TOPOISOMERASE INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to United States Provisional Application No. 63/064,440, filed on August 12, 2020; and to United States Provisional Application No. 63/070,382, filed on August 26, 2020; the contents of each of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to compounds, compositions and methods for treating cancer, specifically pancreatic, breast, lung, gastrointestinal (including colon), lymphoma, or blood cancers.

BACKGROUND OF THE INVENTION

Cancer is a complex genetic disease whereby cells undergo neoplastic transformation and are no longer subject to the same homeostatic regulation as normal cells. Cancer cells are united by their shared traits of uncontrollable cell growth and the ability to invade surrounding tissues. Importantly, they accumulate alterations that confer survival and proliferative advantage and can endure the continuous bombardment of cellular stresses (ex: genomic instability, harsh microenvironments, oncogene activation, etc.) that would usually dictate a cell to undergo apoptosis.

Chemotherapy and radiation, which remain as the standard line of treatment for many cancers, target rapidly dividing cells and, therefore, are not specific towards cancer cells. These uniform treatments cause unbridled toxicity as healthy cells in the blood, skin, stomach, and hair are also impacted. Moreover, different cancers can respond differently to the same treatments. There is a critical need to discover new cancer therapeutics that can distinguish healthy cells from their diseased counterparts to improve anticancer efficacy and reduce side effects.

Cancers pose difficult challenges for the development of novel, effective therapies.

First, the key targets for cancer therapy are often “undruggable”. These proteins do not have binding pockets that are chemically tractable, i.e. they are not amenable to binding by a small molecule/inhibitor and cannot be targeted pharmacologically. Second, drug delivery to cancer cells may be insufficient. Specifically, in many cancers, a complex interplay between cancerous cells and those in the surrounding tissue, or stroma, creates an intricate and dense tumor microenvironment with poor blood circulation, making it difficult to deliver drugs to the tumor. Inadequate supply of the drug can also give rise to subpopulations that are resistant to treatment. These drug-resistant cells could repopulate the tumor with a more aggressive and invasive phenotype.

Third, the complex nature of the disease at the genomic, epigenetic, and metabolic levels converge to activate multiple oncogenic pathways and potentiate crosstalk within these pathways and create intensive intratumoral heterogeneity. Intratumoral heterogeneity makes it difficult to target all cells within the tumor effectively with only one drug.

Fourth, the presence of cancer stem cells, which are believed to be responsible for tumor initiation, metastasis, and drug resistance.

Finally, the ability of cancer to evade the immune system.

Camptothecin is a cytotoxic alkaloid isolated from leaves and barks of Camptotheca accuminata (Nyssaceae), a plant native to China, which has a pentacyclic structure consisting of a characteristic fused 5-ring system of quinoline (rings A and B), pyrroline (ring C), a-pyridone (ring D) and a six-membered lactone (ring E) and is distinguished by displaying a strong inhibitory activity toward biosynthesis of nucleic acid. In addition, camptothecin is characterized by its rapid and reversible action and its lack of any cross-tolerance with existing anti-tumor agents and by exhibiting a strong anti-tumor activity against experimentally transplanted carcinoma such as leukemia L- 1210 in mice or Walker 256 tumor in rats.

Camptothecin

Although camptothecin is still regarded as one of the most potent substances possessing anti-tumor activity, the use of this compound itself for clinical treatments is significantly limited because of high toxicity, poor solubility, and lack of delivery systems. This has led to a search for analogs of camptothecin that are more effective in anti-tumor performance with reduced toxicity and improved solubility.

One camptothecin analog is 7-ethyl-10-hydroxycamptothecin (SN-38), more formally named ((+)-(4S)-4,l l-diethyl-4,9-dihydroxy-lH-pyrano[3',4':6,7]-indolizino[l,2- b]quinoline- 3,14(4H,12H)-dione, which was first disclosed in U.S. Pat. No. 4,473,692. SN-38 is a potent topoisomerase-I inhibitor (Topi), a validated target in many cancers, including pancreatic cancer, breast cancer, and non-small cell lung cancer. It is thought to bind to the enzyme topoisomerase I, the enzyme responsible for relieving torsional strain in DNA by inducing reversible single-strand breaks. The bound SN-38 appears to block religation of the single-strand breaks by topoisomerase- I thereby causing cytotoxicity in mammalian cells which, apparently, cannot otherwise sufficiently repair the breaks. As shown below, SN-38 has two hydroxy groups at the “10” and “20” positions that may be coupled with capping groups to provide derivatives.

SN-38

In addition to SN-38 cytotoxic capacity, recent studies have demonstrated that Topi inhibitors can (1) enhance T cell-mediated cytotoxicity and upregulate expression of MHC class I tumor antigens and (2) work synergistically with approved therapies to target cancer stem cells. SN-38 can therefore act not only on bulk tumor cells, but also the cells that are responsible for recurrence and metastasis while subverting the tumor’s mechanisms for immune evasion.

Unfortunately, SN-38 is not soluble in pharmaceutically acceptable solvents and the lactone ring is unstable at pH 7.4, opening to the inactive carboxylate. This led to the discovery of irinotecan, a water-soluble prodrug.

Irinotecan

The metabolic conversion of irinotecan to SN-38 occurs primarily in the liver by carboxylesterase-mediated cleavage of the carbamate bond between the camptothecin moiety and a dipiperidino side chain. Subsequently, this derivative undergoes conjugation to form the glucuronide metabolite. However, only 3-8% of irinotecan is converted into the active metabolite, SN-38, which results in higher prodrug required, off-target effects, and unpredictable interpatient variability. Irinotecan has approved efficacy in pancreatic cancer, but has significant side effects, including severe neutropenia and GI toxicity.

SN-38 is 1000 times more potent than irinotecan, so it could have a much better therapeutic value if a suitable delivery vehicle can be found. Thus, it is desirable to develop additional Topi inhibitors and therapies with better efficacy and lower adverse effects than irinotecan.

SUMMARY OF THE INVENTION

An object of this invention is to provide Topi inhibitor compounds with improved antitumor efficacy and reduced toxicity.

This invention provides a compound of Formula 1 or a salt thereof

wherein

G ¹ is H or A^B ¹ ,

G ² is H or A ² -B ² , wherein when G ¹ is H, G ² is A ² -B ² and when G ¹ is A^B ¹ , G ² is H;

A ¹ and A ² are each independently an amino acid residue wherein the amino acid is selected from the group consisting of tryptophan, phenylalanine, tyrosine, lysine, arginine, norvaline, 2-/er/-butylglycine, 3-amino-2-naphthoic acid, 6-amino-2- naphthoic acid, 4-amino-l -naphthoic acid, homoarginine, ornithine, proline, histidine, threonine, sulfamoyl-omithine, 2-amino-5-((2-amino-3,4-dioxocyclobut-l-en-l- yl)amino)pentanoic acid, 6-boronoleucine, (l-benzimidazolonyl)alanine, glycine, valine, alanine, beta-alanine, leucine, isoleucine, glutamic acid, aspartic acid, and N- substituted derivatives thereof, wherein when the amino acid comprises glycine, valine, alanine, beta-alanine, leucine, isoleucine, glutamic acid or aspartic acid, it is an N-substituted derivative; and

B ¹ and B ² are independently a direct bond, -OC(=O)-, -NHC(=O)-, -O(CH ₂ ) _n C(=O)-, -NH(CH ₂ ) _n C(=O)-, -O(CH ₂ ) _m OC(=O)-, or -O(CH ₂ CH ₂ O) _r C(=O)-, wherein the bond projecting to the left is attached to A ¹ or A ² , respectively; m is 2, 3, 4, 5, 6, 7, or 8; n is an integer from 1 to 20; and r is 2, 3, 4, 5, 6, 7, or 8.

Embodiments of the compound of Formula 1 include the following. The compound wherein the amino acid residue comprises a residue of a D-amino acid or derivative thereof, an L-amino acid or derivative thereof, or a mixture of a D- or derivative thereof and an L— amino acid or derivative thereof.

The compound wherein B ¹ and B ² are independently a direct bond.

The compound wherein G ¹ is H and G ² is A ² -B ² .

The compound wherein G ¹ is A’-B ¹ and G ² is H.

The compound wherein the amino acid residue comprises a residue of 1- methyltryp tophan .

A notable compound is the compound of Formula 2 or derivative or salt thereof.

This invention also provides a compound of Formula 43 or a salt thereof wherein AAC(=O) comprises an amino acid residue, such as wherein the amino acid is selected from the group consisting of tryptophan, phenylalanine, tyrosine, lysine, arginine, norvaline, 2-/er/-butylglycine, 3-amino-2-naphthoic acid, 6-amino-2-naphthoic acid, 4-amino-l- naphthoic acid, homoarginine, ornithine, proline, histidine, threonine, sulfamoyl-ornithine, 2- amino-5-((2-amino-3,4-dioxocyclobut-l-en-l-yl)amino)pentanoi c acid, 6-boronoleucine, (1- benzimidazolonyl)alanine, glycine, valine, alanine, beta-alanine, leucine, isoleucine, glutamic acid, aspartic acid, and N-substituted derivatives thereof.

Another notable compound is the compound of Formula 3

Another object of this invention is to provide compositions that are more effective in delivering Topi inhibitor compounds with improved antitumor efficacy and reduced toxicity.

This invention also provides a composition comprising a nanoparticle comprising a compound as described above, and wherein the nanoparticle comprises albumin.

Embodiments of the composition include the following.

The composition wherein the composition is substantially free of fatty acids.

The composition wherein the albumin is glycated.

The composition wherein the glycated albumin comprises a reducing sugar selected from the group consisting of glucose, ribose, galactose, fructose, xylose, glyceraldehyde, lactose, maltose, mannose, hexose, heptose, disaccharides, oligosaccharides or combinations thereof.

The composition wherein the albumin is human serum albumin. The composition wherein the composition is substantially free of fatty acids.

The composition wherein the human serum albumin is glycated.

The composition wherein the glycated human serum albumin comprises a reducing sugar selected from the group consisting of glucose, ribose, galactose, fructose, xylose, glyceraldehyde, lactose, maltose, mannose, hexose, heptose, disaccharides, oligosaccharides or combinations thereof.

The invention also provides a method for treating a subject suffering from cancer comprising administering a pharmaceutically effective amount of a compound as described above or a composition as described above.

Embodiments of the method include the following.

The method wherein the cancer comprises pancreatic, breast, lung, gastric, gastrointestinal (such as colon), or bone cancer.

The method wherein the cancer comprises pancreatic cancer.

Embodiments of this invention, including Embodiments of the Summary of the Invention or any other embodiments described herein, can be combined in any manner, and the descriptions of variables in the embodiments pertain not only to the compositions of this invention, but also to the methods or uses of any of the compositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows comparisons of stability of SN-38-l-MethylTryptophan analogs (C20- 1DMT and C10-1DMT) in PBS, according to an embodiment of the disclosed subject matter.

Figure 2 shows comparisons of stability of SN-38-l-MethylTryptophan analogs (C20- 1DMT and C10-1DMT) in methanol, according to an embodiment of the disclosed subject matter.

Figure 3 shows aspects of the viability of SN-38-l-MethylTryptophan analogs (C20- 1DMT) in MTS assays, according to an embodiment of the disclosed subject matter.

Figure 4 shows aspects of protein binding of SN-38 and 1-methyl-D tryptophan, according to an embodiment of the disclosed subject matter.

Figure 5 shows a cut-away schematic of a nanoparticle comprising a SN-38- 1- MethylTryptophan analog (C20-1DMT) in albumin, according to an embodiment of the disclosed subject matter. Figures 6A and 6B shows aspects of the particle size distribution of nanoparticles comprising SN-38-l-MethylTryptophan analog (C20-1DMT) in albumin, according to an embodiment of the disclosed subject matter.

Figures 7A and 7B shows aspects of the particle size distribution of nanoparticles comprising SN-38 in albumin, according to an embodiment of the disclosed subject matter.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” “containing,” “characterized by” or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitation explicitly indicated. For example, a mixture, composition or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such mixture, composition or method.

The transitional phrase “consisting of’ excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

The transitional phrase “consisting essentially of’ is used to define a mixture, composition or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

Where applicants have defined an invention or a portion thereof with an open-ended term such as “comprising,” it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms “consisting essentially of’ or “consisting of.”

Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore “a” or “an” should be read to include one, one or more, or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

Unless stated otherwise, all percentages, parts, ratios, etc., are by weight. When an amount, concentration, or other value or parameter is given as either a range, preferred range or a range defined as being from a list of lower limits or lower preferable values to a list of upper limits or upper preferable values, this is to be understood as specifically disclosing any or all ranges formed from any pair of any lower range limit or preferred value and any upper range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. When the term "about" is used in describing a value or an end-point of a range, the disclosure includes the specific value or endpoint referred to.

The present invention relates to compounds, compositions, formulations, and methods for treating cancer, particularly pancreatic, breast, lung, lymphoma, blood, or gastrointestinal cancers (such as colon cancer); more particularly pancreatic, lung, or colon cancer; or more particularly pancreatic cancer. More specifically, the compounds comprise novel chemical entities in which a DNA-damaging agent is linked to an amino acid moiety via a cleavable bond such as an ester bond. In some instances, the compounds, compositions, and formulations of the present disclosure may be used as a monotherapy. In other aspects, the compounds, compositions, and formulations of the present disclosure may be used in the context of a combination therapy. For example, a compound, composition, or a formulation of the present disclosure may be administered to a patient in combination with a second agent. The second agent may be, for example, one of an anti- PD-1 antibody, an anti-PD-Ll antibody, or a chemotherapeutic. In some aspects, the patient may be receiving a compound, composition, or formulation of the present disclosure, and may have the second agent added to the treatment regimen. In another aspect, the patient may be receiving the second agent, and have a compound, composition, or formulation of the present disclosure added to the treatment regimen. In another aspect, the patient may receive a compound, composition, or formulation of the present disclosure at the same time, or simultaneously with, a second agent.

In some aspects, compounds, compositions, and formulations of the present disclosure may be targeted to specific tissues, including but not limited to pancreas, lung, colon, and tumor tissues. In some aspects, the compounds, compositions, and formulations of the present disclosure may be targeted to or delivered to tumor microenvironments, which may yield enhanced tissue uptake. In some aspects, and particularly with regard to nanoparticle compositions and formulations, the present disclosure provides for an increase in solubility for a compound of the present disclosure relative to the compound without complexing to the nanoparticle. Likewise, a nanoparticle composition or formulation of the present disclosure may yield an increase in payload relative to the compound without complexing to a nanoparticle. The compounds, compositions, and formulations of the present disclosure may be associated with reduced blood cell toxicity, as they allow for reduced concentration in the blood and increased uptake by cells. The compounds, compositions, and formulations of the present disclosure may allow a patient treated by the same to experience no neutropenia, or to have decreased neutropenia relative to other therapeutics.

An amino acid comprises an amine group, a carboxylic acid group, and a unique side chain that defines its difference from other amino acids. As used herein, an “amino acid residue” comprises that portion of the amino acid that remains after the acid portion of the amino acid, or derivative, thereof, is condensed with an active nucleophile and the elements of water are removed.

Table A shows exemplary embodiments of amino acid residues useful in this invention, wherein the bond projecting to the right is attached to B ¹ or B ² in compounds of Formula 1. Amino acids that do not comprise a chiral amine are not characterized as D or L in Table A.

Larger, hydrophobic amino acids such as tryptophan, phenylalanine, tyrosine, leucine, and isoleucine, in either D- or L-forms, are preferable. Unnatural amino acids can also be beneficial for improving active agent pharmacokinetic properties.

Amino acids may be selected from: tryptophan, phenylalanine, tyrosine, leucine, and isoleucine, glutamic acid, aspartic acid, in either D- or L-forms. They also can be selected from unnatural amino acids including norvaline, N-substituted glycines, tButylglycine, napthylanine, ( 1-benzimidazolonyl) alanine.

Notably, the amino acid is not glycine, valine, or beta-alanine unless those amino acids comprise derivatives thereof, such as N-alkyl derivatives, preferably N-methyl derivatives.

A notable amino acid is 1-methyl-D-tryptophan (abbreviated herein as 1DMT), also known as indoximod. However, three major problems hinder indoximod use. It is barely soluble in water, its delivery efficiency is limited, and it has low oral bioavailability.

Issues with treating cancer with chemotherapy compounds include rapid metabolism, excretion, and issues of penetrating physical barrier of tumor stroma resulting in limited exposure at targeted site. Use of either SN-38 or 1-methyl-D-tryptophan in cancer treatment are hindered by poor physio-chemical and pharmacokinetic properties. In certain embodiments the DNA- damaging agent is SN-38 and the amino acid is 1-methyl-D-tryptophan.

The compounds of Formula 1 can be prepared by general methods known in the art of synthetic organic chemistry. One or more of the following methods and variations as described in Schemes 1 through 23 can be used to prepare compounds of Formula 1, including compounds of Formulae 1A through 1R, which are subsets of compounds of Formula 1. The definitions of groups G ¹ , G ² , A ¹ , A ² , B ¹ , B ² , m, n and r in the compounds of Formulae 1 through 38 are as defined above in the Summary of the Invention unless otherwise noted.

The compounds of Formula 43, including the compound of Formula 3, can be prepared by general methods known in the art of synthetic organic chemistry. One or more of the following methods and variations as described in Schemes 24 through 26 can be used to prepare compounds of Formula 43. Scheme 1 shows a general method for protecting the amino group of an amino acid to provide a compound of Formula 5 wherein the amino group is rendered non-nucleophilic so that the acid moiety can be reacted with hydroxy groups on SN-38. Treatment of an amino acid wherein R represents the side chain can be treated with di-/c/7-butyl carbonate to prepare a tertbutyl carbamate (a Boc group). D- or L- amino acids can be protected with equal efficiency to provide protected amines of the general formulae D-5 and L-5. Racemic or non-chiral amino acids may be protected similarly. Notable Boc-protected amino acids are D-5-NMT and L-5-NMT, comprising Boc-protected derivatives of both enantiomers of 1-methyl-tryptophan.

Scheme 1

Z)-amino acid 5.5

The simplest form of conjugation of the amino acid moiety to the SN-38 moiety is an ester (i.e. wherein B ¹ or B ² comprises a direct bond). The hydroxy group at position 10 of SN-38 is more reactive and can be conjugated preferentially over the hydroxy group at position 20.

Scheme 2 shows a general method for preparing SN-38 ester conjugates wherein the amino acid residue is attached at the 10-OH position. Compounds of Formula 1A, a subset of compounds of Formula 1 wherein G ¹ is A^B ¹ , B ¹ is a direct bond and G ² is H are prepared by treating SN-38 with a compound of Formula D-5 or L-5 to prepare compounds of Formula 6 wherein BocAAC=O represents a Boc-protected amino acid residue. Removal of the Boc group from the amino moiety of the amino acid residue provides compounds of Formula 1A, wherein AAC=O represents an amino acid residue.

1A: an SN-38 10-Ester Conjugate

Notable compounds of Formula 1A are SN-38-1OMDT and SN-38-1OMLT prepared using BocMDT and BocMLT respectively.

Scheme 3 shows a method for preparing SN-38 ester conjugates wherein the amino acid residue is attached at the 20-OH position. Treatment of SN-38 with di-tert-butyl carbonate provides the compound of Formula 7, wherein the 10-OH position is blocked by a Boc group. Treatment with a Boc-protected amino acid (BocAA-COOH) provides a compound of Formula 8, which can be deprotected by removal of the t-Boc group to provide a compound of Formula IB.

Scheme 3

IB: an SN-38 20-Ester Conjugate

Notable compounds of Formula IB are SN-38-20MDT, also referred to herein as C20- 1DMT, (the compound of Formula 2) and SN-38-20MET prepared using BocMDT and BocMET respectively.

Other methods of conjugation can be envisioned to strengthen the bond between SN-38 and the amino acid residue. These include, but are not limited to, formation of carbonates or carbamates, such as those wherein B ¹ or B ² comprise -OC(=O)- or -NHC(=O)-.

SN-38 can be first be reacted with phosgene to create a chloroformate, which could then be reacted with an alcohol or amine to provide an extended linker.

As shown in Scheme 4, the compound of Formula 7 can be reacted with phosgene or a phosgene equivalent such as triphosgene to provide the chloroformate of Formula 9. Treatment with a compound of Formula D-5 or L-5 to prepare compounds of Formula 10 wherein BocAAC=O represents a Boc-protected amino acid residue, followed by removal of the Boc group from the amino moiety of the amino acid, wherein AAC=O represents an amino acid residue provides a compound of Formula 1C. A notable compound of Formula 1C comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT.

1C SN-38 Carbonate Conjugate

A carbamate linker can be prepared similarly as shown in Scheme 5, wherein the compound of Formula 9 is reacted with a Boc-protected amino acid amide of Formula 11, followed by removal of the Boc group from the amino moiety of the amino acid, wherein AAC=O represents an amino acid residue provides a compound of Formula ID. A notable compound of Formula ID comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT amide as the compound of Formula 11. Scheme 5

BocMDT amide

Further, an extended cleavable linker can be employed to improve chemical stability, increase flexibility, and/or modify water solubility. For example, as shown in Scheme 6, the compound of Formula 7 can be treated with an a-, 0-, y-, 5- or co-hydroxy carboxylic acid of Formula 13 to provide a compound of Formula 15, an ester with a terminal hydroxy group. The compound of Formula 15 can be condensed with a Boc-protected amino acid of D-5 or L-5 to prepare compounds of Formula 16, followed by removal of the Boc group to provide a compound of Formula IE, a compound of Formula 1 wherein G ¹ is H and B ² comprises -O(CH2) _n C(=O)-. Scheme 6

Alternatively, a compound of Formula 15 can be prepared from the compound of Formula by treatment with a lactone of Formula 14 in the presence of a base as shown in Scheme 7. Scheme 7

A notable compound of Formula IF comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT as the compound of Formula D-5.

Similarly, the Boc-protected SN-38 of formula 7 can be treated with glycine, beta-alanine, GABA or other co-aminocarboxylic acid or derivative thereof such as a lactam as shown in Schemes 8 and 9 to provide an ester with a terminal amino group, a compound of Formula 18, that can be condensed with a Boc-protected amino acid such as D-5 or L-5 to prepare a compound of Formula 19. Removal of the Boc groups provides compounds of Formula 1G wherein G ¹ is H and B ² comprises -NH(CH2) _n C(=O)-. A notable compound of Formula 1G comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT as the compound of Formula D-5.

One can appreciate that it may be necessary to protect the amine group on the compound of Formula 17 prior to coupling with the compound of Formula 7, and then remove the amine protecting group to provide the compound of Formula 18. Preferably, the protecting group on the amine is removable in the presence of the Boc group on the 10-hydroxy of the SN-38 moiety.

Scheme 8 Scheme 9

Another extended linker comprises a diol moiety linking the amino acid residue to the SN- 38 moiety. For example, as shown in Scheme 10, the compound of Formula 9 can be treated with a diol of Formula 21 to provide a compound of Formula 22, a carbonate with a terminal hydroxy group. The compound of Formula 22 can be condensed with a Boc-protected amino acid of D-5 or L-5 to prepare a compound of Formula 23, followed by removal of the Boc group to provide a compound of Formula 1H, a compound of Formula 1 wherein G ¹ is H and B ² comprises - O(CH ₂ ) _m OC(=O)-. A notable compound of Formula 1H comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT as the compound of Formula D-5.

One can appreciate that it may be necessary to protect one of the hydroxy groups on the compound of Formula 21 prior to coupling with the compound of Formula 9, and then remove the protecting group to provide the compound of Formula 22. Preferably, the protecting group on the hydroxy is removable in the presence of the Boc group on the 10-hydroxy of the SN-38 moiety.

Alternatively, as shown in Scheme 11, a compound of Formula 1H may be prepared by treating the compound of Formula 9 with an amino acid ester of Formula 24 to prepare a compound of Formula 24, followed by removal of the Boc groups. A notable compound of Formula 1H comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using the compound of Formula 24a. Scheme 10 Scheme 11

Another extended linker comprises an ethylene glycol oligomer moiety linking the amino acid residue to the SN-38 moiety. For example, as shown in Scheme 12, the compound of Formula 9 can be treated with an ethylene glycol oligomer of Formula 25 to provide a compound of Formula 26, a carbonate with a terminal hydroxy group. The compound of Formula 26 can be condensed with a Boc-protected amino acid of D-5 or L-5 to prepare a compound of Formula 27, followed by removal of the Boc group to provide a compound of Formula 1 J, a compound of Formula 1 wherein G ¹ is H and B ² comprises -O(CH2CH2O) _r C(=O)-. A notable compound of Formula 1 J comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT as the compound of Formula D-5.

One can appreciate that it may be necessary to protect one of the hydroxy groups on the compound of Formula 25 prior to coupling with the compound of Formula 9, and then remove the protecting group to provide the compound of Formula 26. Preferably, the protecting group on the hydroxy is removable in the presence of the Boc group on the 10-hydroxy of the SN-38 moiety.

Alternatively, as shown in Scheme 13, a compound of Formula 1J may be prepared by treating the compound of Formula 9 with an amino acid ester of Formula 28 to prepare a compound of Formula 27, followed by removal of the Boc groups. A notable compound of Formula 1H comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using the compound of Formula 28a.

Scheme 13

As shown in Scheme 14, SN-38 can be reacted with phosgene or a phosgene equivalent such as triphosgene to provide the chloroformate of Formula 29. Treatment with a compound of Formula D-5 or L-5 to prepare a compound of Formula 30 wherein BocAAC=O represents a Boc- protected amino acid residue, followed by removal of the Boc group from the amino moiety of the amino acid, wherein AAC=O represents an amino acid residue provides a compound of Formula IK. A notable compound of Formula IL comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT. Scheme 14

A carbamate linker can be prepared similarly as shown in Scheme 15, wherein the compound of Formula 29 is reacted with a Boc-protected amino acid amide of Formula 11, followed by removal of the Boc group from the amino moiety of the amino acid, wherein AAC=O represents an amino acid residue provides a compound of Formula IL. A notable compound of Formula IM comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT amide as the compound of Formula 11. Scheme 15

As shown in Scheme 16, SN-38 can be treated with an a-, 0-, y-, 5- or co-hydroxy carboxylic acid of Formula 13 to provide a compound of Formula 32, an ester with a terminal hydroxy group. The compound of Formula 32 can be condensed with a Boc-protected amino acid of D-5 or L-5 to prepare compounds of Formula 33, followed by removal of the Boc group to provide a compound of Formula IN, a compound of Formula 1 wherein G ² is H and B ¹ comprises -O(CH2) _n C(=O)-. A notable compound of Formula IN comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT as the compound of Formula D-5.

Scheme 16

Alternatively, a compound of Formula 15 can be prepared from the compound of Formula 7 by treatment with a lactone of Formula 14 in the presence of a base as shown in Scheme 7. Scheme 17

Similarly, as shown in Scheme 18, SN-38 can be treated with glycine, beta-alanine, GABA or other co-aminocarboxylic acid or derivative thereof such as a lactam to provide an ester with a terminal amino group, a compound of Formula 34.

Scheme 18

As shown in Scheme 19, a compound of Formula 34 can be condensed with a Boc- protected amino acid such as D-5 or L-5 to prepare a compound of Formula 35. Removal of the Boc protecting groups using TFA provides compounds of Formula 1G, wherein G ¹ is H and B ² comprises -NH(CH2) _n C(=O)-. A notable compound of Formula 1G comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT as the compound of Formula D-5. Scheme 19

D-5 or L-5

As shown in Scheme 20, the chloroformate of Formula 29 can be treated with a diol of Formula 20 to provide a compound of Formula 35, a carbonate with a terminal hydroxy group. The compound of Formula 35 can be condensed with a Boc-protected amino acid of D-5 or L-5 to prepare a compound of Formula 36, followed by removal of the Boc protecting group using TFA to provide a compound of Formula IQ, a compound of Formula 1 wherein G ² is H and B ¹ comprises -O(CH2) _m OC(=O)-. A notable compound of Formula IQ comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT as the compound of Formula D-5.

Alternatively, as shown in Scheme 21, a compound of Formula IQ may be prepared by treating the compound of Formula 29 with an amino acid ester of Formula 24 to prepare a compound of Formula 36, followed by removal of the Boc groups. A notable compound of Formula IQ comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using the compound of Formula 24a. Scheme 20

Scheme 21

As shown in Scheme 22, the compound of Formula 29 can be treated with an ethylene glycol oligomer of Formula 25 to provide a compound of Formula 37, a carbonate with a terminal hydroxy group. The compound of Formula 37 can be condensed with a Boc-protected amino acid of D-5 or L-5 to prepare a compound of Formula 38, followed by removal of the Boc group to provide a compound of Formula 1R, a compound of Formula 1 wherein G ² is H and B ¹ comprises -O(CH ₂ CH ₂ O) _r C(=O)-. A notable compound of Formula 1R comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using BocMDT as the compound of Formula D-5.

One can appreciate that it may be necessary to protect one of the hydroxy groups on the compound of Formula 20 prior to coupling with the compound of Formula 29, and then remove the protecting group to provide the compound of Formula 18. A notable protecting group may be a Boc group, although others are suitable.

Scheme 22

Alternatively, as shown in Scheme 23, a compound of Formula 1R may be prepared by treating the compound of Formula 29 with an amino acid ester of Formula 28 to prepare a compound of Formula 38, followed by removal of the Boc groups. A notable compound of Formula 1R comprises an N-methyl tryptophan moiety as the amino acid residue, prepared using the compound of Formula 28a. Scheme 23

Any of the compounds of Formula 1, including compounds of Formulae 1A through 1R may be obtained as the free base or a pharmaceutically acceptable salt. Notably, trifluoracetic acid salts of compounds of Formula 1 may be obtained after removal of the Boc protecting groups. In some embodiments, the Boc group may be retained on the amino group of the amino acid residue.

In some embodiments, conjugating to a bulky substituent at the 20-hydroxy group of SN-38 could reduce access to the ester bond to increase its hydrolytic stability. For example, leaving the tert-butoxycarbonyl (Boc)-protection group on the primary amine of 1DMT might provide enough steric hindrance to slow the rate of hydrolysis and increase the stability of C20-1DMT in aqueous solutions. Conjugation to a bulky substituent at the 20-hydroxy group of SN-38 may also stabilize the hydrolytic stability of the lactone.

In another embodiment, boronic acid derivatives of SN-38 may be prepared as shown in Schemes 24, 25 and 26. The compound of Formula 40 can be prepared according to methods described in L. Wang et al., European Journal of Medicinal Chemistry 116 (2016) 84-89. Briefly, triethylamine was added to a solution of N,N-bis(trifluoromethylsulfonyl)aniline and SN-38 in anhydrous DMF to provide the compound of Formula 39, which was added to KOAc and bis(pinacolato)diboron. Pd(dppf)C12 was added dropwise and the reaction was stirred at 80 °C for 12 h to provide the compound of Formula 40.

The compound of Formula 40 can be converted to a compound of Formula 43 as shown generally in the Scheme 25. Esterification with D-5 or L-5 using standard coupling conditions (EDC, DMAP, DCM) provides a compound of Formula 41. Removal of the Boc group in 30% TFA (v/v DCM) provides a compound of Formula 42. Deprotection of the boronate by removal of the pinacol moiety can be carried out using NPUOAc and NaICU in acetone:water (1:1) and at room temperature (e.g. over 24 hr).

One skilled in the art will recognize that the chemical reactions shown in Scheme 25 may be carried out in a different order than that shown. In particular, the deprotection of the boronate group of the compound of Formula 40 may occur prior to the coupling with D-5 or L-5, or the order of removal of the Boc and pinacol groups from the compound of Formula 41 may be reversed. Scheme 25

A notable compound of Formula 43 comprises an N-methyl tryptophan moiety as the amino acid residue, that is, the compound of Formula 3, shown in Scheme 26. The compound of Formula 3 can be prepared using BocMDT as D-5, following procedures described in Schemes 24 and 25.

It is recognized by one skilled in the art that various functional groups can be converted into others to provide different compounds of Formula 1. For a valuable resource that illustrates the interconversion of functional groups in a simple and straightforward fashion, see Larock, R. C., Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2nd Ed., Wiley-VCH, New York, 1999. The above reactions can also in many cases be performed in alternate order.

It is recognized that some reagents and reaction conditions described above for preparing compounds of Formula 1 may not be compatible with certain functionalities present in the intermediates. In these instances, the incorporation of protection/deprotection sequences or functional group interconversions into the synthesis will aid in obtaining the desired products. The use and choice of the protecting groups will be apparent to one skilled in chemical synthesis (see, for example, Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991). One skilled in the art will recognize that, in some cases, after the introduction of a given reagent as depicted in any individual scheme, it may be necessary to perform additional routine synthetic steps not described in detail to complete the synthesis of compounds of Formula 1. One skilled in the art will also recognize that it may be necessary to perform a combination of the steps illustrated in the above schemes in an order other than that implied by the particular order presented to prepare the compounds of Formula 1.

The preceding description relates to coupling SN-38, also known as ((+)-(4S)-4,l 1-diethyl- 4,9-dihydroxy-lH-pyrano[3',4':6,7]-indolizino[l,2-b]quinolin e-3,14(4H,12H)-dione, with amino acid residues. However, SN-38 comprises a chiral center at the “C-20” position. This invention also relates to compounds and compositions comprising the enantiomer of SN-38, i.e. enantio-SN- 38 or ((-)-(4R)-4,l l-diethyl-4,9-dihydroxy-lH-pyrano[3',4':6,7]-indolizino[l,2- b]quinoline- 3,14(4H,12H)-dione coupled with amino acid residues and boronate derivatives of enantio-SN-38. These can be prepared according to the methods described above and in Schemes 1 through 26 by substituting SN-38 with enantio-SN-38.

Notable compounds include compounds 41, 42, 43 and 44 wherein enantio-SN-38 is coupled to D- or L- 1 -methyltryptophan at either the 10- or 20- positions.

Another notable compound is compound 45, in which the 10-hydroxy group is replaced with a boronate group:

As described in Experimental Example 1, the stability of conjugates of SN-38 with amino acid residues, and in particularly, 1 -methyl tryptophan, varies depending to which hydroxy group the amino acid residue is conjugated.

The compounds exhibit increased ability to be encapsulated in nanoformulations to reduce toxicity and improve tumor targeting.

Nanoformulations

The present disclosure also details the process for creating nanoparticles of the conjugates comprising albumin or glycated albumin to ensure effective delivery of the drugs to tumor tissue and protect conjugate from hydrolysis. The nanoparticles are suitable for intravenous injection in aqueous medium.

Another approach to increase drug stability is to use a nanoformulation, which also offers the potential to enhance drug accumulation in cancer tissues and reduce drug distribution in normal tissues. However, there is a misconception in nanomedicine design that the focus is to increase drug exposure in the systemic circulation. The belief is that this increased exposure will lead to improved drug concentration in tumors by the enhanced permeability and retention effect (EPR). EPR is a phenomenon resulting from the abnormal leaky vasculature of tumor tissues and lack of lymphatic drainage. These previous nanomedicines failed to improve cancer tissue penetration and accumulation and consequently did not improve its efficacy. In a review of published studies, Wilhelm et al. found that only 0.7% of the dosed nanoformulation was delivered to the tumor.

EPR is not a sufficient argument for the delivery of curative doses of cancer therapeutics; there needs to be more focus on the innate tissue targeting of the nanomedicine. Nanoparticle formulations do not act as a typical co-solvent; they affect drug disposition via unique drug-carrier interactions. Efficacy of the nanomedicine, therefore, needs to be evaluated by intratumoral distribution, drug release profile within the tumor, and exposure of free drug in circulation.

Albumin is a 585-amino acid protein composed of three homologous domains, each of which has two helical subdomains. The three-dimensional structure of human serum albumin (HSA) is heart-shaped and has several binding pockets that allow it to function as a critical carrier of endogenous (fatty acids, amino acids, hormones, water, ions, etc.) and exogenous (drugs) substances. Literature focuses on two main binding sites in HSA: (1) Sudlow site I, located in subdomain IIA, which binds bulky heterocyclic anions and dicarboxylic acids, and (2) Sudlow site II, situated in subdomain IIIA, which binds aromatic carboxylates. SN-38 binds albumin at site I, whereas L-tryptophan, and likely 1-methyl-D-tryptophan, binds at site II. Our conjugate, C20- 1DMT, may, therefore, bind at two locations to increase loading capacity in the nanoparticle formulation.

In one embodiment, the compositions of the present disclosure involve a nanoparticle formulation using albumin. While previous water-insoluble drugs, such as paclitaxel, have been formulated with HSA, the ability of other water-insoluble drugs to form HSA nanoparticles is not inherently understood or obvious. Docetaxel, for example, shares most of the same structural motifs as paclitaxel and exhibits high binding to albumin, but it does not form a stable nanosuspension with albumin alone. Similarly, SN-38 alone with albumin does not form stable nanoparticles based on this technology even though SN-38 exhibits high binding to albumin in vitro (Synthesis Example 3).

Accordingly, we investigated nanoparticles comprising SN-38 conjugated with amino acid moieties. Specifically, we prepared nanoparticles comprising 20-SN-38-lMethylTryptophan in albumin.

20-SN-38-l-MethylTryptophan (C20-1DMT) in combination with HSA C20-1DMT can be combined with HSA in nanoparticles (Figure 5). The composition may comprise C20-1DMT and HSA at ratio (w/w) from 1:1 to 1:15. The API concentration in C20- 1DMT NP ranges from 0.1 mg/ml to 10 mg/ml.

Figure 5 shows a cut-away schematic of a nanoparticle comprising a SN-38- IMethylTryptophan (C20-1DMT) in albumin, according to an embodiment of the disclosed subject matter. The nanoparticle 500 comprises molecules of SN-38-l-MethylTryptophan (C20- 1DMT) 505 complexed with albumin in a core 510 of the nanoparticle 500. In the embodiment shown in Figure 5, the nanoparticle comprises a shell 520 that may comprise albumin without complexed C20-1DMT. Alternatively, the shell 520 may comprise a composition other than albumin that may provide improved stability or delivery of the nanoparticle to the target site.

C20-1DMT-HSA nanoparticles are generated using pharmaceutical grade solvent in the process with a ratio of organic solvent to aqueous from about 1% to about 20%. The organic solvent is selected from water- miscible and immiscible solvents. The ratio of miscible to immiscible solvents can range from 1:10, 10:1, or 100% miscible or immiscible. Miscible solvents include alcohols such as methanol and ethanol, acetonitrile, dimethyl sulfoxide, acetone, and the like. Immiscible solvents include dichloromethane, chloroform, toluene, ethyl acetate, and the like.

The C20-1DMT-HSA nanoparticle emulsion is generated with high pressure homogenization at pressure from 10,000 to 40,000 psi, at temperatures from about 5 °C to about 50 °C. Solvent removal is achieved via rotary evaporation and water is removed by lyophilization. Sugars (sucrose, mannitol, trehalose) may be added to stabilize the formulation during lyophilization.

C20-1DMT-HSA nanoparticle size may be about 50-200 nm. The nanoparticle is negatively charged (zeta potential).

When C20-1DMT was formulated with HSA (Synthesis Example 4), we were able to form nanoparticles with diameters around 125 nm with low polydispersity (PDI= 0.113) that were stable at room temperature for 4 days (Figures 6A and 6B). Figures 6A and 6B shows aspects of the particle size distribution of nanoparticles comprising C20-1DMT in albumin, according to an embodiment of the disclosed subject matter.

Without being bound by any theory, 1DMT conjugation to SN-38 may increase the hydrophobic surface area and may improve association with albumin to form stable nanoparticles with higher encapsulation efficiency. The C20-1DMT-HSA nanoparticle size is stable in aqueous solution and in blood after dilution. C20-1DMT-HSA nanoparticle size can be manipulated to dissociate in aqueous solution and in blood after dilution.

The C20-1DMT-HSA nanoparticle may contain the drug in the amorphous form or crystal form, preferably in the amorphous form, because the compound is expected to have increased bioavailability in amorphous form over crystalline form.

The C20-1DMT-HSA nanoparticle comprises HSA with monomer, dimer, oligomer with ratios from 60-98%: 1-20%: 1-20%.

Figures 7A and 7B shows aspects of the particle size distribution of nanoparticles comprising SN-38 in albumin, according to an embodiment of the disclosed subject matter.

Formulations of C20-1DMT-HSA nanoparticle may further comprise a pharmaceutical injection medium, such as saline, PBS, water for injection (WFI) or 5 % dextrose solutions.

C20-1DMT in combination with HSA may have special, improved tissue targeting over the free drug. For example, the formulations may target delivery to the pancreas to improve efficacy in treating pancreatic cancer. Targeting to a fatpad may improve efficacy in treating breast cancer. Targeting to the lung may improve efficacy in treating lung cancer. Targeting to bone may improve efficacy in treating metastatic cancers to bone. Targeting to the stomach may improve efficacy in treating gastric cancer. C20-1DMT-HSA nanoparticle can be targeted with specific antibodies, fatty acids, or peptides to further improve tissue targeting.

C20-1DMT in combination with HSA may increase tumor accumulation to improve efficacy. The formulations improve deep tumor penetration, target the tumor microenvironment and enhance intracellular uptake.

In some aspects, the formulations of the present disclosure, including C20-1DMT-HSA formulations, may oppose the effects of indolamine 2,3-deoxygenase 1 (IDO1) by creating a tryptophan sufficiency signal, reactivating mTOR in the context of low tryptophan. This in turn may polarize M0 macrophages away from immune-suppressing M2 tumor associated macrophages (TAMs), which may have protumor activity, to favor immune-stimulating Ml TAMs, which have antitumor activity, or to convert M2 TAMs to Ml TAMs. In other words, C20-1DMT-HSA nanoparticle formulations, and other formulations of the present disclosure, may increase the number of Ml macrophages in tumor microenvironments. In some aspect, the formulations of the present disclosure, including C20-1DMT-HSA formulations, may result in an increase in proliferation of CD8+ T-cells, and differentiation of naive CD4+ to TH17-producing T-cells. In some aspects, these physiological effects may proceed via an aryl hydrocarbon receptor (AhR) dependent manner.

C20-1DMT-HSA nanoparticle efficacy may be mediated by expression level of FcRn (lower FcRn levels on cancer tissues compared to normal will show better response).

C20-1DMT-HSA nanoparticle uptake may be due to increased protein catabolism mediated by Kras+ cancer cells.

Modifications to albumin may further increase association with C20-1DMT and fine-tune nanoparticle performance, including tissue targeting. For instance, glycated albumin has been shown to have a 4.7- to 5.8-fold increase in the association equilibrium constant for L-tryptophan. As 1DMT is a tryptophan mimetic, affinity for glycated albumin may also increase.

Glycation is a non-enzymatic process whereby reducing sugars are added to primary or secondary amines on proteins. Albumin has more than 84 residues that can serve as potential glycation sites: the N-terminus, 59 lysines, and 24 arginines. However, lysine 525 is the most common site for glycation within both in vitro and in vivo glycated HSA samples. Levels of glycated albumin in the blood vary from person to person but are consistently 2- to 5-fold higher in patients with diabetes. The addition of different reducing sugars or the level of glycation (mol sugar/mol albumin) could alter binding capacity of albumin towards C20-1DMT as well as change tissue distribution. Glycation of albumin may also enhance C20-1DMT association, targeting of cancer stem cells, and modify pharmacokinetic properties.

Reducing sugars can be monosaccharides, disaccharides, or oligosaccharides. Reducing sugars used to modify albumin include, and are not limited to glucose, fructose, mannose, maltose, lactose, galactose and xylose.

Glycation levels may range from 1 to 10 mol sugar per mol HSA. Glycation may result in changes to the HSA secondary structure. Binding affinity of C20-1DMT to glycated HSA may be increased.

Glycated albumin will target cancer cells, specifically cancer stem cells through enhanced glucose uptake mediated by the CD44 receptor, a cancer stem cell marker.

Certain sugars used for glycation may imbue nanoformulation with specific targeting ability (e.g. mannose specific transporters are found on fibroblasts and hepatoma cells). C20-1DMT and glycated HSA nanoparticle formation follows the C20-1DMT and HSA nanoparticle protocol described above.

Glycated albumin used in formulation may be from one source or multiple (i.e. one form of sugar or a combination of sugars) to fine-tune drug properties: size, shape, charge, drug release, drug distribution, half-life, etc.

Glycated albumin and unmodified albumin can be combined in precise ratios to optimize nanoparticle characteristics.

With the use of glycated albumin, the potential for boronic acid conjugates also becomes an attractive opportunity to improve binding, stability, and alter drug-release kinetics of the nanoformulation. Compositions comprising glycated albumin allow for use of or combinations with compounds that bind cis-diols, such as boronic acids, which reversibly bind the cis-diol of the sugar moiety. Boronates can be boronic acid-conjugates with anti-cancer therapeutics or siRNA.

Boronates, or compounds that contain boronic acids, have selectivity for cis-diols, such as found in the sugar moieties of glycated albumin. At pH 6.5 or less, the drug conjugate will be released from the glycated carrier. While normal physiological fluid has a pH of 7.4, the tumor microenvironment is slightly acidic which would allow for adequate release of the active pharmaceutical ingredient at the site of action, the tumor. A boronic acid moiety could be added to the C20-1DMT conjugate at the CIO position of SN-38. Or, other boronates, such as FDA approved Velcade, can be combined with C20-1DMT to function synergistically.

Notably, derivatives of SN-38 with a boronic acid group may be useful for formulation with albumin, such as compounds of Formulae 43 and 3. For example, the boronic acid moiety of compounds of Formulae 43 and 3 may comprise a bond with an amino acid side chain in the albumin. In other embodiments, the boronic acid moiety of a compound of Formula 43 can bind to a cis-diol of the albumin, specifically glycated albumin. Specific embodiments of compositions wherein a compound of Formula 43 is bound to a cis-diol of albumin are those comprising a compound of Formula 3. These embodiments include the compound of Formula 4 and the compound of Formula 5, shown below. Cleavage of the boronic acid moiety to release the SN-38 amino acid conjugate (e.g. compounds of Formulae IB through IK, or specifically the compound of Formula 2) is envisioned to occur in vivo by oxidative processes by one of a number of enzymes.

Other boronate derivatives of note include those wherein the boronate is substituted on an amino acid residue, such as 6-borono-leucine (in D- or L-forms) conjugated to either the 10- or 20-hydroxyl groups of SN-38. SN-38-Amino Acid Cyclodextrin Formulation

The SN-38-amino acid conjugate can also be formulated with cyclodextrin to improve stability, increase solubility, and decrease toxicity. Cyclodextrins are FDA-approved hydrophilic rings of starch molecules. They are classified as alpha-, beta-, or gamma-cyclodextrin based on the number of glucose subunits (6, 7, or 8 respectively). Chemical modifications of the hydroxyl group can also alter their binding interactions with drug molecules.

C20-1DMT Cyclodextrin

C20-1DMT cyclodextrin phase solubility curves can be established at pH 5.5 and 7.4.

Cyclodextrins studied include 2-hydroxylpropyl-P-cyclodextrin, sulfobutylether cyclodextrin (dexolve), etc.

Cyclodextrin concentration can be determined by phase solubility curves to give the desired drug concentration. Cyclodextrin use can be the minimal amount required or in excess.

Concentration of the cyclodextrin in an aqueous composition can range from 5% to 40% w/v, such as 10% to 25% w/v. Concentration of the conjugate could be 0.1-10 mg/mL.

After complexation, the complex can be passed through a 0.2 pm filter for sterilization and excess water removed via lyophilization.

Without further elaboration, it is believed that one skilled in the art using the preceding description can utilize the present invention to its fullest extent. The following non-limiting Examples are illustrative of the invention. Steps in the following Examples illustrate a procedure for each step in an overall synthetic transformation, and the starting material for each step may not have necessarily been prepared by a particular preparative run whose procedure is described in other Examples or Steps. Percentages are by weight except for chromatographic solvent mixtures or where otherwise indicated. Parts and percentages for chromatographic solvent mixtures are by volume unless otherwise indicated. iH NMR spectra (CDCI3, 500 MHz unless indicated otherwise) are reported in ppm downfield from tetramethylsilane; “s” means singlet, “d” means doublet, “t” means triplet, “dd” means doublet of doublets, “dt” means doublet of triplets, “q” means quartet, “m” means multiplet, and “br s” means broad singlet.

Synthesis of SN-38 1DMT

We conjugated 1-methyl-D-tryptophan to SN-38 via an ester bond at either the CIO- or C20-position of SN-38 (Synthesis Examples 1 and 2). SYNTHESIS EXAMPLE 1

Preparation of Cl 0-1 DMT

Boc-protected 1-methyl-D-tryptophan (Compound BocMDT) and SN-38 were added to a flask along with ethylene dichloride (EDC) (1.8 equivalents) and N,N-dimethylaminopyridine (DNAP) (0.2 equivalents) in dichloromethane (DCM) overnight (30% yield). Boc-deprotection of 10-(l-methyl-D-tryptophan)-SN-38 took place in DCM with excess trifluoroacetic acid (TFA) (19 equivalents) overnight at room temperature. The reaction mixture was concentrated under reduced pressure to remove excess solvent and TFA. The reaction product was precipitated from DCM using diethyl ether and the resulting solid was collected by filtration and dried under vacuum. Further purification was achieved using HPLC. MW: 592.65 (free base); 706.68 (TFA salt). LCMS m/z 593.2 (M+l), 591.2 (M-l). TLC (95:5 Dichloromethane: Methanol) Rf=0.19. ’ H NMR (DMSO-d ₆ , 400 MHz) 5 8.65 (br s, 3H), 8.23 (d, 1H, J=9.4 Hz), 7.69 (s, 1H), 7.68 (d, 2H, J=8.1 Hz), 7.49 (dt, 2H, J=2.9, 6.0 Hz), 7.39 (s, 1H), 7.33 (s, 1H), 7.23 (t, 1H, J=7.6 Hz), 7.10 (t, 1H, J=7.5 Hz), 6.54 (s, 1H), 5.44 (s, 2H), 5.35 (s, 2H), 4.65 (t, 1H, J=6.8 Hz), 3.5-3.6 (m, 2H), 3.10 (q, 2H, J=7.8 Hz), 1.87 (td, 2H, J=7.1, 14.4 Hz), 1.25 (t, 3H, J=7.6 Hz), 0.88 (t,3H, J=7.2 Hz).

SYNTHESIS EXAMPLE 2

Preparation of C20-1DMT

4.90 g of SN-38 was added to a flask containing di-tert-butyl dicarbonate (BOC2O; 1.1 equiv) and pyridine (10.9 equiv) in DCM and allowed to react overnight at room temperature (86% yield, >95% purity). At 0°C, Boc-protected SN-38 (Compound 7, Scheme 3) and Boc-protected 1-methyl-D-tryptophan (1.32 equiv) (Compound BocMDT) were added to EDC (1.8 equiv) and dimethylamino pyridine (DMAP) (0.2 equiv) in dichloromethane, allowed to warm to room temperature, and reacted overnight (39% yield). Boc deprotections took place using excess TFA (~38 equivalents) in DCM. Crude products were concentrated under reduced pressure to remove solvent and excess TFA. The resulting red oil was dissolved in DCM and diethyl ether was added to precipitate product. Product was collected by filtration and washed with excess ether. MW: 592.65 (free base); 689.67 (TFA salt). LCMS m/z 593.2 (M+l), 591.2 (M-l). TLC (95:5 Ethyl Acetate: Methanol) Rf=0.14. ’ H NMR (DMSO-d ₆ , 400 MHz) 5 10.39 (br s, 1H), 8.40 (br s, 3H), 8.O-8.O (m, 1H), 7.67 (d, 1H, J=7.8 Hz), 7.40 (dd, 2H, J=2.3, 4.7 Hz), 7.33 (d, 1H, J=8.2 Hz), 7.28 (s, 1H), 7.1-7.2 (m, 2H), 7.0-7.1 (m, 1H), 5.53 (s, 2H), 5.2-5.3 (m, 2H), 4.75 (br s, 1H), 3.69 (s, 3H), 3.45 (br dd, 2H, J=5.1 , 14.8 Hz), 3.22 (br dd, 1H, J=8.4, 15.0 Hz), 3.08 (br d, 2H, J=7.8 Hz), 2.13 (dt, 2H, J=6.6, 13.9 Hz), 1.2-1.4 (m, 5H), 0.8-0.9 (m, 4H).

Coupling of 1DMT and SN-38 at the C20 position results in slightly higher yield (39% vs 30%) than at CIO. Coupling of L-l-methyl tryptophan (1LMT)

By the procedures described herein together with methods known in the art, the following compounds of Tables 1 to 87 can be prepared.

Table 1 wherein

G ¹ is H, G ² is A ² -B ² , B ² is a direct bond, and A ² is a residue of

D-tryptophan L-tryptophan D-norv aline L-norv aline

D-N-methyl L-N-methyl tryptophan D-homoarginine L-homoarginine tryptophan D-phenylalanine L-phenylalanine D-ornithine L-omithine

D-tyrosine L-tyrosine D-proline L-proline

D-lysine L-lysine D-histidine L-histidine

D-arginine L- arginine D-N-methylv aline L-N-methylv aline

D-threonine L-threonine D-N-methylalanine L-N-methylalanine

D- L- sulfamoylornithine N-methylglycine N-methylbeta- sulfamoylornithine alanine

D-N-methylleucine L-N-methylleucine D-N-methylglutamic L-N- acid methylglutamic acid

D-N- L-N-methylisoleucine D-N-methylasp artic L-N- methylisoleucine acid methylaspartic acid D-2-/er/-butylglycine L-2-/er/-butylglycine 3-amino-2-naphthoic 4-amino-l- acid naphthoic acid

6-amino-2-naphthoic D-(l- L-(l- acid benzimidazolonyl) benzimidazolonyl) alanine alanine

(2S)-2-Amino-6-boronohexanoic acid (2R)-2-Amino-6-boronohexanoic acid

(6-borono-L-leucine) (6-borono-D-leucine)

The present disclosure also includes Tables 2 through 87, each of which is constructed the same as Table 1 above, except that the Header Row in Table 1 (i.e. “G ¹ is H, G ² is A ² -B ² , B ² is a direct bond, and A ² is”) is replaced with the respective Header Row shown below in Tables 2 through 87. For example, the first entry in Table 2 is a compound of Formula 1 wherein G ¹ is H, G ² is A ² -B ² , B ² is -OC(=O)-, and A ² is D-tryptophan. Tables 3 through 87 are constructed similarly.

SYNTHESIS EXAMPLE 3

SN-38 HSA Nanoformulation Ten mg of SN-38 was dissolved in a 5 mL mixture of chloroform and methanol (10:1). The organic phase was added dropwise to 45 mL aqueous solution containing HSA (0.4% w/v) under high speed stirring (12K rpm). The resultant emulsion was further processed by high pressure homogenization (NanoDeBEE; 30,000 psi) for 12 cycles. The final solution was very polydisperse and was not stable during rotary evaporation (drug crashed out of solution).

SYNTHESIS EXAMPLE 4

C20-1DMT HSA Nanoformulation

25 mg SN-38-1DMT was dissolved in 0.5 mL methanol. The organic phase was added to an aqueous phase containing HSA (0.5% w/v, 25 mL), and the resultant mixture was mixed under high speed (8K rpm) for 5 minutes to form a crude emulsion. This emulsion was further subjected to high-pressure homogenization (NanoDeBEE; 12 cycles at 20,000 psi) with a chiller set at 0°C to maintain solution temperature of 10°C. The organic solvent was removed from the final product via rotary evaporator, sequentially filtered (0.8 pm, 0.45 pm, 0.2 pm) and nanosuspensions were lyophilized for long-term storage. Nanoparticles were resuspended in water or 5% sucrose and were around 125 nm (PDI: 0.113) and 145 nm (PDI: 0.122). Resuspended nanoparticles were stable at rt over 4 days.

SYNTHESIS EXAMPLE 5

Glycation of albumin

20 mL of a 15 mM glucose solution, prepared in PBS with 1 mM sodium azide, was used to dissolve 840 mg HSA. The solution was allowed to incubate in a 37°C water bath for 2-4 weeks, after which it was passed through a size-exclusion desalting column to remove excess glucose and sodium azide. Dialysis was performed to further remove excess glucose. Samples were lyophilized and stored at -80°C until use.

SYNTHESIS EXAMPLE 6

C20-1DMT Glycated HSA Nanoformulation

25 mg SN-38-1DMT was dissolved in 0.5 mL methanol. The organic phase was added to dropwise to an aqueous phase containing glycated HSA (0.5% w/v, 25 mL, 2.34 mol hexose/mol HSA), and the resultant mixture was mixed under high speed (8K rpm) for 5 minutes to form a crude emulsion. This emulsion was further subjected to high-pressure homogenization (NanoDeBEE; 12 cycles at 20,000 psi) with a chiller set at 0°C to maintain solution temperature of 10°C. The organic solvent was removed from the final product via rotary evaporator, sequentially filtered (0.8 m, 0.45 p m, 0.2 pi rn) and nanosuspensions were lyophilized for longterm storage.

SYNTHESIS EXAMPLE 7

C20-1DMT with Cyclodextrin

A phase solubility profile for C20-1DMT with dexolve or another P-cyclodextrin is created by adding excess drug to varying concentrations of cyclodextrin and allowed to equilibrate for 24- 48 hrs. Concentration of drug is measured using LC-MS/MS and then plotted as a function of drug concentration (mM) to cyclodextrin concentration (mM). For formulation, the minimum amount of cyclodextrin is used to solubilize C20-1DMT unless excess cyclodextrin is required for stability, protection from esterases. A Cyclodextrin-C20-1DMT inclusion complex can be formed as needed by adding C20-1DMT to cyclodextrin and allowed to equilibrate for 1-2 days. The solution is sterile filtered before use in animals. Otherwise, the inclusion complex can be lyophilized to form a stabile powder that will be dissolved right before use.

SYNTHESIS EXAMPLE 8

Impact of organic solution on nanoparticle synthesis

In this example are disclosed the results of a trial to determine how varying the total percentage of organic solution changes the average size and size distribution of the nanoparticles of the present disclosure encompassing a range from 7.5% to 15% organic solution. The HSA:Drug ratio was held constant at 4.65:1. The average size and distribution of sizes varies with respect to the organic solution percentage are disclosed. 8, 10, and 12% solutions all produced ranges of nanoparticles with similar average sizes and PDIs.

7.5% organic solution produced the largest range in average sizes of nanoparticles with and the largest range of PDIs as well. The smallest average size of a nanoparticle formed with 7.5% organic solution is 283.2 nm and the largest is 821.8 nm. The PDIs range from 0.256 to 0.552. 8% organic solution produced nanoparticles ranging in size from 225.5 to 389.6 nm and PDIs ranging from 0.122 to 0.613. Similarly the 10% and 12 % organic solutions produced nanoparticles that ranged in size from 231 nm to 332 nm and 226 to 460 nm, respectively. The PDIs ranged from 0.126 to 0.298. and 0.122 to 0.290, respectively. Finally, a 15% organic solution produced a range of nanoparticles with sizes from 256 to 382 nm and PDIs of 0.152 to 0.205. Keeping the organic solution percentage between 8 and 12 percent produced the smallest nanoparticles with the smallest size distribution. The organic solution is comprised of two components, dichloromethane (DCM) and benzyl alcohol (BnOH). The ratio of these two components can also be altered. In the 7.5% organic solution the ratio of DCM:BnOH was 9:1, for all other conditions the ratio was 8:2.

TABLE B: 7.5% organic solution

TABLE C: 8% organic solution

TABLE D: 10% organic solution

TABLE E: 12% organic solution

TABLE F: 15% organic solution

SYNTHESIS EXAMPLE 9

Impact of HSA:Drug ratios on nanoparticle properties

The ratio of essentially fatty acid free human serum albumin (HSA) to active pharmaceutical ingredient (drug) can affect the average size, average size distribution (PDI) of our nanoparticles, and to some extent the stability of the nanoparticles. The organic solution percentage was held constant at 8% for all the experiments represented in the following figure. Of all the experiment summarized below, the HSA:Drug ratio of 8:1 produced the smallest nanoparticles on average with the smallest PDI that were stable at room temperature for at least 48 hours.

An HSA:Drug ratio of 3: 1 produced nanoparticles varying in size from 371.45 nm to 1497 nm in average with the lowest PDI of 0.178 and a highest PDI of 0.304. An HSA:Drug ratio Of 4.65:1 produced nanoparticles with a range of sizes from 283 to 822 nm and a range of PDIs from 0.272 to 0.552. With an HSA:Drug ratio of 5:1, nanoparticles ranging in size from 225.5 to 389.7 with the PDIs ranging from 0.136 to 0.613, were produced. By increasing the HSA:Drug ratio to 6: 1 nanoparticles in the range of 204 to 373 nm with a range of PDIs from 0.093 to 0.495 were produced. The HSA:Drug ratio of 8:1 produced nanoparticles that ranged in size from 184 to 256 nm with a range in PDIs from 0.165 to 0.061. Furthermore the nanoparticles were stable at room temperature for 48 hours. The largest HSA:Drug ratio 12:1 also produced the largest nanoparticles on average. The nanoparticles ranged in size from 698 to 3744 nm with PDIs in the range of 0.155 to 0.504.

TABLE G: HSA:Drug ratio 3:1

TABLE H: HSA:Drug ratio 4.65:1

TABLE I: HSA:Drug ratio 5:1 TABLE J: HSA:Drug ratio 6:1

TABLE K: HSA:Drug ratio 8:1

TABLE L: HSA:Drug ratio 12:1 SYNTHESIS EXAMPLE 10

Impact of type of alcohol on nanoparticle formation

During the synthesis of the present nanoparticles, prechilled water is used to “quench” the BnOH and produce nanoparticles. The drugs disclosed herein show similar solubility in DCM:BnOH and DCM:EtOH systems. The impact of using ethanol instead of benzyl alcohol on stability, average size, and size distribution of nanoparticles was explored. Replacing the benzyl alcohol with ethanol produced nanoparticles that ranged in size from 244.85 nm to 463.05 nm.

TABLE M: Nanoparticles formed in EtOH:DCM, no quench

SYNTHESIS EXAMPLE 11

Impact of source of HSA on nanoparticle formulation

In this trial, “regular” human serum albumin (as opposed to acid-free albumin) was used to create nanoparticles in an effort to determine how doing so might affect the average size and size distribution of nanoparticles. Regular albumin is easier to produce, and essentially fatty acid free albumin requires further purification beyond regular HSA. An HSA:Drug ratio of 8:1 and

using an 8% organic solution produced nanoparticles that ranged in size from 1223 to 1865 nm with PDIs in the range of 0.273 to 0.401.

TABLE N: Size and PDI of nanoparticles made with “regular” HSA

SYNTHESIS EXAMPLE 12

Formulations using hydroxypropyl-beta-cyclodextrin (HP-BCD)

_ In particles made with HP-BCD, dynamic light scattering was employed to determine the size of particles present and how narrowly distributed the size of these particles is. In a 5% HP- BCD formulation (with 5 mg/ml C20-1DMT), 10% EtOH added yielded aggregates around 242 nm in diameter, having PDI of 0.319.

_ HP-BCD may be used to assist in solubilizing C20-1DMT. In some instances, 15% HP- BCD may be used in combination with from 10 mg/ml to 15 mg/ml C20-1DMT. In another aspect, 20% HP-BCD may be used in combination with from 15 mg/ml to 20 mg/ml C20-1DMT. In one aspect, 15% HP-BCD may be used in combination with 10 mg/ml C20-1DMT, wherein 0.22 um filtration of the formulation provides particles with a narrow distribution of sizes, or uniform sizes.

Experimental Example 1 Compound stability in PBS and Methanol

SN-38-1MT conjugates were incubated in PBS at 37 °C. At 30, 60, 90, 120, and 240 minutes, 40 pL was taken from the solution for LC-MS/MS analysis. 5 species were monitored: 1O-SN-38-1DMT , 1O-SN-38-1LMT , 20-SN-38-1DMT , 20-SN-38-1LMT , and SN-38. The same procedure was followed for methanol except it was conducted at room temperature. Although not a typical formulation solvent, methanol was used as a representative for other alcohol solvents. The results are summarized in Figures 1 and 2.

Interestingly, we found that the stability of the bond was affected by the location of conjugation. Stability is greater when the 1 -methyl tryptophan is at conjugated at the C20 position than at the CIO position. At the CIO position, hydrolysis occurred quickly, with only 3% or 30% of the parent drug remaining after 30 minutes incubation in phosphate buffered saline (PBS) or methanol, respectively. Conjugation at the 20-hydroxyl group afforded slightly better stability with hydrolysis occurring in PBS, but not in methanol (Figures 1,2). Without being bound by any theory, stability may be reduced because the hydroxy group at CIO is phenolic. C20-1DMT is stable in methanol for more than 4 hours.

Experimental Example 2

Cell Viability

PANC-1 and BxPC-3 cells were purchased from ATCC. On day 0, cells were plated onto 96-well plates at a density of 3,000 cells/well. After 24 hr incubation, a serial dilution of either SN-38 or SN-38-1DMT was added to each well. Cell viability was measured after 72 hr using the MTS assay. Briefly, MTS reagent was added to cell culture media and allowed to incubate for 2 hr in standard culture conditions. Absorbance was measured at 490 nm (Cytation 5). Data was graphed and IC50 values calculated using GraphPad Prism 8.

Treatment of these pancreatic cancer cell lines, PANC-1 and MIA PaCa-2, with the C20- 1DMT conjugate resulted in similar IC50 values compared to SN-38 alone (Figure 3).

Experimental Example 3

SN-38 and 1DMT plasma protein binding

SN-38 and 1DMT were each dissolved in DMSO at 1 mM, 100 pM, and 10 pM concentrations. 5 pL of each test solution was added to 495 pL human or mouse plasma and allowed to incubate for 5 min at 37°C. 200 pL of this solution was added to the sample chamber and 350 pL of PBS was added to the adjacent chamber. The plate was sealed and incubated for 4 hours at 37°C on an orbital shaker operating at 400 rpm. Following incubation, solution samples (40 pL) from each chamber were transferred to a separate Eppendorf tube. To account for differences in solution components, 40 pL PBS was added to the sample collected from the plasma chamber and 40 pL plasma was added to the sample collected from the buffer chamber. 200 pL of internal standard in ice-cold acetonitrile was added to each tube to precipitate proteins. Tubes were centrifuged for 10 mins at 3500g and supernatant was transferred for LC-MS/MS analysis. % Free = (concentration in buffer chamber/concentration in plasma chamber) x 100%. % Bound= 100% - % Free. The results are summarized in Figure 4.

Experimental Example 4 Pharmacokinetics and biodistribution of C20-1DMT Nanoformulations

C57BL/6 mice are subjected to intravenous (i.v.) injection of C20-1DMT nanoparticles at a dose of 10 mg/kg. 50 pL of blood is drawn and collected in heparinized tubes at 0.5, 1, 2, 4, 6, 8, 12 and 24 h. Plasma is separated, mixed with acetonitrile, vortexed, and centrifuged. Supernatant is taken and injected in LC-MS/MS for analyte detection. Data is used to calculate pharmacokinetic parameters. For tumor and tissue biodistribution, tumor-bearing mice are injected (i.v.) with C20- 1DMT NPs (10 mg/kg). Mice are sacrificed at 0.5, 4, 8, and 12 hr. Tumor and 5 tissues (liver, spleen, kidney, heart, and lung) are collected. Tissues are weighed and homogenized with buffer. 50 pL of the resulting homogenate is added to acetonitrile, vortexed, and centrifuged. Supernatant is taken and injected for LC-MS/MS. Levels of 1DMT, SN-38, and C20-1DMT are measured.

Experimental Example 5

Efficacy studies of C20-1DMT Nanoformulations in flank model

Mice are injected subcutaneously with 2xl0 ⁶ Panc-1 cells (200 pL 50% Matrigel matrix) in the lower right flank to induce tumor xenograft. After tumors reached 20 mm ³ , mice are split into 4 groups: vehicle, C20-1DMT mixture, C20-1DMT nanoparticle, and Irinotecan+ 1DMT. Animals received treatment 3 times a week for 2 weeks via I.V. injection (dosage 10 mg/kg SN- 38 and 1DMT). Tumor volumes are measured twice every week. Mice are euthanized when tumors reached >1500 mm ³ .

Experimental Example 6

Efficacy studies of C20-1DMT Nanoformulations in GEMM

A genetically engineered mouse model of pancreatic cancer, KPC, is used to study efficacy of C20-1DMT nanoformulations. After tumor size reaches 5 mm, mice are injected intravenously with C20-1DMT NP at 10 mg/kg. Dosing was either one time or multiple. Tumor volume was measured daily, and mice are sacrificed when tumor volume reached >1000 mm ³ .

In summary, we have created novel drug conjugates between SN-38 and amino acid moieties such as 1-methyl-D-tryptophan to improve the pharmacokinetic properties of SN-38. We have formed a biocompatible, biodegradable nanoformulation of SN-38- 1DMT for the treatment of cancer, specifically pancreatic cancer. Together SN-38 and 1DMT can kill cancer cells, while remodeling the tumor microenvironment to relieve immunosuppression and allow for the infiltration of cytotoxic T cells. Albumin or cyclodextrin, as the carrier, protects the drug from hydrolysis. Albumin also provides tumor targeting capabilities to the nanoformulation. All aspects of the formulation combine to improve drug targeting, drug efficacy, and reduce toxic side effects.

Experimental Example 7

IC50 determination for irinotecan, SN-38, and C20-1DMT in cancer cells

The half-maximal inhibitory concentration (IC50) of three compounds (irinotecan, SN- 38, and C20-1DMT) was determined for five different cell lines representing three different tissues in which cancer occurs. An exemplary protocol for the colon cancer cell lines SW620 and CT26 is provided; similar protocols were employed for all cell lines, though some were plated at 3000 cells/well.

2000 SW620 or CT26 cells/well were plated in 96- well plates and treated with 1, 5, 10, 20, 100, 500, or 1000 nM irinotecan, SN38, or C20-1DMT for 72 hours. Cells were cultured under 5% CO2 in a cell culture incubator with saturated humidity. After the incubation time, the media was removed from each well and replaced with complete media + MTS reagents according to the manufacturer’s protocol. After a one-hour incubation the absorbance at 490 nm was read on a plate reader, and a percent viability normalized to control was calculated for each treatment condition. From these data, IC50 was calculated using GraphPad Prism 9 software.

Results are provided in Table O. In each case SN38 and C20-1DMT are each at least 100 times more potent than irinotecan. Those concentrations marked as ND could not be determined by the method and are presumed to exceed 100 pM. It will be noted that the only cell line in which irinotecan displayed measurable efficacy was SW620, and in this cell line C20-1DMT provides a 4667-fold reduction (99.97%) compared to irinotecan.

TABLE O