PEGylated BF2-Azadipyrromethene (NIR-AZA) fluorophores, for intraoperative imaging

Accepted Manuscript PEGylated BF2-Azadipyrromethene (NIR-AZA) fluorophores, for intraoperative imaging Dan Wu, Harrison C. Daly, Emer Conroy, Bo Li, William M. Gallagher, Ronan A. Cahill, Donal F. O'Shea PII:

S0223-5234(18)30915-2

DOI:

https://doi.org/10.1016/j.ejmech.2018.10.046

Reference:

EJMECH 10832

To appear in:

European Journal of Medicinal Chemistry

Received Date: 21 August 2018 Revised Date:

18 October 2018

Accepted Date: 19 October 2018

Please cite this article as: D. Wu, H.C. Daly, E. Conroy, B. Li, W.M. Gallagher, R.A. Cahill, D.F. O'Shea, PEGylated BF2-Azadipyrromethene (NIR-AZA) fluorophores, for intraoperative imaging, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.10.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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NIR-AZA

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PEGylated BF2-Azadipyrromethene (NIR-AZA) Fluorophores

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for Intraoperative Imaging

Dan Wu,1 Harrison C. Daly,1 Emer Conroy,2 Bo Li,2 William M. Gallagher,2 Ronan A. Cahill,3

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Donal F. O’Shea*1

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Department of Chemistry, RCSI, 123 St. Stephen’s Green, Dublin 2, Ireland.

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School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin,

Belfield, Dublin 4, Ireland. 3

Department of Surgery, Mater Misericordiae University Hospital, and Section of Surgery and

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Surgical Sciences, School of Medicine, University College Dublin, Ireland.

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EMAIL: [email protected]

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Abstract Clinical imaging utilizing near-infrared fluorescence is growing as an intraoperative aid for the decision-making processes during complex surgical procedures. Existing uses include perfusion

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assessment and lymph node identification with many new applications currently being proposed and developed. While imaging hardware and software have significantly progressed in recent times, suitable NIR-fluorophores remain a limiting factor. In this report, we describe the design, synthesis, photophysical characterization and in vivo imaging assessment of new PEGylated

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NIR-fluorophores based on the BF2-azadipyrromethene fluorophore class. The synthetic route includes PEGylation as the final step, thereby allowing routine access to derivatives substituted with different molecular weights of PEG. Absorption and emission wavelength maxima in PBS

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lie at 690 and 720 nm respectively with quantum yields over 12%. photostability and no light induced singlet oxygen production.

They show excellent

A time-course of NIR-

fluorescence imaging, post i.v. administration, in BALB/c mice showed a rapid and preferential accumulation in the renal excretion pathway within 20 min, indicative of potential clinical usage for intraoperative identification of vial structures along this pathway. Assessment with clinical imaging equipment showed the NIR-AZA fluorophores to be wavelength compatible and

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brighter than currently used methylene blue (MB), and that they have the ability to be imaged simultaneously with indocyanine green (ICG) offering a potential for dual colour clinical

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imaging.

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Introduction: The real-time information provided when utilising near-infrared (NIR) fluorescence guided surgery (FGS) can be regarded as a means of enabling a more informed decision-making process

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during surgical procedures [1]. In current practice, intraoperative fluorescence imaging offers several potential avenues for improving surgical outcomes such as assessment of tissue perfusion and viability, the preservation of vital structures that may be inadvertently damaged or guidance for a more complete resection of malignant tissue [2]. Historically, NIR imaging was first

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developed using indocyanine green (ICG) 1 to map blood perfusion specifically for congenital heart defects. This remains the basis for many of its current uses, albeit for different tissues (Fig. 1) [3]. Today it is employed for assessing vascularization following bowel anastomoses [4],

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reconstructive surgery [5], for lymph node mapping in the bowel mesentery, retroperitoneum [6] and axillary breast tissue [7]. The imaging of hepatocellular carcinoma has shown its potential as a means of tumour margin delineation though, as of yet, it remains to be shown useful for malignant growths in other organs [8]. ICG has a very short 4 min half-life in vivo and is excreted exclusively via the bile [9]. This profile is advantageous for blood perfusion, lymph drainage and liver function assessments but prevents its use for imaging of other structures such

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as the renal excretion pathway or non-liver tumor margins. As such, alternative probes are urgently needed with methylene blue (MB) 2 being investigated by several research teams as it is known to be excreted predominately via the renal pathway (Fig. 1). MB is a phenothiazinium dye that was first prepared in 1876 and is on the World Health Organisation’s list of essential

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medicines [10]. Over the years, it has been investigated for many indications though currently its main clinical use is for the treatment of acquired methemoglobinemia, for which its mode of

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action exploits its redox, rather than its dye, properties [11]. Its characteristic strong blue colour facilitates it use as a histological staining and for colorimetric staining of tissues so that they become visible to the naked eye during surgeries. Recently, it has completed Phase III clinical trials as a means to improve detection and visualization of pre-cancerous lesions and polyps during white light colonoscopy, following oral administration [12]. Additionally, it has been tested off-label as a dye to follow lymphatic flow from primary tumors with the naked eye [13]. As a fused heteroaromatic dye, it has an excited state emission centred at 690 nm though its quantum efficiency is weak at less than 4% [14]. Most recently, efforts have begun to exploit these fluorescence properties with clinical trials for intraoperative imaging of parathyroid tissue 3

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to enhance its differentiation from surrounding soft tissue and identification of ureters during open and laparoscopic surgery [15,16]. Progress on these trials is not yet available, but published reports have described the use of MB for ureter imaging in canine and porcine models with

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individual case studies for ureter imaging reported [17,18,19].

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Fig. 1. Structures and photophysical properties of indocyanine green (ICG) 1, methylene blue (MB) 2 BF2-azadipyrromethenes (NIR-AZA) 3 and 4.

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While MB has achieved EMA and FDA approvals, to date none of these are in conjunction with strong excitation light sources which would be a new feature in its clinical use. This should sound a note of caution as MB is a photosensitizer and is known as an effective producer of singlet oxygen [20]. Photosensitizers are dyes that have the ability to transform their excited state energy (following absorption of light) into reactive oxygen species (ROS) such as singlet oxygen. The singlet oxygen quantum yield of MB has been measured as 0.52 in H2O and 0.58 in MeOH [21]. This ability of MB to produce ROS in vivo has prompted several studies of their photodynamic therapeutic (PDT) characteristics. For example, MB has recently been shown experimentally to be an effective PDT agent for the eradication of anti-microbial infections and 4

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biofilms and an effective promoter of cell death for numerous cancer cell lines [22,23,24] with some cases of patient light sensitivity observed when used for imaging [25]. In addition MB rapidly undergoes metabolic reduction by Flavin reductase to a colourless non-fluorescent leucoMB, the degree to which this reduction occurs is known to be variable which adds another

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uncertainty to its general use [26,27].

As the number of indications for FGS continues to grow it would be advantageous if two different wavelength regions were available, as this would permit dual colour techniques to be

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employed. A logical approach would be to centre one of these wavelengths on existing hardware technologies for the fluorophore ICG (λmax fluorescence 818 nm) and add capacity for a second wavelength in the 700-750 nm range. Both wavelength regions offer the benefits of light

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penetration through tissue, high sensitivity and good signal to background noise.

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continuous in line software image analysis, potential applications such as intraoperative anatomical or physiological differentiation by colour coding or identification of smaller metastatic deposits seem close to hand. The interpretation of dynamic multi-colored image sequences utilizing artificial intelligence (AI) and machine learning methods could help pioneer

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the way for autonomous surgeries [28].

Our research efforts have focused on the BF2-azadipyrromethene (NIR-AZA) fluorophore class as they have excellent photophysical characteristics including substituent determined emission maxima between 675 and 800 nm, high quantum yields and exceptional photostability [29]. For

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example, NIR-AZA 3 has emission max at 789 nm and has shown promising results for lymphatic imaging in ex vivo human colonic tissue specimens with clinical instrumentation (Fig.

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1). A minor change in substituents as shown in NIR-AZA 4 produces emission max at 727 nm, derivatives of which have been employed successfully for real-time cellular microscopy and small animal imaging [30,31]. To date, the NIR-AZA class of fluorophore has shown no cellular or in vivo toxicity in the concentration ranges used for imaging, which is a positive indicator for clinical translation [32,33].

NIR-fluorescence based approaches for vital structure identification during operational procedures are potentially important contributors to improving surgical patient outcomes. An important example of this is ureter preservation during lower abdominal surgeries [34,35]. In practice, following i.v. administration at the optimal time point during the surgical procedure, a 5

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rapid renal excretion of fluorescent probe would allow ureter positioning to be controlled for the remainder of the procedure.

Iatrogenic ureteral injury is a serious complication of lower

abdominal surgery and is one of the most serious complications of gynaecologic surgery. Postoperatively recognised ureteral injuries are associated with significant morbidity, loss of

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kidney function and formation of ureterovaginal fistulas. Although such adverse events are rare, with incidents reported from 0.7% to 10%, the severity of ensuing complications would strongly encourage the development of a means to further reduce their occurrence. Clear identification of the ureter in the abdominal region undergoing surgery at the outset of the procedure would be a

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consistently reliable way to avoid unintentional ureteral damage. Additionally, if an assessment for ureteral damage could be performed routinely near completion of the procedure this would

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allow for direct in situ repair if needed thereby limiting post-operative complications. Ideally, probe administration, imaging and clinical decision-making should occur without significantly impeding the normal workflow of the team within the operating room. The focus of this work was to tailor the properties of the NIR-AZA class to have good aqueous solubility and fluorescence quantum yields, and to be compatible with clinical instrumentation that utilises wavelengths in the 670-750 nm spectral range. In this first study, a robust synthetic

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route was developed that could allow the late stage refinement of biological properties as required. As proof of principal, an initial in vivo mouse study was conducted to provide an assessment of fluorescence intensity distributions over time, specifically monitoring the renal pathway, in addition to lymph nodes and tumors. An in vitro imaging assessment was carried

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out to investigate the potential simultaneous use of the new probes in conjunction with ICG.

Synthesis

For this program of work a water-soluble NIR-AZA fluorophore with wavelengths comparable to MB but with superior quantum yield and an ability to promote its excretion via the renal pathway was required. The strategy adopted was to covalently link two poly(ethylene-glycol) (PEG) units onto the fluorophore. Drug functionalization with PEG has played an important and successful role in drug delivery with numerous examples of PEG substituted peptide and small molecule drugs [36,37,38]. It was anticipated that the PEG monomer unit of [O(CH2)2]n would 6

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both solubilise and shield the fluorophore from the surrounding aqueous media. We reasoned that substitution with PEG units would permit the refinement of the pharmacokinetic profile which is strongly influenced by the molecular mass of the PEG used. Studies have shown that PEGylated substrates with mass range of 5-10,000 Da remain for relatively short times in the

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circulatory system and are excreted effectively via urine, while those of 20-30,00 Da range tend to slow down excretion and as such are more favoured as drug delivery vectors [39,40]. For this study, a synthetic route was designed such that the PEG units were introduced at the last step

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thereby allowing maximum flexibility to introduce different sized PEG chains.

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Fig. 2. General structure of PEGylated NIR-AZA 5.

Three amino-PEG derivatives of sizes 3, 5 and 10 kDa were chosen for covalent attachment at two positions on the fluorophore resulting in the final NIR-AZAs as 6 kDa, 10 kDa and 20 kDa PEG conjugates. The synthesis route commenced with an alkylation of bis-phenol substituted

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NIR-AZA 6 [41] with tert-butyl bromoacetate and NaH in dry THF under reflux for 3 h to give the bis-ester substituted derivative 7 in a yield of 83%. Hydrolysis of the ester groups was

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achieved with trifluoroacetic acid (TFA) in dichloromethane (DCM) over 3 h at room temperature, providing the di-carboxylic acid derivative 8 in high yield. Next activated di-ester 9 was produced by an 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) mediated coupling of 8 with N-hydroxysuccinimide. NMR and HRMS analysis confirmed that both carboxylic acids were converted to esters. Coupling reactions proceeded efficiently following the addition of amino-substituted PEG chains of molecular weight 3, 5 or 10 kDa to a DMSO solution containing 9 with reactions monitored by HPLC. In each case the final product 5a-c was isolated in high yields of 80-83% following purification by elution through a Sep Pak C18 reverse-phase column (Scheme 1). 7

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Scheme 1. Synthesis route to PEG substituted NIR-AZAs 5a-c.

Photophysical properties of NIR-AZAs 5a-c were recorded and analysed in comparison with MB

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2. The absorption wavelengths of 5a-c in PBS were all 690 nm and emission maxima at 721(±1) nm showing that the fluorophores readily made aqueous solutions and the length of the PEG chain did not alter these key photophysical characteristics (Fig. 3, Table 1 and SI). Fluorescence

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quantum yields for 5a-c were comparable at 12% and considerably higher than MB (Table 1, entries 1-4). As would be expected, in organic solvent chloroform a small 10 nm hypsochromic shift was observed for both absorbance and emission maxima for all three derivatives with a higher quantum yields of 0.33 (Table 1, entries 5, 6, 7).

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Fig. 3. Normalized absorption (left) and emission (right) spectra of MB 2 (black traces), 5a (blue traces) 5b (red traces), and 5c (dark yellow traces) in PBS at 5 µM.

Table 1. Photophysical property of 2, 5a-c, 7, 8 in PBS and CHCl3.

λmax abs Ext. coeff. λmax flu (nm)a Φflub (nm)a (M-1cm-1) 1 PBS 670 49,500 690 0.016c/0.04d,e 2 2 PBS 690 62,000 722 0.12 5a 3 PBS 690 69,000 720 0.12 5b 4 PBS 690 70,000 720 0.12 5c 5 CHCl3 679 83,000 710 0.32 5a 6 CHCl3 680 86,000 709 0.33 5b 680 86,000 708 0.33 7 CHCl3 5c 8 CHCl3 681 87,000 709 0.34 7 9 CHCl3 681 89,000 711 0.30 8 5 µM. b MB used as reference standard. c in PBS, d in MeOH, e reference 14. Comp

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Entry

The strength of fluorophore emissions are known to be non-linear with respect to concentration due to inner filter effects, which leads to reabsorption of emitted photons. Typically, as concentration of a fluorophore increases, so does its fluorescence intensity until a maximum is reached and then it decreases again due to inner filter effects. To identify this profile, plots of concentration versus integrated emission intensity were constructed for 2 and 5a-c (Fig. 4, SI). Similar profiles were obtained for each of the NIR-AZA derivatives 5a-c with strong intensities observed between concentrations of 1 and 20 µM and maximum intensities at approximately 10 9

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µM. These results are encouraging as they provide a sufficiently broad concentration range for imaging within the renal excretion pathway. A similar profile was observed for MB 2 though the

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absolute intensity was less due to its lower quantum yield.

Fig. 4. Relationships between concentration and emission intensities for 5b and MB 2. Intensities measure in PBS at room temperature. 5b (red trace), 2 (black trace). See SI for plots of 5a, c.

As the acidity and alkalinity of urine can vary significantly between pH 4.5 to 8, the stability of

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emission intensities of 5a-c were checked in this range.

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characteristics was anticipated for this pH range as NIR-AZA fluorophores do not contain acidic or basic sites. This was confirmed by pH titrations of PBS solutions of 5a-c in which the

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emission intensities remained constant (Fig. 5).

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Fig. 5. Insensitivity of emission intensity of 5a-c to varying pH. Measurements taken in PBS at 5 µM fluorophore concentration. 5a (triangles), 5b (solid circle), 5c (open circle). 10

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Fluorescence imaging requires the use of incident light at the wavelength of absorption of the fluorophore to promote its excitation into the excited state. Relaxation from this excited state can occur via the desired pathway by emission of a photon of light, or by loss of energy due to rotational or vibrational motions, or by energy transfer to other adjacent molecules such as

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oxygen. For imaging purposes, the production of highly reactive singlet oxygen would be very undesirable as it is a strong oxidizing agent and known to inflict cellular and tissue damage. In order to test for light induced singlet oxygen production by 5a-c a series of comparative experiments were carried out with MB 2 which is known to have a relatively high singlet oxygen To determine if singlet oxygen is being produced at fluorophore

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quantum yield [21].

concentrations that would be in the range for in vivo imaging, the rate of disappearance of the

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known singlet oxygen trap 1,3-diphenylisobenzofuran (DPBF) in irradiated aqueous methanol solutions of either MB 2 or 5a-c was measured (SI) [42]. This was achieved experimentally by following intensity changes of the 410 nm absorbance band of DPBF over 1 h at in light irradiated solutions containing 10 µM concentration of fluorophore. To mimic light wavelengths used for excitation in a clinical therapeutic setting, a fibre optic delivered light of wavelength 630 (30) nm was employed. A 150 W halogen bulb light source was used at 10% power with

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each experiment repeated in triplicate. Control experiments included the irradiation of DPBF alone and maintaining solutions of fluorophore and DPBF in the dark, both of which showed no change in the intensity of the 410 nm DPBF absorbance band (SI). As anticipated, MB 2 showed a strong production of singlet oxygen with DPBF entirely consumed within 20 min (Fig. 6 A, B).

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In contrast, probes 5a-c showed no singlet oxygen production under identical conditions (Fig. 6 C, D). This is a positive indicator that phototoxicity due to singlet oxygen generation would not

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be a concern with these probes.

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Fig. 6. Photooxidation of DPBF (5 x 10-5 M) with MB 2 and 5b (1 x10-5 M) in PBS/MeOH (1:3). Panels A and B: Spectral and plotted changes in DPBF absorption band of irradiated solution containing MB 2. Panels C and D: Spectral and plotted changes in DPBF absorption

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band of irradiated solution containing 5b. See SI for control experiments, 5a and 5c data.

Next, the photostability of 5a-c was tested in comparison with MB 2 as a reference probe.

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Aqueous solutions at 10 µM concentration were irradiated using the same set up described above for singlet oxygen production testing but at 100% halogen bulb power, with the emission

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intensity of the fluorophores monitored over time.

As expected, MB showed significant

photobleaching with 50% decrease in fluorescence intensity within 2 h (Fig. 7). In contrast, the NIR-AZA derivatives 5a-c all behaved in a similar manner with virtually no change in fluorescence intensity throughout the irradiation. These results are again a positive indicator for their in vivo imaging use and consistent with previous findings for related NIR-AZA compounds [43].

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Fig. 7. Comparative photobleaching experiments for MB 2 (black trace) and 5b (red trace) (1 x10-5 M) in PBS. See SI for 5a, c data.

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In vivo studies

Of the three derivatives synthesised, NIR-AZA 5b was selected for an initial in vivo study using BALB/c mice as it had the required photophysics and with a total of 10 kDa PEGylated mass would be expected to favour a rapid renal excretion. Following a single i.v. tail injection of 5b (2 mg/kg) into three mice, images were acquired every 10 min for the first h, after which organs

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were collected from an individual animal and the remainder followed, with repeated imaging, for a further 24 h. Most encouragingly, a rapid increase in fluorescence from the bladder region, indicative of accumulation of 5b, was observed in all three animals within 20 min. This was maintained with some increase in intensity for 60 min (Fig. 8, see SI for additional animal

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Fig. 8. Time-course of NIR-fluorescence imaging for 60 min post i.v. administration of 5b using a MDA-MB-231 subcutaneous tumor model in BALB/c mice. One representative mouse shown (see SI for images of other two mice). Panel A: NIR fluorescence at indicated time points post i.v. administration of 5b. Panel B: NIR-fluorescence images from panel A with intensity scale adjusted illustrating areas of highest fluorescence. Blue rings indicated ROI positions from which fluorescence intensity values were determined from background liver and bladder at each time point (n=3). Excit. 660–690 nm, emis. 710–730 nm.

Plotting two equal sized ROIs centred on the bladder and liver showed a good signal to

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background fluorescence ratio in favour of the bladder, which increased steadily over the first 60 min (Fig. 9, Panel A). An ex-vivo imaging of resected organs 60 min followwing administration

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of 5b confirmed emission from the bladder, kidneys, liver, lungs and lymph node (SI).

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Fig. 9. Fluorescence intensity analysis of 5b in vivo over time. Panel A: Comparative NIRfluorescence intensity from a bladder (black bars) and liver (grey bars) ROIs as shown in Fig 8 over a 60 min time-period (n=3). Panel B: NIR-Fluorescence intensity values taken from the entire animal at time points over 24 h (n=2).

Imaging of two mice was continue for 24 h which showed at later time points a significant reduction in fluorescence intensity occurred with over 50% reduction in one animal within 5 h and at 10 h in the other (Fig. 9 Panel B, Fig. 10 and SI).

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Fig. 10. Time-course of NIR-fluorescence imaging for 24 h post i.v. administration of 5b using a MDA-MB-231 subcutaneous tumor model in one representative mouse (see SI for images of other mouse). Excit. 660-690 nm, emis. 710-730 nm. Panel A: NIR fluorescence at indicated time points post i.v. administration of 5b. Panel B: NIR-fluorescence images from panel A with intensity scale adjusted illustrating areas of highest fluorescence.

Analysis of fluorescence profile from tumor ROIs showed it to be fluorescent throughout the monitoring time though no significant intensity bias with respect to other tissues observed until 9 h (SI). This would be expected, as retention of PEGylated compounds within tumors is

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recognised as the enhanced permeation and retention effect though this requires a long time-

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period to allow clearance from normal tissues allowing the retained probe to be visualised [44]. As part of our probe assessment, it was important to test compatibility of fluorophores 5 with clinically used instrumentation. Most clinical instrumentations are currently spectroscopically positioned for ICG 1 wavelengths but recently new clinical instrumentation has been developed which can image simultaneously in two NIR-colour zones [45]. The Stryker PINPOINT system has capability to image in MB spectral zones, so we opted to use this instrument to compare the absolute brightness of 5b and MB 2. Well plates containing 15 µM aqueous solutions of both were imaged simultaneously and image analysis software used to quantify the relative brightness with results showing that 5b was over 40% brighter than MB (Fig. 11). 15

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Fig. 11. Comparative brightness study of 5b and 2 at 15 µM concentration using the Stryker PINPOINT instrument. Duplicate identical solutions in two wells for all images. Fluorescence showing in white for clarity.

To date, new NIR-probes (other than MB) designed and tested for ureter imaging have used wavelengths close to those of the currently clinically used ICG [46, 47]. A restriction of using a

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probe with the same emission wavelengths as ICG is that they would not be distinguishable from each other if used at the same time. Simultaneous dual wavelength imaging is not, as of yet, in clinical use though clear advantages could be anticipated if different tissues could be colour coded with probes from different NIR-spectral regions. As such, a preliminary analysis of the

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potential of co-utilising 5b and ICG 1 was investigated. There is approximately a 100 nm difference in absorption λ max for these fluorophores allowing for two distinct excitation

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wavelengths at 690 nm for 5b and 790 nm for 1 (Fig. 12, panel A). Following excitation of solutions of each fluorophore at these wavelengths, clearly distinct emission profiles for both 5b and 1 are obtainable (Fig. 12, panel B).

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Fig. 12. Illustration of potential dual wavelength imaging of 5 and ICG 1 (5 µM in DMSO). Panel A: Normalized overlaid absorption spectra and positions of excitation for 5b (red trace, 690 nm) and ICG 1 (green trace, 790 nm). Panel B: Normalized overlaid fluorescence spectra of 5b (red trace) and ICG 1 (green trace). DMSO was used as solvent as ICG has very low fluorescence quantum yields in water or PBS.

Mixing equal concentration solutions of 1 and 5b and sequentially exciting the mixture at 690 and 790 nm showed that both emission profiles were producible from the mixture. There was

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little spectral interference between the fluorophores, with only a small emission observed at 820 nm (from ICG) when irradiating the solution at 690 nm which can be attributed to the weak absorption of ICG at 690 nm (Fig. 13, panel A). Using the dual wavelength imaging system from Stryker, which can alternate between detecting emissions centred at approximately 700 nm

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and 800 nm, well plates containing solutions of 5b and 1 were imaged using both wavelength zones. Images showed that 5b was only detected with 700 nm emission settings and ICG only

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with 800 nm settings (Fig. 13, panels B and C). Physically mixing of the solutions of 5b and 1 and reimaging alternatively using both wavelength settings showed the solutions were fluorescent in both wavelength zones (panels D and E). While as of yet only illustrative, these results show that the spectral practicalities of clinical imaging in multi-colour are achievable and worthy of further pursuit.

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Fig. 13. Potential for dual colour imaging of 5b with 1 using the Stryker PINPOINT instrument. Panel A: Emission spectra from a mixed solution of 1 and 5b (5 µM for both) in EtOH, sequentially excited at 690 nm and 790 nm. Panel B: Imaging of 700 nm emission from separate wells containing 5b and 1. Panel C: Imaging of 800 nm emission from separate wells containing 5b and 1. Panel D: Imaging of 700 nm emission from wells contained mixed solution of 5b and 1. Panel E: Imaging of 800 nm emission from wells containing mixed solution of 5b and 1. Duplicate identical solutions in two wells for all images. Fluorescence showing in white for clarity.

Conclusion

A general synthetic route to NIR-AZA fluorophores with PEGylated substituents of varying molecular weight has been developed. Aqueous NIR absorption and emission properties range between 690 - 720 nm, which positions them in the correct spectral range for use in fluorescence-guided surgery. Excellent photophysical properties include good quantum yield in aqueous solutions, near-perfect pH- and photostabilities and no detectable singlet oxygen 18

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generation. A time course of NIR-fluorescence imaging in BALB/c mice showed a rapid and preferential accumulation in the renal excretion pathway within 20 min of i.v. administration, indicative of potential clinical usage for intraoperative identification of vial structures along this pathway. Imaging of solutions of 5 and ICG with an instrument designed for simultaneous dual

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colour imaging proved successful. Taken together, the positive results obtained for PEGylated NIR-AZAs are sufficiently encouraging to warrant further assessment in more clinically relevant models, which will be reported on in due course. Additional on-going work includes dual colour

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tissue imaging and the application of AI and machine learning techniques to clinical imaging.

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Experimental Protocols General

All solvents were purified and degassed before use. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm silica gel coated aluminum plates using UV light (254 nm) as visualizing agent. Unless specified, all reagents were used as received without

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further purifications. Chromatographic separations were carried out with silica gel and alumina 90 using flash-column techniques.

1

H NMR and

13

C NMR spectra were recorded at 400 MHz

and 100 MHz respectively, and calibrated using residual non-deuterated solvent as an internal reference. Chemical shifts are reported in parts-per-million (ppm). ESI mass spectra were

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acquired using a microTOF-Q spectrometer interfaced to a Dionex UltiMate 3000 LC in positive and negative modes as required. MicroTof control 3.2. APCI experiments were carried out on a microTOF-Q III spectrometer interfaced to a Dionex UltiMate 3000 LC. Analytical reverse

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phase HPLC was carried out using a Shimadzu Prominence LC system using YMC-triart phenyl column 150 mm x 4.6 mm I.D.S - 5 mm, 12 nm. Absorbance spectra were recorded with a Varian cary 50 scan UV-visible spectrophotometer. Fluorescence spectra were recorded with a Varian cary eclipse fluorescence spectrophotometer. Organic solvents for absorbance and fluorescence experiments were of HPLC quality and milipore filter HPLC grade water was used. SigmaPlot, MestreNova, Chem-Draw and Living Image software were used for data analysis. In vivo imaging experiments conducting using an IVIS spectrum noninvasive quantitative

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molecular imaging system from Caliper life sciences. Images in Figure 11 and 13 taken with a Stryker PINPOINT fluorescence imaging system. Chemistry of

di-t-butyl

2,2'-(((5,5-difluoro-1,9-diphenyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-

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Synthesis

f][1,3,5,2]triazaborinine-3,7-diyl)bis(4,1-phenylene))bis(oxy))diacetate 7.

A solution of 6 (300 mg, 0.57 mmol) and NaH (60% oil dispersion) (91 mg, 3.8 mmol) was stirred in dry THF (28 mL) and treated with t-butylbromoacetate (440 mg, 2.28 mmol) at 0 °C

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under N2. The mixture was allowed warm to rt and heated at reflux for 3 h. The mixture was cooled in an ice bath to 0 °C, saturated aqueous NH4Cl (30 mL) was added and the product

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extracted with ethyl acetate (2 × 40 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and the filtrate concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using CH2Cl2 as eluent affording 7 (358 mg, 83%, mp 121-123 °C) as a red metallic solid. 1H NMR (400 MHz, CDCl3): δ 8.01-7.85 (m, 8H), 7.35-7.23 (m, 6H), 6.94-6.80 (m, 6H), 4.45 (s, 4H), 1.40 (s, 18H) ppm. 13C NMR (100 MHz, CDCl3): δ 167.6, 160.2, 158.0, 145.3, 143.2, 132.4, 131.7, 129.3, 129.3, 128.5, 124.9,

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118.7, 114.8, 82.7, 65.6, 28.1 ppm. HRMS (APCI): calcd. for C44H41BF2N3O6 [M-H]- 756.3056; found 758.3207. Synthesis

of

di-tert-butyl

2,2'-(((5,5-difluoro-1,9-diphenyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-

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f][1,3,5,2]triazaborinine-3,7-diyl)bis(4,1-phenylene))bis(oxy))diacetate 8 [48]. A solution of 7 (200 mg, 0.26 mmol) in dry CH2Cl2 (18 mL) was treated with trifluoroacetic acid (2 mL), the mixture stirred at rt for 3 h under nitrogen and the solvent removed under reduced

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pressure. The residue was treated with CH2Cl2 (5 mL), the suspension placed under sonication for 5 min, filtered and washed with CH2Cl2 (40 mL) to yield the product as a dark red metallic solid 8 (142 mg, 85%, mp 183-184 °C). 1H NMR (400 MHz, DMSO-d6): δ 8.26-8.03 (m, 8H), 7.64-7.42 (m, 8H), 7.13 (d, J = 8.4 Hz, 4H), 4.86 (s, 4H), (OH not observed) ppm.

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C NMR

(100 MHz, DMSO-d6): δ 170.3, 160.9, 157.9, 145.0, 142.7, 132.3, 132.2, 130.1, 129.6, 129.2, 124.1, 120.4, 115.4, 65.0 ppm. HRMS (APCI): calcd. for C36H25BF2N3O6 [M-H]- 644.1810; found 644.1835. Synthesis of NIR-AZA 9. 20

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Compound 8 (160 mg, 0.25 mmol), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (154 mg, 0.99 mmol) and N-hydroxysuccinimide (288 mg, 2.5 mmol) was dissolved in anhydrous DMSO (4 mL) and stirred at rt for 3 h under N2 atmosphere. The solution was partitioned between with CH2Cl2 (50 ml) and 0.5 M HCl (50 ml). The organic phase was

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washed with H2O (50 ml), brine (50 ml), dried over Na2SO4, filtered and evaporated to dryness, keeping the temperature of the bath below 30°C. The product 9 was obtained as a dark green solid without further purification (188 mg, 90%), mp 137-139 °C. 1H NMR (400 MHz, DMSOd6): δ 8.24-8.13 (m, 8H), 7.66 (s, 2H), 7.59-7.44 (m, 6H), 7.23 (d, J = 9.0 Hz, 4H), 5.53 (s, 4H), 19

F NMR (376 MHz, DMSO-d6): -130.34 (q, J = 32.2 Hz) ppm. HRMS

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2.86 (s, 8H) ppm.

(APCI): calcd. for C44H33BF2N5O10 [M+H]+ 840.2289; found 840.2295.

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Synthesis of NIR-AZA 5a.

A mixture of 9 (20 mg, 0.0238 mmol) and O-(2-aminoethyl)polyethylene glycol 3,000 (CAS 32130–27–1) (139 mg, 0.0464 mmol) was dissolved in anhydrous DMSO (2 mL) and stirred at rt for 1 h under a N2 atmosphere. The solvent was removed by lyophilisation and the crude product was partitioned between CH2Cl2 (20 mL) and H2O (20 mL). The aqueous phase was extracted

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with DCM (2×20 mL). The organic layers were combined, washed with aqueous HCl (20 mL, pH 5), water (20 mL), brine (20 mL), dried over anhydrous Na2SO4, filtered and evaporated to dryness. The residue was dissolved in HPLC grade water (10 mL), passed through a Sep Pak C18 reverse-phase column, and freeze-dried. The product 5a was obtained as a dark green solid

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(133 mg, 80%), mp 56-58 °C. 1H NMR (400 MHz, CDCl3): δ 8.20-7.99 (m, 8H), 7.55-7.41 (m, 6H), 7.13-6.98 (m, 8H), 4.59 (s, 4H), 3.65 (s, 820H) ppm. 19F NMR (376 MHz, CDCl3): -131.73

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(q, J = 31.7 Hz) ppm.

Synthesis of NIR-AZA 5b.

A mixture of 9 (20 mg, 0.0238 mmol) and O-(2-aminoethyl)polyethylene glycol 5,000 (CAS 32130–27–1) (232 mg, 0.0464 mmol) was dissolved in anhydrous DMSO (2 mL) and stirred at rt for 1 h under a N2 atmosphere. The solvent was removed by lyophilisation and the crude product partitioned between CH2Cl2 (20 mL) and H2O (20 mL). The aqueous phase was extracted with DCM (2×20 mL). The organic layers were combined, washed with aqueous HCl (20 mL, pH 5), water (20 mL), brine (20 mL), dried over anhydrous Na2SO4, filtered and evaporated to dryness. 21

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The residue was dissolved in HPLC grade water (10 mL), passed through a Sep Pak C18 reversephase column, and freeze-dried. The product 5b was obtained as a dark green solid (217 mg, 83%), mp 62-64 °C. 1H NMR (400 MHz, CDCl3): δ 8.12-8.02 (m, 8H), 7.49-7.39 (m, 6H), 7.087.01 (m, 6H), 4.60 (s, 4H), 3.65 (s, 1370H) ppm (NH not observed).

F NMR (376 MHz,

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CDCl3): -131.45 (q, J = 31.7 Hz) ppm.

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Synthesis of NIR-AZA 5c.

A mixture of 9 (10 mg, 0.0119 mmol) and O-(2-aminoethyl)polyethylene glycol 10,000 (CAS

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32130–27–1) (232 mg, 0.0232 mmol) was dissolved in anhydrous DMSO (1 mL) and stirred at rt for 1 h under a N2 atmosphere. The solvent was removed by lyophilisation and the crude was

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partitioned between DCM (20 mL) and H2O (20 mL). The aqueous phase was extracted with DCM (2×20 mL). The organic layers were combined, washed with aqueous HCl (20 mL, pH 5) water (20 mL), brine (20 mL), dried over anhydrous Na2SO4, filtered and evaporated to dryness. The residue was dissolved in HPLC grade water (10 mL), passed through a Sep Pak C18 reversephase column, and freeze-dried. The product 5c was obtained as a dark green solid (199 mg, 80%), mp 69-71 °C. 1H NMR (400 MHz, CDCl3): δ 8.15-7.96 (m, 8H), 7.50-7.40 (m, 6H), 7.08-

32.0 Hz) ppm.

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F NMR (376 MHz, CDCl3): -131.70 (q, J =

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6.99 (m, 8H), 4.59 (s, 4H), 3.65 (s, 2733H) ppm.

Comparative Singlet-Oxygen Generation Measurements.

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Aerated PBS:MeOH (1:3) solutions, at 20 °C, of methylene blue 2 or 5a-c (1×10-5 M) and 1,3diphenylisobenzofuran (5×10-5 M) were irradiated with a fiber optic delivered and filtered (620 ± 30 nm) light from a 150 W halogen lamp at 10% power for 20 min. Aliquots (2 mL) were

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removed from the reaction mixture at 1 min intervals, and a UV-visible spectrum was recorded. Reaction of 1,3-diphenylisobenzofuran with singlet oxygen was monitored by the reduction in the intensity of the absorption band at 410 nm over time. Irradiation of PBS:MeOH (1:3) DPBF solution (5 × 10-5 M) in the absence of 2 gave no reduction in intensity of the 410 nm absorption band. Photobleaching experiments

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PBS solutions, at 20 °C, of methylene blue 2 (1 × 10-5 M) or 5a-c (4 × 10-6 M) were irradiated with a fiber optic delivered and filtered (620 ± 30 nm) light from a 150 W halogen lamp at 100% power. Photo-stability was determined by measurement of the reduction in fluorescence output

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for the 690 nm emission band for 2 or 720 nm emission bands for 5a-c over 3 h. Emission spectra of mixed solution containing 1 and 5b.

A stock solution of 1 and 5b (1 mM) were prepared in EtOH. 10 µL of stock solution 1 and 5b were mixed in a quartz fluorescence cuvette and diluted to 2 mL with EtOH to give the final

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concentration of 5 µM each for 1 and 5b. Emission spectra were recorded using excitation wavelength of at 690 nm and 790 nm with the slit of 2.5 nm.

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In vivo Imaging Study

MDA-MB-231, human breast adenocarcinoma cell line, was obtained from Caliper Life Sciences. Cells were maintained as a monolayer culture in minimum essential medium containing 10% (v/v) FBS and supplemented with 1% (v/v) L-glutamine, 50 Uml-1 penicillin, 50 µl ml-1 streptomycin, 1% (v/v) sodium pyruvate and 1% (v/v) non-essential amino acids. All cells were

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maintained in 5% CO2 (v/v) and 21% O2 (v/v) at 37 °C. Balb/C mice (Harlan) were housed in the biomedical facility (UCD) in individually ventilated cages in temperature and humidity controlled rooms with a 12 h light-dark cycle. Two to five million MDA-MB-231 cells in 100 ml of a DPBS:Matrigel (50:50) solution were injected subcutaneously behind the fore limb of the 5-

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week-old mice using a 25-g needle. Tumors reached an average diameter of 6 mm before imaging experiments. All animal protocols were approved by University College Dublin’s local

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Animal Research Ethics Committee and under the license from the Department of Health and Children. 5b was dissolved in PBS (200 ml) was administered through the lateral tail vein at a concentration of 2 mg kg-1. Optical imaging was performed with an IVIS Spectrum small-animal in-vivo imaging system (Caliper LS) with integrated isoflurane anaesthesia. Images were acquired at regular intervals post injection of 5b with excitation 675 nm (30 nm band-pass filter) and emission 720 nm (20 nm band-pass filter) narrow band-pass filters and were analysed using Living Image Software v3.0 (Caliper LS). Competing financial interests

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DOS declares the following competing financial interest. Patents have been filed on BF2azadipyrromethene based NIR fluorophores (EP2493898 and US8907107) in which he has a financial interest.

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Acknowledgements DOS gratefully acknowledges Science Foundation Ireland grant number 17/TIDA/4936 for financial support. Thanks to Dr. Gary Hessman (Trinity College Dublin) for MALDI mass

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analysis. Appendix A. Supplementary data

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Highlights

PEGylated NIR-AZA fluorophores

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Excellent photostability and no light induced singlet oxygen production

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Preferential accumulation in the renal excretion pathway

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Intraoperative identification of vial structures

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Dual colour clinical imaging

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1