, 117.1, 65.8, 54.8, 50.7, 45.7, 44.7, 44.6, 44.2, 36.7, 25.5, 25.2, 23.7, 22.6, 19.1. LRMS [M+H]+ 494.4.

Example 97 Preparation ofNI,N1'—((5-(3-(isobutylamino)propoxy)-I,3-phenylene)bis(methylene))bis(N3- (3-(octylamino)propyl)propane—I,3-diamine), hydrochloride salt (CZ-110) (711.

HN—\_/NH Step 1: tert-Butyl (3-hydr0xypr0pyl) (isobutylkarbamate. 3-(l's0- Butylamino)propanol (7.92 g, 60.5 mmol) and THF (100 mL) were added to a round- bottom ﬂask. To the solution was added aqueous NaOH (10%, 100 mL) followed by the slow addition of di-tert-butyl dicarbonate (11.86 g, 54.4 mmol). The reaction mixture was stirred for 12 h. The reaction mixture was extracted with CHZClz (3 x 100 mL). The combined organics were dried over NagSO4, ﬁltered, and concentrated under reduced pressure to afford the desired product (12.0 g, 86%), which was used without further ation.

Step 2: 3-((tert—But0xycarb0nyl) (isobunzl)amin0)pr0pyl methanesulfonate. tert- Butyl (3-hydroxypropyl)(isobutyl)carbamate (0.73 g, 3.15 ), triethylamine (0.64 g, 6.30 mmol) and CH2C12 (30 mL) were added to a round-bottom ﬂask. To the solution was added methanesulfonyl chloride (0.54 g, 4.72 mmol) and the reaction mixture was stirred for 5 h.

The reaction mixture was concentrated under reduced re and puriﬁed by column chromatography (hexanes/EtOAC) to afford the desired t (0.63 g, 64%) as a yellow oil.

Step 3: Dimethyl 5-(3-((tert—but0xycarb0nyl)(isobutyl)amin0)pr0p0xy)is0phthalate: Dimethyl 5-hydroxyisophthalate (0.30 g, 1.44 mmol) cesium carbonate (0.95 g, 2.88 mmol) and CH3CN (25 mL) were stirred for 30 min. rt-Butoxycarbonyl)(isobutyl)amino)- propyl methanesulfonate (0.63 g, 2.02 mmol) was added, and the reaction mixture was stirred for 16 h. The on mixture was concentrated under reduced pressure and partitioned between EtOAc (50 mL) and H20 (50 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (50 mL). The combined organics were dried over NazSO4, ﬁltered, and concentrated under reduced pressure to afford the desired t, which was used without further puriﬁcation.

[0548] Step 4: tert-Butyl 5-bis(hydroxymethpr/aenoxy)pr0pyl)(isobugibcarbamate.

To a solution of dimethyl 5-(3-((tert-butoxycarbonyl)(isobutyl)amino)propoxy)isophthalate (0.60 g, 1.44 mmol) in THF (10 mL) was added LiAlH4 (0.30 g, 7.89 mmol). The reaction mixture was stirred for 8 h and subsequently quenched with 2N HCl (10 mL) and extracted with EtZO (2 x 25 mL) and EtOAc (2 x 25 mL). The combined organic layers were dried over Na2SO4, ﬁltered, and concentrated under reduced pressure to afford the desired product (0.28 g, 54%) as an oil, which was used without r puriﬁcation.

Step 5: tert-Butyl (3-(3,5-dz‘f0rmylphenoxy)pr0pyl) (isobutyDCCIrbamate. utyl (3-(3,5-bis(hydroxymethyl)phenoxy)propyl)(isobutyl)carbamate (0.29 g, 0.78 mmol) and CH2C12 (20 mL) were added to a round-bottom ﬂask. To the solution was added PCC (0.42 g, 1.94 mmol) and the reaction was stirred for 16 h. The reaction mixture was trated under reduced pressure. Puriﬁcation by column chromatography (90% hexanes/EtOAc) afforded the desired product (0.15 g, 20% 3 steps) as a white semi-solid which was used without further puriﬁcation.

Step 6: NI,NI/-((5-(3-(iso-Butylaminwpropoxy}I,3-phenylene)bis(methylene))- bis(N3- 'n0pr0py0pr0pane-1,3-dz'amz'ne). tert-Butyl 5-diformylphenoxy)propyl)- (isobutyl)carbamate (0.15 g, 0.42 mmol) and MeOH (10 mL) were added to a round-bottom ﬂask. To the solution was added tert—butyl (3-((3-aminopropyl)amino)propyl)carbamate (0.20 g, 0.84 mmol), and the reaction mixture was stirred for 24 h. Sodium borohydride (0.06 g, 1.69 mmol) was added and the reaction e stirred for 1 h. The reaction mixture was concentrated under reduced pressure to afford a white solid. Aqueous NaOH (10%, 50 mL) and EtOAc (50 mL) were added, the layers separated, and the aqueous layer was extracted with EtOAc (50 mL). The organic layers were combined, dried over Na2S04, ﬁltered, and concentrated under reduced pressure to afford the d product as a clear oil, which was used without further puriﬁcation.

Step 7: NI,NI/-((5-(3-(iso-Butylamm0ni0)pr0p0xy)-1,3- phenylene)bis(methylene))bis(N3-(3-amm0ni0pr0pyl)pr0pane-I,3-dz'aminium) chloride. To N1,N1'-((5 -(3 -Butylamino)propoxy)-1,3 -phenylene)bis(methylene))bis(N3-(3 - aminopropyl)propane—l,3—diamine) from step 6 was added methanolic HCl (50 mL, 1.0M).

The reaction e was stirred for 1 h, the reaction mixture was concentrated under reduced pressure and the solid collected by vacuum ﬁltration. The solid was washed with EtzO (10 mL) and hot MeOH (10 mL) to afford the desired product (0.14 g, 43%) as a white solid. 1H NMR (500 MHz, D20) 6 ppm 7.21 (s, 1H), 7.19 (s, 2H), 4.30 (s, 4H), 4.23 (t, J: 5.5 Hz, 2H), 3.29 (t, J: 7 Hz, 2H), 3.25-3.19 (m, 12H), 3.12 (t, J: 7.5 Hz, 4H), 2.95 (d, J: 7 Hz, 2H), 2.25—2.03 (m, 11H), 1.01 (d, J: 7.5 Hz, 6H). 13C NMR (125 MHZ, D20) 158.9, 133.0, 123.7, 117.1, 65.8, 54.8, 50.7, 45.7, 44.7, 44.6, 44.2, 36.7, 25.5, 25.2, 23.7, 22.6, 19.1. LRMS [M+H]+494.4.

Step 8: To N1,N1'-((5-(3-(isoButylammonio)propoxy)-1,3-phenylene)bis- (methylene))bis(]\73-(3 -ammoniopropyl)propane-1,3-diaminium) hydrochloride salt was added s NaOH (10%, 100 mL) and 75% CHClg/isopropanol (100 mL). The layers were separated, and the aqueous layer was extracted with 75% CHClg/isopropanol (4 x 100 mL). The organic layers were combined, dried over Na2SO4, ﬁltered, and concentrated under reduced pressure to afford a clear oil, which was used without further puriﬁcation.

Step 9: To a round-bottom ﬂask was added the crude N1,N1'-((5-(3- (isobutylammonio)propoxy)- 1 ,3 -phenylene)bis(methylene))bis(l\l3-(3 - ammoniopropyl)propane-1,3-diaminium) (0.91 g, 1.84 mmol) and MeOH (20 mL). To the solution was added octanal (0.47 g, 3.68 mmol) and the reaction mixture was d at rt for 24 h. Sodium borohydride (0.28 g, 7.36 mmol) was added and the reaction mixture was stirred for 1 h. The reaction mixture was concentrated under reduced pressure to afford a white solid. Aq. NaOH (10%, 100 mL) and EtOAc (100 mL) were added and the mixture stirred for 1 h. The layers were separated and the aqueous layer was extracted with EtOAc (100 mL). The combined organics were dried over , ﬁltered, and concentrated under reduced pressure to afford the desired product as a clear oil which was used without further puriﬁcation.

Step 10: N1,N1'-((5-(3-(isobatylamino)propoxy)-I,3-plzenylene)bis(methylene))— bis(N3-(3-(octylamino)propyl)propane-I,3—diamine), hydrochloride salt. To the crude N1,N1'- ((5 -(3 -(isobutylamino)propoxy)-1,3 -phenylene)bis(methylene))bis(N3-(3 - (octylamino)propyl)propane-1,3-diamine) was added methanolic HCl (100 mL, 1.0M). The reaction mixture was stirred at rt for 1 h. The reaction mixture was concentrated under d pressure, and the solid was collected by vacuum ﬁltration and washed with Et20 (50 mL) and hot MeOH (50 mL) to afford the desired t as a white solid (0.75 g, 42%). 1H NMR (500 MHz, D20) 5 7.21 (s, 1H), 7.20 (s 2H), 4.30 (s, 4H), 4.24 (t, J = 5 Hz, 2H), 3.30 (t, J = 7 Hz, 2H), 3.26-3.14 (m, 16H), 3.07 (t, J = 7 Hz, 4H), 2.96 (d, J = 7.5 Hz, 2H), 2.26- 2.03 (m, 11H), 1.69 (p, J = 6.5 Hz, 4H), 1.38—1.28 (m, 20H), 1.02 (d, J = 6.5 Hz, 6H), 0.87 (t, J = 5.5 Hz, 6H). 13C NMR (125 MHZ,D20)8158.9,133.0,123.7, 117.1, 65.8, 54.8, 50.8, 47.9, 45.7, 44.6, 44.6, 44.2, 44.1, 30.9, 28.1, 28.1, 25.6, 25.5, 25.4, 25.2, 22.6, 22.6, 19.1, 13.4.

Example 98 Antimicrobial activity ofpolyamines in agar media A 0.5 McFarland standard of each bacteria was made, then dual lawns of bacteria spread onto the surface of tryptic soy agar (TSA). 500 uL of the suspended gel were placed on the center of each lawn. Plates were incubated at 37 C for 24 hours, then imaged.

Images of the resulting gels are shown in FIGS. 30A to 301 show the effects of the polyamines CZ-86, CZ-100, and CZ-110 on various bacterial cell cultures. The data shown indicated that when the polyamine compounds were mixed with the gel, the compounds ed out of the gel and created zones of inhibition in the ial lawns. The gel alone showed no signs of antimicrobial activity (B).

The hydrogel that was used for those images is a LifeCore gel with 1% sodium hyaluronate (1% Sodium Hyaluronate Solution Part #82). The CZ compound was suspended in water at 1% (w/w) concentration and combined with the re gel 1:1 for a final product that contained 0.5% sodium hyaluronate and 0.5% CZ compound.

Example 99 CZ-86 and CZ-90p01yaml'ne hemolysz's activity The polyamines CZ—86 and CZ—90 were tested for hemolytic activity. The difference between the hemolytic indexes of both polyamines and the negative control was 0.00 t. This places both test articles in the non-hemolytic range according to the grade outlined below in Table 6A.

Table 6A: Hemol ic Index and Grade: Hemol ic Index Hemol tic Grade S1i_ht1 Hemol ic All test method acceptance criteria were met. The test procedures listed below were followed without deviation.

Table 6B: Results Test Article / Optical Average tic Average Corrected Control Density Optical Index Hemolytic Hemolytic Density Index (% Index (% Hemolysis) Hemolysis) Negative Control Test e / Average Hemolytic Average Corrected Control l Index Hemolytic Hemolytic y Index (% Index (% Hemolysis) Hemolysis) Positive 97.068 101.7 Control 105.638 102.255 Phosphate Buffered Saline (PBS) Blank Table 6C: Hemo_lobin Standard: Reression Outut Constant 0.00056 Standard Error ofY Estimate 0.00604 R2 0.99961 De _rees of Freedom X ient s 1.44547 Standard Error of Coefﬁcient 0.01164 Acceptance Criteria: The negative control must produce a corrected hemolytic index of less than 2%. The positive control must produce a corrected hemolytic index of greater than 5%.

Procedure: The PBS used in testing was calcium and magnesium free. The method has been validated using human blood from one donor. This is in compliance with ISO 10993-4 which states due to differences in blood activity, human blood should be used where possible. Furthermore, g unmatched human blood from multiple donors may cause red blood cell agglutination which in turn could cause hemolysis; this supports the use of a single donor.

The blood was drawn using vacutainers containing 0.1 M sodium citrate at a ratio of 9:1 (3.2% anticoagulant to blood). The blood in this test was used within four hours ofblood draw. The collected blood was refrigerated until testing was performed.

A hemoglobin standard was diluted with Drabkin's reagent to give solutions at concentrations of 0.80, 0.60, 0.40, 0.30, 0.20, 0.10, 0.02, and 0.01 mg/ml. These ons were d to stand at room temperature for a minimum of ﬁve minutes. The absorbance was read on a ophotometer at 540 nanometers (nm). A standard curve was determined with the absorbance values and the standard trations of hemoglobin.

Human blood was fuged at 700-800 x g for 15 minutes. A 1 mL aliquot of the plasma was added into 1 mL of Drabkin's reagent, and placed at room temperature for a minimum of 15 minutes. The absorbance was read on a spectrophotometer at 540 nm. The obin concentration was ined from the standard curve and then multiplied by a factor of 2 to obtain the total plasma free obin. The plasma free hemoglobin was less than 2 mg/mL (actual value 0.221 mg/mL). A 20 uL aliquot of blood was added to 5 mL of Drabkin's reagent in duplicate and allowed to stand at room temperature for a m of 15 minutes. The absorbance was read on a spectrophotometer at 540 nm then multiplied by 251 to account for the dilution.

[0564] Based on the plasma hemoglobin and the blood absorbance against Drabkin's reagent, the blood was diluted out to 10 :: 1 mg/mL with PBS. To verify the blood dilution, a 300 [LL aliquot of this blood was added to 4.5 mL of Drabkin's reagent in triplicate and allowed to stand at room temperature for a minimum of 15 minutes. The absorbance was read on a ophotometer at 540 nm then multiplied by 16 to account for the dilution.

[0565] Glass test tubes were labeled appropriately. Both samples were prepared the same way. The samples were allowed to thaw prior to testing. To each test tube, 7 mL of the test article and 1 mL of the diluted blood were added. The controls consisted of the appropriate amount of control material, 7 mL of PBS and 1 mL of blood. Three tubes were prepared for each test article and control. The tubes were incubated at 37 :: 2°C for 3 hours :: 5 s.

Tubes were gently ed twice at 30 minute intervals throughout the incubation period. A non-hemolytic ve control, a tic positive control and a PBS blank were included.

After incubation, test articles were centrifuged at 700-800 K g for 15 minutes and 1 mL of the supernatant ﬂuid was combined with 1 mL of Drabkin's reagent and allowed to stand at room temperature for a minimum of 15 minutes. Following the centrifugation phase, the supernatant of both test articles visually appeared clear and were particulate free. The PBS blank and the negative control supernatant visually appeared clear and were particulate free. The supernatant of the positive control visually appeared red and were ulate free.

The test articles and controls were read at 540 nm in a spectrophotometer.

The hemolytic index (percent hemolysis) was interpreted using the following equation: Hemoglobin Released (mg/mL) H emo y 1C1 t' I 11dex —_ x 100 Hemoglobin t (mg/mL) Where: Hemoglobin Released (mg/mL) = (Optical Density x X ient+ Constant) x 16 Hemoglobin Present (mg/ml) = Diluted Blood 10 :: 1 mg/mL

[0568] The corrected hemolytic index was calculated by subtracting the hemolytic index of the PBS blank solution from the hemolytic index of the test article and controls.

The test article is compared to the ve control by subtracting the hemolytic index of the negative control from the hemolytic index of the test article.

Table 6D: Test ters: BloodT 6 Used: Nitrile Glove Material, tested at 3 cmZ/mL Pol u-ro lene s tested at 0.2 ; ams/mL Total Hemo lobin Kit: Stanbio, 80 m/dL Incubation Time: Incubation Temerature: 37 :: 2°C Example 100 Minimum Elation Media WEN!) elution test 5 Summary: The Minimal Essential Media (MEM) Elution test was designed to determine the cytotoxicity of extractable substances. The test article was added to cell monolayers and incubated. The cell monolayers were examined and scored based on the degree of cellular destruction. All test method acceptance criteria were met. The test ure(s) listed above were followed without ion.

Table 7A: Results of Test Results Scores mum“ O‘CZ'ZS Pass/Fail Initial Fail 4 4 4 4 Concentration 2.5 m_/mL ————— ————— ————— ————— Table 7B: Controls: Scores Amount . Tested / Extraction Identiﬁcation Extraction #1 #2 #3 Average Ratio Solvent Amount Negative Control - 0.2 g/mL 4 g / 20 mL Pol IOI'O lene Pellets Media Control “nun—— Positive Control - 4 4 4 4 0.2 g/mL 4 g / 20 mL Latex Natural Rubber Acceptance Criteria: The United States Pharmacopeia & National Formulary (USP <87>) states that the test article meets the requirements, or receives a passing score (Pass) if the vity grade is not greater than grade 2 or a mild reactivity. The ANSI/AAMI/ISO rd states that the ement of a numerical grade greater than 2 is considered a cytotoxic effect, or a failing score (Fail).

Acceptance criteria were based upon the ve and media controls ing "0" reactivity grades and positive controls receiving a 3-4 reactivity grades ate to severe).

The test was considered valid as the control results were within acceptable parameters.

The cell monolayers were examined microscopically. The wells were scored as to the degree of discernable morphological cytotoxicity on a ve scale of 0 to 4: Table 7C: Standards for Grades Conditions of All Reactivity Cultures No cell lysis, None 0 intrac o lasmic _ranules.

Not more than 20% Slight 1 rounding, occasional lysed cells. rounding, no extensive cell 1 sis. roundin; andl sed cells. destruction.

[0574] The results from the three wells were averaged to give a ﬁnal cytotoxicity score.

Procedure: The test article was added to 1X Minimal Essential Media + 5% bovine serum at an initial concentration of 2.5 mg/ml. This initial concentration was then diluted 1: 2, 1:4, 1:8, and 1:16. Multiple well cell e plates were seeded with a veriﬁed quantity of industry standard L-929 cells (ATCC CCL-l) and incubated until approximately 80% conﬂuent. The test articles and l extracts were added to the cell monolayers in cate. The cells were incubated at 37 :: 1°C with 5 :: 1% C02 for 48 :: 3 hours.

Example 101 Minimum Elation Media (MEM) elution test 0fCZ-86 Summary: The polyamine compound CZ-86 was added to cell monolayers and incubated according to the procedure described in e 100.

Table 8A: Results , _ Results mm of (31-86 Pass/Fan 400 m ————— ————_— ————_— ——-_‘--_-_ ——-_-_-_-_ Table 8B: Controls: Amount Tested / tion ﬁcation Extraction Average Ratlo Solvent Amount Negative Control - 4 g / 20 mL Polypropylene Pellets Media Control -_ 20 ml Positive Control - 4 4 4 g / 20 mL Latex Natural Rubber The cell monolayers were examined microscopically. The wells were scored as to the degree of discemable morphological cytotoxicity on a relative scale of 0 to 4: Table 8C: Standards for Grades Cultures No cell lysis, None 0 intracytoplasmic granules.

Less than or equal to 20% Slight 1 rounding, occasional lysed cells. than or eual to 50% rounding, no extensive cell lysis.

Greater than 50% to less Moderate 3 than 70% rounding and l sed cells.

Nearly complete Severe 4 destruction of the cell la ers.

Example 102 Minimum Elation Media (MEA/I) elutian test 0fCZ—52 and CZ—100 Summary: The test articles shown below was added to cell monolayers and ted according to the procedure of Example 100.

Table 9A: s Results Scores Identiﬁcation Pass/Fail I(a)#2 Avera 1 e a:>—I ’— 4 4; A 4 m(/3 VJ [\J [\J [\J m(/1 (/1 a:(ll (/1 wA-h DJ-h-h ;_a ;_a Hang-l; #-l> N [vb-DA N-b-b-b N-b NA N-b #-l> AAOO 4; A ##-I>OO --_—____ Code for Test Article: 1) Chlorhexidine gluconate 2) Glutaraldehyde 3) CZ-52 4) CZ—lOO ) ycin 6) Benzethonium chloride Table 9B: Controls: es Amount Tested Extraction Identiﬁcation -A / Extractlon. verage Ratio Solvent Amount Negative Control - 0.2 g/mL 4 g / 20 mL Pol r0 1 lene Pellets Media Control “nun—— Positive Control - 4 4 4 4 0.2 g/mL 4 g / 20 mL Latex Natural Rubber The cell monolayers were examined microscopically. The wells were scored as to the degree of discernable morphological xicity on a relative scale of 0 to 4: Table 9C: Standards for Grades Conditions of All Cultures Reactivi Grade intrac o lasmic es. rounding, occasional lysed cells. rounding, no extensive cell 1 sis. roundin andl sed cells. destruction.

Example 103 Minimum Elation Media (MEM) elution test 0fCZ-58, CZ-62, CZ—65 and CZ-66

[0580] Summary: The test articles shown below was added to cell yers and incubated according to the procedure of Example 100.

Table 10A: Results Identiﬁ- Results Scores Dil“tion cation Pass/Fail #1 #2 #3 Avera_e b—KN-h-hb—tb—KUJA-hl—tb—KN l—‘N-b-bl—‘l—‘N-b-bl—‘l—‘LQ-b-bl—‘l—‘N j—A h—K Table 10B: Controls: Amount Tested Identiﬁcation / Extraction e E11323?“ Solvent Amount Negative Control - 0 Wm Pol pro o lene Pellets 0 N/A Positive Control - 4 0.2gmL 4g/20mL Latex Natural Rubber The cell monolayers were examined microscopically. The wells were scored as to the degree of discernable morphological cytotoxicity on a relative scale of 0 to 4: Table 10C: Standards for Grades Conditions of All Cultures Reactivi Grade No cell lysis, intrac o lasmic es.

Not more than 20% rounding, Slight 1 occasional l sed cells.

Not more than 50% rounding, Mild 2 no extensive cell 1 sis.

Not more than 70% rounding Moderate 3 and lysed cells.

Nearly complete cell Severe destruction.

Example 104 Minimum Elation Media (MEM) elution test 0fCZ—92, CZ—96 and CZ-99 Summary: The test articles shown below was added to cell monolayers and incubated according to the procedure of Example 100.

Table llA: Results ﬁ- Results Dilution catlon e /mL Fail 4 4 CZ-92 2 2 0 0 CZ-96 0 0 0 0 0 0 CZ—99 Amount Tested Extraction Identiﬁcation / Extraction Ratio Solvent Amount Negative Control - 4 g / 20 mL Polypropylene s Media Control 20 ml ve Control - 4 g / 20 mL Latex Natural Rubber The cell monolayers were examined microscopically. The wells were scored as to the degree of discernable morphological cytotoxicity on a ve scale of 0 to 4: Table llC: Standards for Grades Conditions of All Cultures No cell lysis, None intrac o lasmic _ranules.

Less than or equal to 20% Slight rounding, occasional lysed cells.

Greater than 20% to less than or equal to 50% rounding, no extensive celll sis.

Greater than 50% to less than Moderate 3 70% rounding and lysed cells.

Nearly complete destruction Severe 4 ofthe cell la ers.

While this invention has been described in certain embodiments, the present ion can be further modiﬁed within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the ion using its general principles. Further, this ation is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims.

Claims

CLAIMS :

1. A non-therapeutic method for dispersing or killing a biofilm, the method comprising a step of contacting the biofilm with an anti-biofilm composition, thereby dispersing or killing the biofilm; wherein the anti-biofilm composition comprises a biocidal ine compound selected from the group consisting of Ra Ra A1 A5 A1 A5 A3 Ra , Ra A3 Ra , and a salt thereof; wherein: A1, A2, A3, and A5 are each an independently selected CR5; each Ra is an independently selected group of Formula V: R1a R1b N R2c R4 Rm R2d R3 ; each R1a and R1b is a member independently selected from hydrogen and alkyl; each R2a, R2b, R2c and R2d is a member independently selected from the group consisting of en, alkyl, and alkyl; each R3 is a member independently selected from the group consisting of -Z1-R4, -Z1-Y1-R4, -Z1-Y1-Y2-R4, and -Y2-Y3-R4; each Y1, Y2, and Y3 is an independently ed group of Formula IA: Rm R2d each Z1 and Z2 is an independently selected -N(R4)-; each Rm is -CH2-; 16584570_1 (GHMatters) P41348NZ01 each m is an integer independently selected from 1 to 2; each R4 is a member independently ed from the group consisting of hydrogen, alkyl, fluoroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, arylalkyl, cycloalkylalkyl, and heteroarylalkyl; or, alternatively, for an -N(R4)2 group, one of the two R4 in the group is a member selected from the group consisting of -(CO)OR6a, -(CO)N(R6a)(R6b), and -C(NR6a)N(R6b)(R6c); or, alternatively, for an -N(R4)2 group, the two R4 groups join to form a heterocyclic ring; each R5 is a member independently selected from the group consisting of en, alkyl, hydroxyl, alkoxy, aminoalkoxy, alkylamino, alkylaminoalkoxy, alkenyl, alkynyl, aryl, aryloxy, arylamino, cycloalkyl, lkoxy, cycloalkylalkoxy, cycloalkylamino, cycloalkylalkylamino, heterocyclyl, heterocycyloxy, heterocycylamino, halo, haloalkyl, fluoroalkyloxy, heteroaryl, heteroaryloxy, heteroarylamino, arylalkyl, kyloxy, arylalkylamino, heteroarylalkyl, heteroarylalkyloxy, arylalkylamino, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; and each R6a, R6b, and R6c is a member independently selected from hydrogen and alkyl; wherein if R4 is -(CO)OR6a, R6a is alkyl; wherein the polyamine compound comprises at least six primary or secondary amino groups.

2. The method of claim 1, wherein: each R1a and R1b is hydrogen; and each R2a, R2b, R2c and R2d is hydrogen.

3. The method of claim 1 or 2, wherein: each Ra is an independently selected H(CH2)n]pNHR4; each n is an integer ndently selected from 3 or 4; and each p is an integer independently selected from 1 to 3.

4. The method of any one of claims 1 to 3, wherein the biocidal polyamine compound is a hydrochloride salt.

5. The method of any one of claims 1 to 4, wherein the biocidal polyamine compound is selected from the group ting of 16584570_1 (GHMatters) P41348NZ01 Ra Ra Ra Ra R5 , Ra , and a salt f; wherein: each Ra is an independently selected -CH2[NH(CH2)n]pNHR4; each n is independently selected from 3 or 4; each p is ndently selected from 1 to 3; each R4 is a member independently selected from the group consisting of hydrogen, alkyl, fluoroalkyl, l, alkynyl, aryl, cycloalkyl, heteroaryl, arylalkyl, cycloalkylalkyl, and heteroarylalkyl; and R5 is a member independently selected from the group consisting of hydrogen, alkyl, hydroxyl, alkoxy, aminoalkoxy, alkylamino, alkylaminoalkoxy, l, alkynyl, aryl, aryloxy, arylamino, cycloalkyl, cycloalkoxy, cycloalkylalkoxy, cycloalkylamino, cycloalkylalkylamino, heterocyclyl, heterocycyloxy, heterocycylamino, halo, haloalkyl, alkyloxy, heteroaryl, heteroaryloxy, heteroarylamino, arylalkyl, arylalkyloxy, arylalkylamino, heteroarylalkyl, heteroarylalkyloxy, heteroarylalkylamino, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl; n the polyamine nd comprises at least six primary or secondary amino groups.

6. The method of any one of claims 1 to 5, wherein: each Ra is an independently selected -CH2[NH(CH2)n]pNHR4; each n is 3; and each p is independently selected from 1 to 3.

7. The method of any one of claims 1 to 6, wherein each R4 is a member independently selected from the group consisting of hydrogen, alkyl, fluoroalkyl, alkenyl, alkynyl, arylalkyl, and cycloalkylalkyl.

8. The method of any one of claims 1 to 7, wherein each R4 is a member independently selected from the group ting of hydrogen, alkyl, arylalkyl, and cycloalkylalkyl. 16584570_1 (GHMatters) P41348NZ01

9. The method of any one of claims 1 to 8, wherein each R4 is a member independently ed from the group consisting of hydrogen, isopropyl, isobutyl, hexyl, octyl, and exylmethyl.

10. The method of any one of claims 5 to 9, wherein each R4 is not hydrogen.

11. The method of claim 10, wherein each R4 is independently selected from the group consisting of butyl, isobutyl, hexyl, and octyl.

12. The method of any one of claims 1 to 11, wherein each R5 is selected from the group consisting of hydrogen, alkyl, hydroxyl, alkoxy, aminoalkoxy, alkylamino, alkylaminoalkoxy, aryl, aryloxy, cycloalkyl, cycloalkoxy, lkylalkoxy, halo, fluoroalkyl, fluoroalkyloxy, heteroaryl, arylalkyl, arylalkyloxy, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl.

13. The method of any one of claims 1 to 12, n each R5 is a member independently selected from the group consisting of hydrogen, hydroxyl, alkoxy, alkylaminoalkoxy, cycloalkoxy, cycloalkylalkoxy, halo, heteroaryl, arylalkyloxy, and hydroxyalkyl.

14. The method of any one of claims 5 to 11, wherein R5 is hydroxyl, alkoxy, cycloalkoxy, heterocycyloxy, arylalkyloxy, heteroaryloxy, or heteroarylalkyloxy.

15. The method of any one of claims 5 to 12, wherein R5 is ed from the group ting of yl, alkoxy, cycloalkoxy, and arylalkyloxy.

16. The method of claim 5, wherein the biocidal polyamine compound is selected from the group ting of Ra Ra R5 and a salt thereof; wherein: each Ra is an independently selected -CH2[NH(CH2)n]pNHR4; each n is independently selected from 3 or 4; 16584570_1 (GHMatters) P41348NZ01 each p is independently selected from 1 to 3; each R4 is a member independently selected from the group consisting of hydrogen, alkyl, arylalkyl, and cycloalkylalkyl; and R5 is selected from the group consisting of hydrogen, hydroxyl, alkoxy, alkylaminoalkoxy, cycloalkoxy, cycloalkylalkoxy, halo, heteroaryl, arylalkyloxy, and hydroxyalkyl; wherein the ine compound comprises at least six primary or secondary amino

17. The method of claim 16, wherein each n is 3.

18. The method of claim 16 or 17, n each R4 is a member independently selected from the group consisting of hydrogen, isopropyl, yl, hexyl, octyl, and cyclohexylmethyl.

19. The method of any one of claims 16 to 18, wherein each R4 is not hydrogen.

20. The method of claim 19, wherein each R4 is independently selected from the group ting of butyl, isobutyl, hexyl, and octyl.

21. The method of claim 16, wherein R5 is selected from the group consisting of hydroxyl, alkoxy, cycloalkoxy, and arylalkyloxy.

22. The method of claim 5, wherein the biocidal polyamine compound is selected from the group consisting of Ra Ra Ra and a salt f; wherein: each Ra is an independently selected -CH2[NH(CH2)n]pNHR4; each n is 3; each p is independently selected from 1 to 3; and 16584570_1 (GHMatters) P41348NZ01 each R4 is a member independently selected from the group consisting of hydrogen, alkyl, arylalkyl, and cycloalkylalkyl; wherein the polyamine compound comprises at least six y or secondary amino groups.

23. The method of claim 22, wherein each R4 is independently selected from the group consisting of hydrogen, butyl, isobutyl, hexyl, and octyl.

24. The method of claim 5, wherein the biocidal polyamine compound is selected from the group consisting of Ra Ra Ra and a salt f; wherein: each Ra is an independently selected -CH2[NH(CH2)n]pNHR4; each n is 3; each p is ndently selected from 1 to 3; and each R4 is a member independently selected from the group consisting of alkyl, arylalkyl, and cycloalkylalkyl; wherein the polyamine compound comprises at least six primary or ary amino groups.

25. The method of claim 24, wherein each R4 is independently selected from the group consisting of butyl, isobutyl, hexyl, and octyl.

26. The method of any one of claims 1 to 4, wherein the al polyamine compound is selected from the group consisting of: 16584570_1 (GHMatters) P41348NZ01 H H H2N N H2N N H BocHN N HN N HN N H H HN N N NH2 N NH2 H H . 6 HCl . 6 HOBz N NHBoc , , H , HN NH2 H HN N H2N N H NH2 NH NH NH HN N N H2N H . N HN N NH2 H 6 HCl , H , , H NH2 H H N N N HN H2N N NH HN H N NH2 H2N HN HN NH H2N HN H2N NH BocHN . N NH NH 9 HCl H NH2 . . mixture of mono / di / tri-Boc 9 HCl , HO 6 HCl , , H H N N H BocHN N NH H2N N NH H H H2N N NH H2N HN H2N HN H2N HN N NH N NH N NH H H H NHBoc . H2N . . 8 HCl x HOBz H2N x decanoic acid , , , 16584570_1 ters) P41348NZ01 HN N HN NH NH NH N HN NH2 H O N S N N N N H H H2N H H H NH2 H2N . 8 HCl NH2 . NH2 · 6 HCl 6 HCl , , , N NH N NH H H NH NH O N N O NH H N N NH2 H H H2N H2N NH2 . . 6 HCl H2N 6 HCl , , , HN NH HN NH N N O H H O N HN NH HN HN N H H O N H2N H2N NH2 . . 6 HCl H . 6 HCl 6 HCl , , , N N H H N N NH HN H H NH HN . 6 HCl · 6 HCl NH N NH2 H2N , , 16584570_1 ters) P41348NZ01 H H N N H H N N N N H H NH HN NH NH H2N HN NH N 6 HCl · 6 HCl , , N N H H HN NH NH HN HN NH HN NH NH N N N H H H NH HN H2N NH2 · 6 HCl · 6 HCl · 6 HCl , , , N N HN NH HN NH H H NH HN N N O N N H H H H HN HN NH HN NH2 H2N · 6 HCl · 6 HCl , , , HN NH N N N N H H H H O N N N N N N H H H H H H Br NH2 H2N NH HN NH HN NH . . · 7 HCl 6 HCl 6 HCl , , , 70_1 (GHMatters) P41348NZ01 N NH NH N N H H N NH HN N N H H N N H H · 6 HCl · 6 HCl , , and N N H H NH HN N HN 6 HCl

27. The method of claim 16, wherein the biocidal polyamine nd is selected from the group consisting of: H H H2N N H2N N H H2N N HN N HN N H H HN N N NH2 N NH2 H H . 6 HCl . 6 HOBz N NH2 , , H , N NH2 HN N H H HN NH HN NH NH HN NH2 N S N N N H H . H H NH2 H2N . 8 HCl HO 6 HCl NH2 · 6 HCl , , , 16584570_1 (GHMatters) P41348NZ01 N NH N NH H N NH H NH H NH O N O NH H O NH H2N H NH2 . NH2 . H2N 6 HCl 6 HCl H2N , , , HN NH HN NH N N O H H O N HN HN H N N N H H H NH OH H2N H2N NH2 NH2 . . N 6 HCl 6 HCl 6 HCl H , , , N N H H N N N N NH HN H H H H NH HN NH HN . 6 HCl O O · 6 HCl NH N H2N H2N NH2 H2N 6 HCl , , , N N HN NH H H HN NH NH HN HN NH N N H H NH N N N NH HN H2N NH2 · 6 HCl · 6 HCl · 6 HCl , , , 70_1 (GHMatters) P41348NZ01 N N H H NH HN HN NH HN NH NH N O H N N O N N H H H H NH HN NH2 H2N · 6 HCl · 6 HCl , , , N N H H HN NH NH HN N N O H H O N N N N H H HN HN H H NH2 H2N NH HN NH . · 6 HCl · 7 HCl 6 HCl , , N NH N N NH H H O N N H H H Br N N H H NH HN 6 HCl · 6 HCl , , , N N H H N N H H NH HN NH HN N N N HN H H . · 6 HCl 6 HCl , and .

28. The method of claim 27, wherein the biocidal polyamine compound is selected from the group ting of: 16584570_1 (GHMatters) P41348NZ01 N NH2 H HN NH N NH HN NH H H O N S N N H H H2N H . NH2 H2N . NH2 HO 6 HCl · 6 HCl 6 HCl , , , N NH N NH H H NH NH O N N O NH H H N N NH2 H H H2N H2N NH2 . . 6 HCl H2N 6 HCl , , , HN NH HN NH N N O H H O N HN NH HN HN N H H O N H2N H2N NH2 . . 6 HCl H . 6 HCl 6 HCl , , , HN NH HN NH N N H H O NH HN HN NH N N O H H · 6 HCl N N NH HN NH2 H2N H2N NH2 · 6 HCl · 6 HCl , , , 16584570_1 ters) P41348NZ01 N N H H NH HN HN NH HN NH NH N O N N O H N N H H H H NH HN NH2 H2N · 6 HCl · 6 HCl , , , N N H H HN NH NH HN N N HN HN H H NH2 H2N · 6 HCl · 7 HCl , , N NH N N NH H H O N N H H H Br N N H H NH HN 6 HCl · 6 HCl , and .

29. The method of claim 19, wherein the biocidal polyamine nd is selected from the group consisting of: 16584570_1 (GHMatters) P41348NZ01 HN NH N N N N H H H H NH HN NH HN N HN . 6 HCl NH NH N H NH N . N 6 HCl H · 6 HCl , , N N H H HN NH NH HN HN NH NH N O N N H N N H H H H NH HN NH HN · 6 HCl · 6 HCl , , , , N N H H NH HN N N N N O H H H H N N N N H H H H HN HN Br NH HN NH HN . . · 6 HCl 6 HCl 6 HCl , , , 16584570_1 ters) P41348NZ01 N NH NH N N H H N NH HN N N H H N N H H · 6 HCl · 6 HCl , , and N N H H NH HN N HN 6 HCl

30. The method of claim 21, wherein the biocidal polyamine compound is selected from the group ting of: N NH N NH H N NH H NH H NH O N O NH H O NH H2N H NH2 . NH2 . H2N 6 HCl 6 HCl H2N , , , HN NH HN NH N N O H H O N HN HN N H N N H H H NH OH H2N H2N NH2 . N NH2 . . 6 HCl 6 HCl 6 HCl H , , , 16584570_1 (GHMatters) P41348NZ01 HN NH N N N N H H H H NH HN O NH HN N N O O H H · 6 HCl NH HN H2N H2N NH2 H2N 6 HCl , , , N N HN NH H H NH HN N N H H HN HN NH2 H2N · 6 HCl · 6 HCl , and .

31. The method of claim 22 or 23, wherein the biocidal ine compound is selected from the group consisting of: 16584570_1 (GHMatters) P41348NZ01 H2N N NH HN H NH2 HN N NH2 NH NH NH H2N HN N NH N H2N H H . N HN H NH2 . 6 HCl 9 HCl , , , N H H2N N NH H H2N N NH H2N HN H2N HN N NH H N NH H2N . x HOBz H2N . x decanoic acid , and .

32. The method of claim 24 or 25, wherein the biocidal polyamine compound is selected from the group consisting of: HN N NH NH N HN and a salt thereof.

33. The method of any one of claims 1 to 32, wherein the biofilm comprises an antibiotic-resistant bacterial species.

34. An anti-biofilm composition comprising a biocidal polyamine compound used in the method of any one of claims 16 to 32. 70_1 ters) P41348NZ01

35. The anti-biofilm composition of claim 34, wherein the biocidal ine compound is selected from the group consisting of Ra Ra R5 and a salt thereof; wherein: each Ra is an independently selected -CH2[NH(CH2)n]pNHR4; each n is independently ed from 3 or 4; each p is independently selected from 1 to 3; each R4 is a member independently ed from the group consisting of hydrogen, alkyl, kyl, and cycloalkylalkyl; and R5 is selected from the group consisting of hydrogen, hydroxyl, alkoxy, alkylaminoalkoxy, cycloalkoxy, cycloalkylalkoxy, halo, heteroaryl, arylalkyloxy, and hydroxyalkyl; wherein the polyamine compound comprises at least six primary or secondary amino

36. The anti-biofilm composition of claim 34, n the biocidal polyamine compound is selected from the group consisting of Ra Ra Ra and a salt thereof; wherein: each Ra is an independently selected -CH2[NH(CH2)n]pNHR4; each n is 3; each p is independently selected from 1 to 3; and each R4 is a member independently selected from the group consisting of hydrogen, alkyl, arylalkyl, and cycloalkylalkyl; wherein the polyamine nd comprises at least six primary or secondary amino groups. 16584570_1 (GHMatters) P41348NZ01

37. The anti-biofilm composition of claim 34 or 36, wherein the biocidal polyamine compound is selected from the group consisting of Ra Ra Ra and a salt thereof; wherein: each Ra is an independently selected -CH2[NH(CH2)n]pNHR4; each n is 3; each p is independently selected from 1 to 3; and each R4 is a member independently selected from the group consisting of alkyl, arylalkyl, and cycloalkylalkyl; n the polyamine compound comprises at least six primary or secondary amino groups.

38. The anti-biofilm composition of any one of claims 34 to 37, wherein the al polyamine compound is selected from the group consisting of: H H H2N N H2N N H H2N N HN N HN N H H HN N N NH2 N NH2 H H . 6 HCl . 6 HOBz N NH2 , , H , N NH2 HN N H H HN NH HN NH NH HN NH2 N S N N N H H . H H NH2 H2N . 8 HCl HO 6 HCl NH2 · 6 HCl , , , 16584570_1 (GHMatters) P41348NZ01 N NH N NH H N NH H NH H NH O N O NH H O NH H2N H NH2 . NH2 . H2N 6 HCl 6 HCl H2N , , , HN NH HN NH N N O H H O N HN HN H N N N H H H NH OH H2N H2N NH2 NH2 . . N 6 HCl 6 HCl 6 HCl H , , , N N H H N N N N NH HN H H H H NH HN NH HN . 6 HCl O O · 6 HCl NH N H2N H2N NH2 H2N 6 HCl , , , N N HN NH H H HN NH NH HN HN NH N N H H NH N N N NH HN H2N NH2 · 6 HCl · 6 HCl · 6 HCl , , , 16584570_1 ters) P41348NZ01 N N H H NH HN HN NH HN NH NH N O H N N O N N H H H H NH HN NH2 H2N · 6 HCl · 6 HCl , , , N N H H HN NH NH HN N N O H H O N N N N H H HN HN H H NH2 H2N NH HN NH . · 6 HCl · 7 HCl 6 HCl , , N NH N N NH H H O N N H H H Br N N H H NH HN 6 HCl · 6 HCl , , , N N H H N N HN NH2 H H NH HN NH2 NH NH HN N N N HN N H2N H H . H . · 6 HCl 6 HCl 6 HCl , , , 16584570_1 ters) P41348NZ01 H H N N H2N N NH H2N N NH H H HN N NH NH H2N HN H2N HN N NH N NH N HN H H H NH2 . H2N . 9 HCl x HOBz , , , H2N N NH H HN N NH NH H2N HN N NH H N HN H2N . H x decanoic acid , and .

39. A non-therapeutic method for inhibiting formation of a biofilm, the method comprising a step of treating planktonic bacteria with the anti-biofilm ition of any one of claims 34 to 38, thereby inhibiting incorporation of the planktonic bacteria into the biofilm.

40. The anti-biofilm composition of any one of claims 34 to 38, further comprising a carrier.

41. The anti-biofilm composition of claim 40, wherein the ition is a tablet, a pill, a troche, a capsule, an aerosol spray, a solution, a suspension, a gel, a paste, a cream, a foam, a wash on, a dressing, a wound gel, or a synthetic tissue.

42. The anti-biofilm composition of claim 40, wherein the composition is coated on or impregnated into a surface of a medical device. 16584570_1 ters) P41348NZ01

43. The anti-biofilm composition of claim 42, wherein the medical device is a clamp, a s, a scissors, a skin hook, tubing, a needle, a retractor, a scaler, a drill, a chisel, a rasp, a saw, a catheter, an orthopedic , an cial heart valve, a prosthetic joint, a voice prosthetic, a stent, a shunt, a pacemaker, a surgical pin, a respirator, a ventilator, and an endoscope.

44. The anti-biofilm composition of claim 40, wherein the anti-biofilm composition is selected from the group consisting of a sanitizing wipe, a cleanser, a toilet bowl insert, a shampoo, a bath additive, a liquid soap, a solid soap, a lotion, a cream, a deodorant, a moist cleaning cloth, an oil, and a powder.

45. The anti-biofilm composition of claim 40, wherein the anti-biofilm composition is a paint, a pipe coating, a flush solution, or pipeline flush solution.

46. Use of an iofilm composition in the manufacture of a medicament for therapeutically sing or killing a biofilm on a surface of a subject, wherein the ofilm composition comprises: (i) a pharmaceutically acceptable carrier; and (ii) a therapeutically effective amount of a biocidal polyamine compound used in the method of any one of claims 1 to 32; wherein the anti-biofilm composition is formulated for topical, oral, transmucosal, vaginal, or rectal administration.

47. The use of claim 46, wherein the biofilm comprises an antibiotic-resistant bacterial species.

48. The use of claim 46 or 47, wherein the anti-biofilm ition is a tablet, a pill, a troche, a capsule, an aerosol spray, a solution, a sion, a cream, a foam, a liquid, a gel, a paste, or a powder.

49. The use of any one of claims 46 to 48, wherein the surface is a dermal surface, a mucosal surface, an oral surface, a urinary tract surface, a vaginal tract surface, or a lung surface. 16584570_1 (GHMatters) P41348NZ01

50. The use of any one of claims 46 to 49, wherein the surface is skin or soft tissue that has been compromised by dermatitis, ulcers, a burn injury, or trauma.

51. The use of any one of claims 46 to 50, n the anti-biofilm composition is for treating a patient with a biofilm-related disorder.

52. The use of claim 51, wherein the biofilm-related disorder is an infection, pneumonia, cystic fibrosis, otitis media, a urinary tract disorder, a periodontal disease, iectasis, dental caries, or acne. 73919818V.1 70_1 (GHMatters) P41348NZ01